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Journal articles on the topic 'Viral gene segments'
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1
Wendel, Isabel, Dennis Rubbenstroth, Jennifer Doedt, Georg Kochs, Jochen Wilhelm, Peter Staeheli, Hans-Dieter Klenk, and Mikhail Matrosovich. "The Avian-Origin PB1 Gene Segment Facilitated Replication and Transmissibility of the H3N2/1968 Pandemic Influenza Virus." Journal of Virology 89, no. 8 (January 28, 2015): 4170–79. http://dx.doi.org/10.1128/jvi.03194-14.
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ABSTRACTThe H2N2/1957 and H3N2/1968 pandemic influenza viruses emerged via the exchange of genomic RNA segments between human and avian viruses. The avian hemagglutinin (HA) allowed the hybrid viruses to escape preexisting immunity in the human population. Both pandemic viruses further received the PB1 gene segment from the avian parent (Y. Kawaoka, S. Krauss, and R. G. Webster, J Virol 63:4603–4608, 1989), but the biological significance of this observation was not understood. To assess whether the avian-origin PB1 segment provided pandemic viruses with some selective advantage, either on its own or via cooperation with the homologous HA segment, we modeled by reverse genetics the reassortment event that led to the emergence of the H3N2/1968 pandemic virus. Using seasonal H2N2 virus A/California/1/66 (Cal) as a surrogate precursor human virus and pandemic virus A/Hong Kong/1/68 (H3N2) (HK) as a source of avian-derived PB1 and HA gene segments, we generated four reassortant recombinant viruses and compared pairs of viruses which differed solely by the origin of PB1. Replacement of the PB1 segment of Cal by PB1 of HK facilitated viral polymerase activity, replication efficiency in human cells, and contact transmission in guinea pigs. A combination of PB1 and HA segments of HK did not enhance replicative fitness of the reassortant virus compared with the single-gene PB1 reassortant. Our data suggest that the avian PB1 segment of the 1968 pandemic virus served to enhance viral growth and transmissibility, likely by enhancing activity of the viral polymerase complex.IMPORTANCEDespite the high impact of influenza pandemics on human health, some mechanisms underlying the emergence of pandemic influenza viruses still are poorly understood. Thus, it was unclear why both H2N2/1957 and H3N2/1968 reassortant pandemic viruses contained, in addition to the avian HA, the PB1 gene segment of the avian parent. Here, we addressed this long-standing question by modeling the emergence of the H3N2/1968 virus from its putative human and avian precursors. We show that the avian PB1 segment increased activity of the viral polymerase and facilitated viral replication. Our results suggest that in addition to the acquisition of antigenically novel HA (i.e., antigenic shift), enhanced viral polymerase activity is required for the emergence of pandemic influenza viruses from their seasonal human precursors.
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Kohl, Alain, Anice C. Lowen, Vincent H. J. Léonard, and Richard M. Elliott. "Genetic elements regulating packaging of the Bunyamwera orthobunyavirus genome." Journal of General Virology 87, no. 1 (January 1, 2006): 177–87. http://dx.doi.org/10.1099/vir.0.81227-0.
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The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) comprises three segments of negative-sense, single-stranded RNA. The RNA segments are encapsidated by the viral nucleocapsid (N) protein and form panhandle-like structures through interaction of complementary sequences at their 5′ and 3′ termini. Transcription and replication of a BUN genome analogue (minireplicon), comprising the viral non-coding sequences flanking a reporter gene, requires just the viral RNA polymerase (L protein) and N protein. Here, sequences of Bunyamwera serogroup M segment RNAs were compared and conserved elements within nt 20–33 of the 3′ and 5′ non-coding regions that can affect packaging of minireplicons into virions were identified. RNA-folding models suggest that a conserved sequence within nt 20–33 of the 5′ end of the genome segments maintains conserved structural features necessary for efficient transcription. Competitive packaging experiments using M, L and S segment-derived minireplicons that encode different reporter genes showed variable packaging efficiencies of the three segments. Packaging of a particular segment appeared to be independent of the presence of other segments and, for the S segment, packaging efficiency was unaffected by the inclusion of viral coding sequences in the minireplicon.
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Jan, Fuh-Jyh, Carmen Fagoaga, Sheng-Zhi Pang, and Dennis Gonsalves. "A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing." Journal of General Virology 81, no. 8 (August 1, 2000): 2103–9. http://dx.doi.org/10.1099/0022-1317-81-8-2103.
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We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R2 plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R1 plants were resistant and resistance was not observed in R2 plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.
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Froggatt, Heather M., Kaitlyn N. Burke, Ryan R. Chaparian, Hector A. Miranda, Xinyu Zhu, Benjamin S. Chambers, and Nicholas S. Heaton. "Influenza A virus segments five and six can harbor artificial introns allowing expanded coding capacity." PLOS Pathogens 17, no. 9 (September 27, 2021): e1009951. http://dx.doi.org/10.1371/journal.ppat.1009951.
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Influenza A viruses encode their genomes across eight, negative sense RNA segments. The six largest segments produce mRNA transcripts that do not generally splice; however, the two smallest segments are actively spliced to produce the essential viral proteins NEP and M2. Thus, viral utilization of RNA splicing effectively expands the viral coding capacity without increasing the number of genomic segments. As a first step towards understanding why splicing is not more broadly utilized across genomic segments, we designed and inserted an artificial intron into the normally nonsplicing NA segment. This insertion was tolerated and, although viral mRNAs were incompletely spliced, we observed only minor effects on viral fitness. To take advantage of the unspliced viral RNAs, we encoded a reporter luciferase gene in frame with the viral ORF such that when the intron was not removed the reporter protein would be produced. This approach, which we also show can be applied to the NP encoding segment and in different viral genetic backgrounds, led to high levels of reporter protein expression with minimal effects on the kinetics of viral replication or the ability to cause disease in experimentally infected animals. These data together show that the influenza viral genome is more tolerant of splicing than previously appreciated and this knowledge can be leveraged to develop viral genetic platforms with utility for biotechnology applications.
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Duponchel, Sarah, Cécile Troupin, Lan Trang Vu, Aurélie Schnuriger, Germain Trugnan, and Antoine Garbarg-Chenon. "Transfection of exogenous rotavirus rearranged RNA segments in cells infected with a WT rotavirus results in subsequent gene rearrangements." Journal of General Virology 95, no. 9 (September 1, 2014): 2089–98. http://dx.doi.org/10.1099/vir.0.065573-0.
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Group A rotaviruses, members of the family Reoviridae, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 dsRNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was used previously to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segment 5 or 11 into cells infected with a WT helper rotavirus (bovine strain RF) resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of WT or exogenous segments is a major hindrance to the use of this reverse genetics approach.
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Jan, Fuh-Jyh, Carmen Fagoaga, Sheng-Zhi Pang, and Dennis Gonsalves. "A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance." Microbiology 81, no. 1 (January 1, 2000): 235–42. http://dx.doi.org/10.1099/0022-1317-81-1-235.
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We showed previously that transgenic plants with the green fluorescent protein (GFP) gene fused to segments of the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) displayed post-transcriptional gene silencing of the GFP and N gene segments and resistance to TSWV. These results suggested that a chimeric transgene composed of viral gene segments might confer multiple virus resistance in transgenic plants. To test this hypothesis and to determine the minimum length of the N gene that could trans-inactivate the challenging TSWV, transgenic plants were developed that contained GFP fused with N gene segments of 24–453 bp. Progeny from these plants were challenged with: (i) a chimeric tobacco mosaic virus containing the GFP gene, (ii) a chimeric tobacco mosaic virus with GFP plus the N gene of TSWV and (iii) TSWV. A number of transgenic plants expressing the transgene with GFP fused to N gene segments from 110 to 453 bp in size were resistant to these viruses. Resistant plants exhibited post-transcriptional gene silencing. In contrast, all transgenic lines with transgenes consisting of GFP fused to N gene segments of 24 or 59 bp were susceptible to TSWV, even though the transgene was post-transcriptionally silenced. Thus, virus resistance and post-transcriptional gene silencing were uncoupled when the N gene segment was 59 bp or less. These results provide evidence that multiple virus resistance is possible through the simple strategy of linking viral gene segments to a silencer DNA such as GFP.
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Marsh, Glenn A., Raúl Rabadán, Arnold J. Levine, and Peter Palese. "Highly Conserved Regions of Influenza A Virus Polymerase Gene Segments Are Critical for Efficient Viral RNA Packaging." Journal of Virology 82, no. 5 (December 19, 2007): 2295–304. http://dx.doi.org/10.1128/jvi.02267-07.
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ABSTRACT The genome of the influenza A virus is composed of eight different segments of negative-sense RNA. These eight segments are incorporated into budding virions in an equimolar ratio through a mechanism that is not fully understood. Two different models have been proposed for packaging the viral ribonucleoproteins into newly assembling virus particles: the random-incorporation model and the selective-incorporation model. In the last few years, increasing evidence from many different laboratories that supports the selective-incorporation model has been accumulated. In particular, different groups have shown that some large viral RNA regions within the coding sequences at both the 5′ and 3′ ends of almost every segment are sufficient for packaging foreign RNA sequences. If the packaging regions are crucial for the viability of the virus, we would expect them to be conserved. Using large-scale analysis of influenza A virus sequences, we developed a method of identifying conserved RNA regions whose conservation cannot be explained by population structure or amino acid conservation. Interestingly, the conserved sequences are located within the regions identified as important for efficient packaging. By utilizing influenza virus reverse genetics, we have rescued mutant viruses containing synonymous mutations within these highly conserved regions. Packaging of viral RNAs in these viruses was analyzed by reverse transcription using a universal primer and quantitative PCR for individual segments. Employing this approach, we have identified regions in the polymerase gene segments that, if mutated, result in reductions of more than 90% in the packaging of that particular polymerase viral RNA. Reductions in the level of packaging of a polymerase viral RNA frequently resulted in reductions of other viral RNAs as well, and the results form a pattern of hierarchy of segment interactions. This work provides further evidence for a selective packaging mechanism for influenza A viruses, demonstrating that these highly conserved regions are important for efficient packaging.
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Gao, Qinshan, Man-Seong Park, and Peter Palese. "Expression of Transgenes from Newcastle Disease Virus with a Segmented Genome." Journal of Virology 82, no. 6 (January 16, 2008): 2692–98. http://dx.doi.org/10.1128/jvi.02341-07.
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ABSTRACT Paramyxoviruses belong to the Paramyxoviridae family of the order Mononegavirales. They have a nonsegmented negative-stranded RNA genome and can cause a number of diseases in humans and animals. We generated a recombinant Newcastle disease virus (NDV) possessing a two-segmented genome. Each genomic segment is flanked by authentic NDV 3′ and 5′ noncoding termini allowing for efficient replication and transcription. A reporter gene encoding green fluorescent protein (GFP) was inserted into one segment, and a red fluorescent protein dsRed gene was inserted into the other segment in order to easily detect the replication and transcription of segments in infected cells. The rescued viruses grew well and were stable in embryonated chicken eggs over multiple passages. We were able to detect the expression of both reporter genes in the same cell infected with the virus possessing a segmented genome, and viral particles can contain either one or two types of RNA segments. We also rescued a two-segmented virus expressing GFP and the severe acute respiratory syndrome-associated coronavirus spike S protein, which is about 200 kDa. The chimeric virus extends the coding capacity of NDV by 30%, suggesting that the two-segmented NDV can be used for development of vaccines or gene therapy vectors carrying long and multiple transgenes.
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Gao, Qinshan, Edward W. A. Brydon, and Peter Palese. "A Seven-Segmented Influenza A Virus Expressing the Influenza C Virus Glycoprotein HEF." Journal of Virology 82, no. 13 (April 30, 2008): 6419–26. http://dx.doi.org/10.1128/jvi.00514-08.
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ABSTRACT Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.
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Muramoto, Yukiko, Ayato Takada, Ken Fujii, Takeshi Noda, Kiyoko Iwatsuki-Horimoto, Shinji Watanabe, Taisuke Horimoto, Hiroshi Kida, and Yoshihiro Kawaoka. "Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions." Journal of Virology 80, no. 5 (March 1, 2006): 2318–25. http://dx.doi.org/10.1128/jvi.80.5.2318-2325.2006.
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ABSTRACT The genome of influenza A viruses comprises eight negative-strand RNA segments. Although all eight segments must be present in cells for efficient viral replication, the mechanism(s) by which these viral RNA (vRNA) segments are incorporated into virions is not fully understood. We recently found that sequences at both ends of the coding regions of the HA, NA, and NS vRNA segments of A/WSN/33 play important roles in the incorporation of these vRNAs into virions. In order to similarly identify the regions of the PB2, PB1, and PA vRNAs of this strain that are critical for their incorporation, we generated a series of mutant vRNAs that possessed the green fluorescent protein gene flanked by portions of the coding and noncoding regions of the respective segments. For all three polymerase segments, deletions at the ends of their coding regions decreased their virion incorporation efficiencies. More importantly, these regions not only affected the incorporation of the segment in which they reside, but were also important for the incorporation of other segments. This effect was most prominent with the PB2 vRNA. These findings suggest a hierarchy among vRNA segments for virion incorporation and may imply intersegment association of vRNAs during virus assembly.
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Soldan, Samantha S., Matthew L. Plassmeyer, Meghan K. Matukonis, and Francisco González-Scarano. "La Crosse Virus Nonstructural Protein NSs Counteracts the Effects of Short Interfering RNA." Journal of Virology 79, no. 1 (January 1, 2005): 234–44. http://dx.doi.org/10.1128/jvi.79.1.234-244.2005.
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ABSTRACT Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system.
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Michalakis, Yannis, and Stéphane Blanc. "The Curious Strategy of Multipartite Viruses." Annual Review of Virology 7, no. 1 (September 29, 2020): 203–18. http://dx.doi.org/10.1146/annurev-virology-010220-063346.
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Multipartite virus genomes are composed of several segments, each packaged in a distinct viral particle. Although this puzzling genome architecture is found in ∼17% of known viral species, its distribution among hosts or among distinct types of genome-composing nucleic acid remains poorly understood. No convincing advantage of multipartitism has been identified, yet the maintenance of genomic integrity appears problematic. Here we review recent studies shedding light on these issues. Multipartite viruses rapidly modify the copy number of each segment/gene from one host species to another, a putative benefit if host switches are common. One multipartite virus functions in a multicellular way: The segments do not all need to be present in the same cell and can functionally complement across cells, maintaining genome integrity within hosts. The genomic integrity maintenance during host-to-host transmission needs further elucidation. These features challenge several virology foundations and could apply to other multicomponent viral systems.
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Catchpole, A. P., L. J. Mingay, E. Fodor, and G. G. Brownlee. "Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene." Journal of General Virology 84, no. 3 (March 1, 2003): 507–15. http://dx.doi.org/10.1099/vir.0.18795-0.
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The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U–A replacing G–C at nucleotide positions 12′–11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.
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Doucet, Daniel, Anic Levasseur, Catherine Béliveau, Renée Lapointe, Don Stoltz, and Michel Cusson. "In vitro integration of an ichnovirus genome segment into the genomic DNA of lepidopteran cells." Journal of General Virology 88, no. 1 (January 1, 2007): 105–13. http://dx.doi.org/10.1099/vir.0.82314-0.
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Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.
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Bonneau, K. R., B. A. Mullens, and N. J. MacLachlan. "Occurrence of Genetic Drift and Founder Effect during Quasispecies Evolution of the VP2 and NS3/NS3A Genes of Bluetongue Virus upon Passage between Sheep, Cattle, andCulicoides sonorensis." Journal of Virology 75, no. 17 (September 1, 2001): 8298–305. http://dx.doi.org/10.1128/jvi.75.17.8298-8305.2001.
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ABSTRACT Bluetongue virus (BTV) is the cause of an insect-transmitted virus infection of ruminants that occurs throughout much of the world. Individual gene segments differ between field strains of BTV; thus, we hypothesized that key viral genes undergo genetic drift during alternating passage of BTV in its ruminant and insect hosts. To test this hypothesis, variation in the consensus sequence and quasispecies heterogeneity of the VP2 and NS3/NS3A genes of a plaque-purified strain of BTV serotype 10 was determined during alternating infection of vector Culicoides sonorensis and a sheep and calf. Consensus sequences were determined after reverse transcriptase-nested PCR amplification of viral RNA directly from ruminant blood and homogenized insects, and quasispecies heterogeneity was determined by the sequencing of clones derived from directly amplified viral RNA. Comparison of these sequences to those of the original BTV inoculum used to initiate the cycle of BTV infection demonstrated, for the first time, that individual BTV gene segments evolve independently of one another by genetic drift in a host-specific fashion, generating quasispecies populations in both ruminant and insect hosts. Furthermore, a unique viral variant was randomly ingested by C. sonorensis insects that fed on a sheep with low-titer viremia, thereby fixing a novel genotype by founder effect. Thus, we conclude that genetic drift and founder effect contribute to diversification of individual gene segments of field strains of BTV.
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Inagaki, Arisa, Hideo Goto, Satoshi Kakugawa, Makoto Ozawa, and Yoshihiro Kawaoka. "Competitive Incorporation of Homologous Gene Segments of Influenza A Virus into Virions." Journal of Virology 86, no. 18 (June 27, 2012): 10200–10202. http://dx.doi.org/10.1128/jvi.01204-12.
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By using two reporter protein-encoding virus-like RNAs derived from identical viral RNA (vRNA) segments, we assessed their incorporation efficiency into single progeny virions. Most plaques formed by the recombinant viruses that were generated in cells positive for both reporter genes expressed only one or the other protein. These results suggest that two virus-like RNAs encoding different reporter proteins compete for incorporation into virions, and individual influenza virions incorporate single, but not multiple, copies of homologous vRNA segments.
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Wasilenko, Jamie L., Chang Won Lee, Luciana Sarmento, Erica Spackman, Darrell R. Kapczynski, David L. Suarez, and Mary J. Pantin-Jackwood. "NP, PB1, and PB2 Viral Genes Contribute to Altered Replication of H5N1 Avian Influenza Viruses in Chickens." Journal of Virology 82, no. 9 (February 27, 2008): 4544–53. http://dx.doi.org/10.1128/jvi.02642-07.
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ABSTRACT The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.
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Nelson, Martha I., Susan E. Detmer, David E. Wentworth, Yi Tan, Aaron Schwartzbard, Rebecca A. Halpin, Timothy B. Stockwell, et al. "Genomic reassortment of influenza A virus in North American swine, 1998–2011." Journal of General Virology 93, no. 12 (December 1, 2012): 2584–89. http://dx.doi.org/10.1099/vir.0.045930-0.
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Revealing the frequency and determinants of reassortment among RNA genome segments is fundamental to understanding basic aspects of the biology and evolution of the influenza virus. To estimate the extent of genomic reassortment in influenza viruses circulating in North American swine, we performed a phylogenetic analysis of 139 whole-genome viral sequences sampled during 1998–2011 and representing seven antigenically distinct viral lineages. The highest amounts of reassortment were detected between the H3 and the internal gene segments (PB2, PB1, PA, NP, M and NS), while the lowest reassortment frequencies were observed among the H1γ, H1pdm and neuraminidase segments, particularly N1. Less reassortment was observed among specific haemagglutinin–neuraminidase combinations that were more prevalent in swine, suggesting that some genome constellations may be evolutionarily more stable.
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Kobayashi, Takeshi, James D. Chappell, Pranav Danthi, and Terence S. Dermody. "Gene-Specific Inhibition of Reovirus Replication by RNA Interference." Journal of Virology 80, no. 18 (September 15, 2006): 9053–63. http://dx.doi.org/10.1128/jvi.00276-06.
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ABSTRACT Mammalian reoviruses contain a genome of 10 segments of double-stranded RNA (dsRNA). Reovirus replication and assembly occur within distinct structures called viral inclusions, which form in the cytoplasm of infected cells. Viral nonstructural proteins μNS and σNS and core protein μ2 play key roles in forming viral inclusions and recruiting other viral proteins and RNA to these structures for replication and assembly. However, the precise functions of these proteins in viral replication are poorly defined. Therefore, to better understand the functions of reovirus proteins associated with formation of viral inclusions, we used plasmid-based vectors to establish 293T cell lines stably expressing small interfering RNAs (siRNAs) specific for transcripts encoding the μ2, μNS, and σNS proteins of strain type 3 Dearing (T3D). Infectivity assays revealed that yields of T3D, but not those of strain type 1 Lang, were significantly decreased in 293T cells stably expressing μ2, μNS, or σNS siRNA. Stable expression of siRNAs specific for any one of these proteins substantially diminished viral dsRNA, protein synthesis, and inclusion formation, indicating that each is a critical component of the viral replication machinery. Using cell lines stably expressing μNS siRNA, we developed a complementation system to rescue viral replication by transient transfection with recombinant T3D μNS in which silent mutations were introduced into the sequence targeted by the μNS siRNA. Furthermore, we demonstrated that μNSC, which lacks the first 40 amino residues of μNS, is incapable of restoring reovirus growth in the complementation system. These results reveal interdependent functions for viral inclusion proteins and indicate that cell lines stably expressing reovirus siRNAs are useful tools for the study of viral protein structure-function relationships.
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Charpentier, Charlotte, Tamara Nora, Olivier Tenaillon, François Clavel, and Allan J. Hance. "Extensive Recombination among Human Immunodeficiency Virus Type 1 Quasispecies Makes an Important Contribution to Viral Diversity in Individual Patients." Journal of Virology 80, no. 5 (March 1, 2006): 2472–82. http://dx.doi.org/10.1128/jvi.80.5.2472-2482.2006.
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ABSTRACT Although recombination during human immunodeficiency virus type 1 (HIV-1) replication in vitro and in vivo has been documented, little information is available concerning the extent that recombination contributes to the diversity of HIV-1 quasispecies in the course of infection in individual patents. To investigate the impact of recombination on viral diversity, we developed a technique that permits the isolation of contemporaneous clonal viral populations resulting from single infectious events by plasma-derived viruses, thereby permitting the assessment of recombination throughout the viral genomes, including widely separated loci, from individual patients. A comparison of the genomic sequences of clonal viruses from six patients, including patients failing treatment with antiretroviral therapy, demonstrated strong evidence for extensive recombination. Recombination increased viral diversity through two distinct mechanisms. First, evolutionary bottlenecks appeared to be restricted to minimal segments of the genome required to obtain selective advantage, thereby preserving diversity in adjacent regions. Second, recombination between adjacent gene segments appeared to generate diversity in both pol and env genes. Thus, the shuffling of resistance mutations within the genes coding for the protease and reverse transcriptase, as well as recombination between these regions, could increase the diversity of drug resistance genotypes. These findings demonstrate that recombination in HIV-1 contributes to the diversity of viral quasispecies by restricting evolutionary bottlenecks to gene segments and by generating novel genotypes in pol and env, supporting the idea that recombination may be critical to adaptive evolution of HIV in the face of constantly moving selective pressures, whether exerted by the immune system or antiretroviral therapy.
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Holwerda, Melle, Laura Laloli, Manon Wider, Lutz Schönecker, Jens Becker, Mireille Meylan, and Ronald Dijkman. "Establishment of a Reverse Genetic System from a Bovine Derived Influenza D Virus Isolate." Viruses 13, no. 3 (March 18, 2021): 502. http://dx.doi.org/10.3390/v13030502.
Full textAbstract:
The ruminant-associated influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5′ and 3′ rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5′ and 3′ NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5′ and 3′ non-coding regions (NCR) highlighted that the terminal 3′ end of all segments harbours an uracil instead of a cytosine nucleotide, similar to other influenza viruses. Subsequent analysis on the functionality of the 5′ and 3′ NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5′ and 3′ NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin, as well as by using an in vitro model recapitulating the natural replication site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.
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Le Sage, Valerie, Adalena Nanni, Amar Bhagwat, Dan Snyder, Vaughn Cooper, Seema Lakdawala, and Nara Lee. "Non-Uniform and Non-Random Binding of Nucleoprotein to Influenza A and B Viral RNA." Viruses 10, no. 10 (September 25, 2018): 522. http://dx.doi.org/10.3390/v10100522.
Full textAbstract:
The genomes of influenza A and B viruses have eight, single-stranded RNA segments that exist in the form of a viral ribonucleoprotein complex in association with nucleoprotein (NP) and an RNA-dependent RNA polymerase complex. We previously used high-throughput RNA sequencing coupled with crosslinking immunoprecipitation (HITS-CLIP) to examine where NP binds to the viral RNA (vRNA) and demonstrated for two H1N1 strains that NP binds vRNA in a non-uniform, non-random manner. In this study, we expand on those initial observations and describe the NP-vRNA binding profile for a seasonal H3N2 and influenza B virus. We show that, similar to H1N1 strains, NP binds vRNA in a non-uniform and non-random manner. Each viral gene segment has a unique NP binding profile with areas that are enriched for NP association as well as free of NP-binding. Interestingly, NP-vRNA binding profiles have some conservation between influenza A viruses, H1N1 and H3N2, but no correlation was observed between influenza A and B viruses. Our study demonstrates the conserved nature of non-uniform NP binding within influenza viruses. Mapping of the NP-bound vRNA segments provides information on the flexible NP regions that may be involved in facilitating assembly.
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Baranovskaya, I. L., M. V. Sergeeva, A. S. Taraskin, A. A. Lozhkov, and A. V. Vasin. "Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo." Microbiology Independent Research Journal 8, no. 1 (March 18, 2021): 1–9. http://dx.doi.org/10.18527/2500-2236-2021-8-1-1-9.
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The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.
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Schnepf, Nathalie, Claire Deback, Axelle Dehee, Elyanne Gault, Nathalie Parez, and Antoine Garbarg-Chenon. "Rearrangements of Rotavirus Genomic Segment 11 Are Generated during Acute Infection of Immunocompetent Children and Do Not Occur at Random." Journal of Virology 82, no. 7 (January 23, 2008): 3689–96. http://dx.doi.org/10.1128/jvi.01770-07.
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ABSTRACT Group A rotaviruses are the main cause of viral gastroenteritis in infants. The viral genome consists of 11 double-stranded RNA (dsRNA) segments. Dysfunction of the viral RNA polymerase can lead to gene rearrangements, which most often consist of partial sequence duplication of a dsRNA segment. Gene rearrangements have been detected in vivo during chronic infection in immunodeficient children or in vitro during passages at a high multiplicity of infection in cell culture, suggesting that these replication conditions lead to selective advantages favoring the recovery of viruses with rearranged genes. During acute rotavirus infection, the replication level is high, but the occurrence of rearrangement events has never been reported. By the use of a reverse transcription-PCR assay specifically designed to detect small numbers of copies of rearranged forms of segment 11 in a high background of its standard counterpart, we detected 12 rearrangement events among 161 cases (7.5%) of acute rotavirus infection in immunocompetent children. Strikingly, in all but one case, rearrangement took place at the same location within the short direct repeat AUGU sequence. For the unique case with a different rearrangement pattern, the rearrangement occurred within the direct repeat ACAAGUC that was specific for this isolate. In conclusion, we report the occurrence of segment 11 rearrangements during acute rotavirus infection in immunocompetent children. We show that under such conditions of infection, the viral RNA polymerase generates rearrangements which occur not at random but within direct repeats which might constitute hot spots for RNA recombination.
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Ikegami, Tetsuro, C. J. Peters, and Shinji Makino. "Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System." Journal of Virology 79, no. 9 (May 1, 2005): 5606–15. http://dx.doi.org/10.1128/jvi.79.9.5606-5615.2005.
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ABSTRACT Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-α/β) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-α/β production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.
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Katz, R. A., B. R. Cullen, R. Malavarca, and A. M. Skalka. "Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control." Molecular and Cellular Biology 6, no. 2 (February 1986): 372–79. http://dx.doi.org/10.1128/mcb.6.2.372.
Full textAbstract:
Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation.
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Katz, R. A., B. R. Cullen, R. Malavarca, and A. M. Skalka. "Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control." Molecular and Cellular Biology 6, no. 2 (February 1986): 372–79. http://dx.doi.org/10.1128/mcb.6.2.372-379.1986.
Full textAbstract:
Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation.
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Shepherd, Frances, Diana Herrera-Ibata, Elizabeth Porter, Nitipong Homwong, Richard Hesse, Jianfa Bai, and Douglas Marthaler. "Whole Genome Classification and Phylogenetic Analyses of Rotavirus B strains from the United States." Pathogens 7, no. 2 (April 18, 2018): 44. http://dx.doi.org/10.3390/pathogens7020044.
Full textAbstract:
Rotaviruses (RVs) are a major etiological agent of acute viral gastroenteritis in humans and young animals, with rotavirus B (RVB) often detected in suckling and weaned pigs. Group A rotavirus classification is currently based on the two outer capsid proteins, VP7 and VP4, and the middle layer protein, VP6. Using RVB strains generated in this study and reference sequences from GenBank, pairwise identity frequency graphs and phylogenetic trees were constructed for the eleven gene segments of RVB to estimate the nucleotide identity cutoff values for different genotypes and determine the genotype diversity per gene segment. Phylogenetic analysis of VP7, VP4, VP6, VP1–VP3, and NSP1–NSP5 identified 26G, 5P, 13I, 5R, 5C, 5M, 8A, 10N, 6T, 4E, and 7H genotypes, respectively. The analysis supports the previously proposed cutoff values for the VP7, VP6, NSP1, and NSP3 gene segments (80%, 81%, 76% and 78%, respectively) and suggests new cutoff values for the VP4, VP1, VP2, VP3, NSP2, NSP4, and NSP5 (80%, 78%, 79%, 77% 83%, 76%, and 79%, respectively). Reassortment events were detected between the porcine RVB strains from our study. This research describes the genome constellations for the complete genome of Group B rotaviruses in different host species.
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Poon, Leo L. M., Polly W. Y. Mak, Olive T. W. Li, Kwok Hung Chan, Chung Lam Cheung, Edward S. Ma, Hui-Ling Yen, Dhanasekaran Vijaykrishna, Yi Guan, and J. S. Malik Peiris. "Rapid Detection of Reassortment of Pandemic H1N1/2009 Influenza Virus." Clinical Chemistry 56, no. 8 (August 1, 2010): 1340–44. http://dx.doi.org/10.1373/clinchem.2010.149179.
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BACKGROUND Influenza viruses can generate novel reassortants in coinfected cells. The global circulation and occasional introductions of pandemic H1N1/2009 virus in humans and in pigs, respectively, may allow this virus to reassort with other influenza viruses. These possible reassortment events might alter virulence and/or transmissibility of the new reassortants. Investigations to detect such possible reassortants should be included as a part of pandemic influenza surveillance plans. METHODS We established a real-time reverse-transcription (RT)-PCR–based strategy for the detection of reassortment of pandemic H1N1/2009 virus. Singleplex SYBR green–based RT-PCR assays specific for each gene segment of pandemic H1N1/2009 were developed. These assays were evaluated with influenza viruses of various genetic backgrounds. RESULTS All human pandemic H1N1 (n = 27) and all seasonal human (n = 58) isolates were positive and negative, respectively, for all 8 segments. Of 48 swine influenza viruses isolated from our ongoing surveillance program of influenza viruses in swine, 10 were positive in all reactions. All 8 viral segments of these 10 samples were confirmed to be of pandemic H1N1 origin, indicating that these were caused by zoonotic transmissions from human to pigs. The 38 swine viruses that were nonpandemic H1N1/2009 had 1–6 gene segments positive in the tests. Further characterization of these nonpandemic H1N1/2009 swine viruses indicated that these PCR-positive genes were the precursor genes of the pandemic H1N1/2009 virus. CONCLUSIONS Our results demonstrated that these assays can detect reintroductions of pandemic H1N1/2009 virus in pigs. These assays might be useful screening tools for identifying viral reassortants derived from pandemic H1N1/2009 or its precursors.
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Kobayashi, Takeshi, Laura S. Ooms, James D. Chappell, and Terence S. Dermody. "Identification of Functional Domains in Reovirus Replication Proteins μNS and μ2." Journal of Virology 83, no. 7 (January 28, 2009): 2892–906. http://dx.doi.org/10.1128/jvi.01495-08.
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ABSTRACT Mammalian reoviruses are nonenveloped particles containing a genome of 10 double-stranded RNA (dsRNA) gene segments. Reovirus replication occurs within viral inclusions, which are specialized nonmembranous cytoplasmic organelles formed by viral nonstructural and structural proteins. Although these structures serve as sites for several major events in the reovirus life cycle, including dsRNA synthesis, gene segment assortment, and genome encapsidation, biochemical mechanisms of virion morphogenesis within inclusions have not been elucidated because much remains unknown about inclusion anatomy and functional organization. To better understand how inclusions support viral replication, we have used RNA interference (RNAi) and reverse genetics to define functional domains in two inclusion-associated proteins, μNS and μ2, which are interacting partners essential for inclusion development and viral replication. Removal of μNS N-terminal sequences required for association with μ2 or another μNS-binding protein, σNS, prevented the capacity of μNS to support viral replication without affecting inclusion formation, indicating that μNS-μ2 and μNS-σNS interactions are necessary for inclusion function but not establishment. In contrast, introduction of changes into the μNS C-terminal region, including sequences that form a putative oligomerization domain, precluded inclusion formation as well as viral replication. Mutational analysis of μ2 revealed a critical dependence of viral replication on an intact nucleotide/RNA triphosphatase domain and an N-terminal cluster of basic amino acid residues conforming to a nuclear localization motif. Another domain in μ2 governs the capacity of viral inclusions to affiliate with microtubules and thereby modulates inclusion morphology, either globular or filamentous. However, viral variants altered in inclusion morphology displayed equivalent replication efficiency. These studies reveal a modular functional organization of inclusion proteins μNS and μ2, define the importance of specific amino acid sequences and motifs in these proteins for viral replication, and demonstrate the utility of complementary RNAi-based and reverse genetic approaches for studies of reovirus replication proteins.
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Troupin, Cécile, Axelle Dehée, Aurélie Schnuriger, Patrice Vende, Didier Poncet, and Antoine Garbarg-Chenon. "Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses." Journal of Virology 84, no. 13 (April 28, 2010): 6711–19. http://dx.doi.org/10.1128/jvi.00547-10.
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ABSTRACT Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.
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Boussier, Jeremy, Sandie Munier, Bernadette Crescenzo-Chaigne, Sylvie Behillil, Vincent Enouf, Sylvie van der Werf, and Nadia Naffakh. "Detection and Quantification of Influenza Virus Defective Viral Genomes from NGS Datasets Obtained after RT or RT-PCR Product Sequencing." Proceedings 50, no. 1 (June 17, 2020): 84. http://dx.doi.org/10.3390/proceedings2020050084.
Full textAbstract:
Like most RNA viruses, influenza viruses (IAV) generate defective viral genomes (DVGs) during viral replication. Although there is accumulating evidence of a biological impact of DVGs, the molecular mechanisms leading to their production remain to be unveiled. Various next-generation sequencing (NGS) technologies and detection methods can be used to characterize DVGs. Here, we developed a bioinformatics pipeline called DG-seq to quickly identify and quantify DVGs in influenza viral stocks and compared two processing methods for NGS, with or without PCR amplification. To evaluate the performance of the DG-seq pipeline, we used either synthetic in-vitro-transcribed DVGs mixed with the full set of synthetic full-length genomic RNAs, or biological RNA samples extracted in duplicate from three IAV stocks: mutant viruses with a K635A or a R638A mutation in the PA subunit of the polymerase that impairs viral transcription, and their wild-type (WT) counterpart. Viral genomic RNAs were reverse-transcribed and either directly subjected to Illumina sequencing (RT-seq) or PCR-amplified prior to sequencing (RT-PCR-seq). Both methods displayed a good reproducibility between batches, with a lower detection rate but a more accurate quantification of DVGs in RT-seq samples. The PA mutants produced more DVGs than the WT virus, derived mostly from the polymerase gene segments, but also from the NA and HA segments, suggesting that an imbalance between transcription and replication can promote DVG production. Breakpoints occurred near the segment extremities, with no hotspot identified. Interestingly, we observed short direct A/T-rich repeats adjacent to the breakpoint ends at a significantly higher frequency than in the random case. This work provides the first comparison of DVG detection and quantification from NGS data obtained in the presence or absence of PCR amplification and gives novel insight into the mechanisms of influenza virus DVG production.
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Wang, Fangzheng, Guanqun Liu, Yao Lu, Magda Hlasny, Qiang Liu, and Yan Zhou. "Acquisition of Avian-Origin PB1 Facilitates Viral RNA Synthesis by the 2009 Pandemic H1N1 Virus Polymerase." Viruses 12, no. 3 (February 28, 2020): 266. http://dx.doi.org/10.3390/v12030266.
Full textAbstract:
The constant crosstalk between the large avian reservoir of influenza A viruses (IAV) and its mammalian hosts drives viral evolution and facilitates their host switching. Direct adaptation of an avian strain to human or reassortment between avian-origin gene segments with that of human strains are the two mechanisms for the emergence of pandemic viruses. While it was suggested that the 1918 pandemic virus is of avian origin, reassortment of 1918 human isolates and avian influenza viruses led to the generation of 1957 and 1968 pandemic viruses. Interestingly, the avian PB1 segment, which encodes the catalytic subunit of IAV polymerase, is present in the 1957 and 1968 pandemic viruses. The biological consequence and molecular basis of such gene exchange remain less well understood. Using the 2009 pandemic H1N1 virus as a model, whose polymerase contains a human-origin PB1 subunit, we demonstrate that the acquisition of an avian PB1 markedly enhances viral RNA synthesis. This enhancement is also effective in the absence of PB2 adaptive mutations, which are key determinants of host switching. Mechanistically, the avian-origin PB1 does not appear to affect polymerase assembly but imparts the reassorted pandemic polymerase-augmented viral primary transcription and replication. Moreover, compared to the parental pandemic polymerase, the reassorted polymerase displays comparable complementary RNA (cRNA)-stabilizing activity but is specifically enhanced in progeny viral RNA (vRNA) synthesis from cRNA in a trans-activating manner. Overall, our results provide the first insight into the mechanism via which avian-origin PB1 enhances viral RNA synthesis of the 2009 pandemic virus polymerase.
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Pilewski, J. M., J. F. Engelhardt, J. E. Bavaria, L. R. Kaiser, J. M. Wilson, and S. M. Albelda. "Adenovirus-mediated gene transfer to human bronchial submucosal glands using xenografts." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 4 (April 1, 1995): L657—L665. http://dx.doi.org/10.1152/ajplung.1995.268.4.l657.
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The cystic fibrosis (CF) transmembrane conductance regulator has been localized to both submucosal glands and surface epithelium, suggesting that both glandular and surface epithelium may be important targets for gene therapy. To determine the distribution and efficiency of recombinant adenovirus-mediated gene transfer to human airway submucosal glands, an in vivo model was developed by heterotopically transplanting human bronchial segments from both normal and CF lung tissue into severe combined immunodeficient mice. A serotype 5 E1-deleted recombinant adenovirus containing a lacZ reporter gene driven by the cytomegalovirus promoter (H5.010CMVlacZ) was used to infect the xenografts. Transgene expression was correlated with three factors: 1) viral titer, 2) penetration of microspheres, and 3) dwell time of the viral instillate. At viral titers ranging from 10(8) to 10(11) plaque forming units/ml, expression of the lacZ gene was observed in a subpopulation of epithelial cells within approximately 40% of submucosal glands, with more efficient gene transfer to the ducts than the secretory tubules. Within individual glands, gene transfer varied from < 1% to approximately 20% of submucosal cells, including duct, mucous, and serous cells. Adenovirus-sized fluorescent microspheres were found to penetrate only some of the submucosal glands, suggesting that the gene transfer efficiency to submucosal tubules is due to limited viral particle penetration rather than tropism. Gene transfer to surface epithelial cells was inefficient. However, the percentage of transduced surface epithelial cells increased from < 1% to 5–10% as the dwell time of viral solution was increased from 5 min to 1 h, indicating that the time allowed for virus binding and entry is important for gene transfer efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)
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Bézier, Annie, Faustine Louis, Séverine Jancek, Georges Periquet, Julien Thézé, Gabor Gyapay, Karine Musset, et al. "Functional endogenous viral elements in the genome of the parasitoid wasp Cotesia congregata : insights into the evolutionary dynamics of bracoviruses." Philosophical Transactions of the Royal Society B: Biological Sciences 368, no. 1626 (September 19, 2013): 20130047. http://dx.doi.org/10.1098/rstb.2013.0047.
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Bracoviruses represent the most complex endogenous viral elements (EVEs) described to date. Nudiviral genes have been hosted within parasitoid wasp genomes since approximately 100 Ma. They play a crucial role in the wasp life cycle as they produce bracovirus particles, which are injected into parasitized lepidopteran hosts during wasp oviposition. Bracovirus particles encapsidate multiple dsDNA circles encoding virulence genes. Their expression in parasitized caterpillars is essential for wasp parasitism success. Here, we report on the genomic organization of the proviral segments (i.e. master sequences used to produce the encapsidated dsDNA circles) present in the Cotesia congregata parasitoid wasp genome. The provirus is composed of a macrolocus, comprising two-thirds of the proviral segments and of seven dispersed loci, each containing one to three segments. Comparative genomic analyses with closely related species gave insights into the evolutionary dynamics of bracovirus genomes. Conserved synteny in the different wasp genomes showed the orthology of the proviral macrolocus across different species. The nudiviral gene odv-e66-like1 is conserved within the macrolocus, suggesting an ancient co-localization of the nudiviral genome and bracovirus proviral segments. By contrast, the evolution of proviral segments within the macrolocus has involved a series of lineage-specific duplications.
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Postnikova, Yulia, Anastasia Treshchalina, Elizaveta Boravleva, Alexandra Gambaryan, Aydar Ishmukhametov, Mikhail Matrosovich, Ron A. M. Fouchier, Galina Sadykova, Alexey Prilipov, and Natalia Lomakina. "Diversity and Reassortment Rate of Influenza A Viruses in Wild Ducks and Gulls." Viruses 13, no. 6 (May 27, 2021): 1010. http://dx.doi.org/10.3390/v13061010.
Full textAbstract:
Influenza A viruses (IAVs) evolve via point mutations and reassortment of viral gene segments. The patterns of reassortment in different host species differ considerably. We investigated the genetic diversity of IAVs in wild ducks and compared it with the viral diversity in gulls. The complete genomes of 38 IAVs of H1N1, H1N2, H3N1, H3N2, H3N6, H3N8, H4N6, H5N3, H6N2, H11N6, and H11N9 subtypes isolated from wild mallard ducks and gulls resting in a city pond in Moscow, Russia were sequenced. The analysis of phylogenetic trees showed that stable viral genotypes do not persist from year to year in ducks owing to frequent gene reassortment. For comparison, similar analyses were carried out using sequences of IAVs isolated in the same period from ducks and gulls in The Netherlands. Our results revealed a significant difference in diversity and rates of reassortment of IAVs in ducks and gulls.
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Zamoto-Niikura, Aya, Kaori Terasaki, Tetsuro Ikegami, C. J. Peters, and Shinji Makino. "Rift Valley Fever Virus L Protein Forms a Biologically Active Oligomer." Journal of Virology 83, no. 24 (October 7, 2009): 12779–89. http://dx.doi.org/10.1128/jvi.01310-09.
Full textAbstract:
ABSTRACT Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) causes mosquito-borne epidemic diseases in humans and livestock. The virus carries three RNA segments, L, M, and S, of negative or ambisense polarity. L protein, an RNA-dependent RNA polymerase, encoded in the L segment, and N protein, encoded in the S segment, exert viral RNA replication and transcription. Coexpression of N, hemagglutinin (HA)-tagged L, and viral minigenome resulted in minigenome replication and transcription, a finding that demonstrated HA-tagged L was biologically active. Likewise L tagged with green fluorescent protein (GFP) was biologically competent. Coimmunoprecipitation analysis using extracts from cells coexpressing HA-tagged L and GFP-tagged L showed the formation of an L oligomer. Bimolecular fluorescence complementation analysis and coimmunoprecipitation studies demonstrated the formation of an intermolecular L-L interaction through its N-terminal and C-terminal regions and also suggested an intramolecular association between the N-terminal and C-terminal regions of L protein. A biologically inactive L mutant, in which the conserved signature SDD motif was replaced by the amino acid residues GNN, exhibited a dominant negative phenotype when coexpressed with wild-type L in the minigenome assay system. Expression of this mutant L also inhibited viral gene expression in virus-infected cells. These data provided compelling evidence for the importance of oligomerization of RVFV L protein for its polymerase activity.
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Clarke, Penny, Suzanne M. Meintzer, Christian Widmann, Gary L. Johnson, and Kenneth L. Tyler. "Reovirus Infection Activates JNK and the JNK-Dependent Transcription Factor c-Jun." Journal of Virology 75, no. 23 (December 1, 2001): 11275–83. http://dx.doi.org/10.1128/jvi.75.23.11275-11283.2001.
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ABSTRACT Viral infection often perturbs host cell signaling pathways including those involving mitogen-activated protein kinases (MAPKs). We now show that reovirus infection results in the selective activation of c-Jun N-terminal kinase (JNK). Reovirus-induced JNK activation is associated with an increase in the phosphorylation of the JNK-dependent transcription factor c-Jun. Reovirus serotype 3 prototype strains Abney (T3A) and Dearing (T3D) induce significantly more JNK activation and c-Jun phosphorylation than does the serotype 1 prototypic strain Lang (T1L). T3D and T3A also induce more apoptosis in infected cells than T1L, and there was a significant correlation between the ability of these viruses to phosphorylate c-Jun and induce apoptosis. However, reovirus-induced apoptosis, but not reovirus-induced c-Jun phosphorylation, is inhibited by blocking TRAIL/receptor binding, suggesting that apoptosis and c-Jun phosphorylation involve parallel rather than identical pathways. Strain-specific differences in JNK activation are determined by the reovirus S1 and M2 gene segments, which encode viral outer capsid proteins (ς1 and μ1c) involved in receptor binding and host cell membrane penetration. These same gene segments also determine differences in the capacity of reovirus strains to induce apoptosis, and again a significant correlation between the capacity of T1L × T3D reassortant reoviruses to both activate JNK and phosphorylate c-Jun and to induce apoptosis was shown. The extracellular signal-related kinase (ERK) is also activated in a strain-specific manner following reovirus infection. Unlike JNK activation, ERK activation could not be mapped to specific reovirus gene segments, suggesting that ERK activation and JNK activation are triggered by different events during virus-host cell interaction.
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Wang, Shixia, Te-hui W. Chou, Pavlo V. Sakhatskyy, Song Huang, John M. Lawrence, Hong Cao, Xiaoyun Huang, and Shan Lu. "Identification of Two Neutralizing Regions on the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Produced from the Mammalian Expression System." Journal of Virology 79, no. 3 (February 1, 2005): 1906–10. http://dx.doi.org/10.1128/jvi.79.3.1906-1910.2005.
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ABSTRACT The Spike (S) protein of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) plays important roles in viral pathogenesis and potentially in the development of an effective vaccine against this virulent infectious disease. In this study, the codon-optimized S gene of SARS-CoV was synthesized to construct DNA vaccine plasmids expressing either the full-length or segments of the S protein. High titer S-specific immunoglobulin G antibody responses were elicited in rabbits immunized with DNA against various segments of the S protein. Two neutralizing domains were identified on the S protein, one at the N terminus (Ser12-Thr535) and the other near the C terminus (Arg797-Ile1192).
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Thu Hang, Nguyen Thi, Hoang Thi Thu Hang, Nguyen Hung Chi, Vu Huyen Trang, Chu Hoang Ha, and Nguyen Trung Nam. "DESIGNING AND CLONING NA GENE OF INFLUENZA A/H5N1 VIRUS INTO pHW2000 VECTOR FOR PREPARATION OF A CANDIDATE VACCINE MASTERSEED STRAIN." Vietnam Journal of Biotechnology 16, no. 2 (December 17, 2018): 369–76. http://dx.doi.org/10.15625/1811-4989/16/2/13450.
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The influenza A/H5N1 virus is an RNA virus belonging to the family of Orthomyxoviridae. The highly pathogenic influenza A/H5N1 virus exhibit the ability to cause high mortality in poultry and infect humans. Technology for vaccine seed strain production of influenza A virus using reverse genetics requires the creation of recombinant vectors carrying viral genomic segments. To create recombinant pHW2000 vectors containing the neuraminidase (NA) gene segment encoding an important surface antigen of influenza A virus, two N1 NA gene structures were designed based on the NA gene sequences of two subtypes of highly pathogenic influenza A/H5N1 clade (clade 1.1 and clade 2.3.2.1c) and then inserted into pHW2000 vector. These two clades of highly pathogenic avian influenza viruses that are still circulating in Vietnam, with antigen homology and genetic relationships to many strains of influenza A viruses, have been suggested to be used for producing vaccines against emerging avian influenza A/H5N1 virus. Each NA gene construct consists of 1453 nucleotides in which two ends of the gene are two non-coding regions (46 nucleotides and 57 nucleotides) containing primer binding site and cleavage site of BsaI. In the middle of each NA gene is one region of 1350 nucleotides encoding 449 amino acids, ensuring catalytic function and antigenicity of NA protein. Two NA segments corresponding to the two clades of influenza A viruses were successfully cloned into pHW2000 vectors for the generation of two recombinant vectors pHW2000-NA clade 1.1 and pHW2000-NA clade 2.3.2.1c. These recombinant vectors will be used for production of candidate avian influenza vaccine strains using reverse genetics technique.
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Monkewich, Sandra, Han-Xin Lin, Marc R. Fabian, Wei Xu, Hong Na, Debashish Ray, Olena A. Chernysheva, Peter D. Nagy, and K. Andrew White. "The p92 Polymerase Coding Region Contains an Internal RNA Element Required at an Early Step in Tombusvirus Genome Replication." Journal of Virology 79, no. 8 (April 15, 2005): 4848–58. http://dx.doi.org/10.1128/jvi.79.8.4848-4858.2005.
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ABSTRACT The replication of positive-strand RNA viral genomes involves various cis-acting RNA sequences. Generally, regulatory RNA sequences are present at or near genomic termini; however, internal replication elements (IREs) also exist. Here we report the structural and functional characterization of an IRE present in the readthrough portion of the p92 polymerase gene of Tomato bushy stunt virus. Analysis of this element in the context of a noncoding defective interfering RNA revealed a functional core structure composed of two noncontiguous segments of sequence that interact with each other to form an extended helical conformation. IRE activity required maintenance of several base-paired sections as well as two distinct structural features: (i) a short, highly conserved segment that can potentially form two different and mutually exclusive structures and (ii) an internal loop that contains a critical CC mismatch. The IRE was also shown to play an essential role within the context of the viral genome. In vivo analysis with novel RNA-based temperature-sensitive genomic mutants and translationally active subgenomic viral replicons revealed the following about the IRE: (i) it is active in the positive strand, (ii) it is dispensable late in the viral RNA replication process, and (iii) it is functionally inhibited by active translation over its sequence. Together, these results suggest that IRE activity is required in the cytosol at an early step in the viral replication process, such as template recruitment and/or replicase complex assembly.
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Danthi, Pranav, Mark W. Hansberger, Jacquelyn A. Campbell, J. Craig Forrest, and Terence S. Dermody. "JAM-A-Independent, Antibody-Mediated Uptake of Reovirus into Cells Leads to Apoptosis." Journal of Virology 80, no. 3 (February 1, 2006): 1261–70. http://dx.doi.org/10.1128/jvi.80.3.1261-1270.2006.
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ABSTRACT Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein σ1 and membrane penetration protein μ1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a σ1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L × T3D reassortant viruses revealed that the μ1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of σ1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the μ1 protein plays an essential role in stimulating proapoptotic signaling.
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Lehner, P. J., E. C. Wang, P. A. Moss, S. Williams, K. Platt, S. M. Friedman, J. I. Bell, and L. K. Borysiewicz. "Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 79–91. http://dx.doi.org/10.1084/jem.181.1.79.
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The major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response is important in the clearance of viral infections in humans. After influenza A infection, a peptide from the matrix protein, M58-66, is presented in the context of the MHC allele HLA-A0201 and the resulting CTL response is detectable in most HLA-A0201 subjects. An initial study suggested that M58-66-specific CTL clones show conserved T cell receptor (TCR) alpha and beta gene segments. We have addressed the significance of this observation by determining the expression of V beta 17 during the development of M58-66-specific CTL lines in 21 unrelated HLA-A0201 subjects, and analyzing TCR usage by M58-66-specific CTL clones. TCR V beta 17 was the dominant V beta segment used and CD8 V beta 17 expansion correlated with M58-66-specific lysis. Limiting dilution analysis from five subjects showed the M58-66 CTL precursor frequency to vary between 1/54,000 and less than 1/250,000, and that up to 85% of the matrix peptide (M58-66)-specific CTL used the V beta 17 gene segment. The M58-66 specific CTL response was dependent on previous viral exposure and specific V beta 17 expansion, as it was not found in cord blood, despite a readily expandable V beta 17+ CD8+ T cell subpopulation. Sequence analysis of 38 M58-66-specific V beta 17 transcripts from 13 subjects revealed extensive conservation in the CDR3 region including conservation of an arginine-serine motif. To test the dependence of this CTL response on the V beta 17 gene segment, peripheral blood lymphocytes were depleted of CD8+ TCR V beta 17+ cells, before the generation of M58-66-specific CTL. In most cases such depletion blocked or severely reduced the generation of the M58-66-specific response, and under limiting dilution conditions could abolish M58-66-specific CTL precursors. These studies reveal the dependence of this natural human immune response on a particular TCR gene segment.
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Oh, Jangsuk, John G. Julias, Andrea L. Ferris, and Stephen H. Hughes. "Construction and Characterization of a Replication-Competent Retroviral Shuttle Vector Plasmid." Journal of Virology 76, no. 4 (February 15, 2002): 1762–68. http://dx.doi.org/10.1128/jvi.76.4.1762-1768.2002.
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ABSTRACT We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.
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Gilbertson, Brad, Tian Zheng, Marie Gerber, Anne Printz-Schweigert, Chi Ong, Roland Marquet, Catherine Isel, Steven Rockman, and Lorena Brown. "Influenza NA and PB1 Gene Segments Interact during the Formation of Viral Progeny: Localization of the Binding Region within the PB1 Gene." Viruses 8, no. 8 (August 20, 2016): 238. http://dx.doi.org/10.3390/v8080238.
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Eifan, Saleh A., and Richard M. Elliott. "Mutational Analysis of the Bunyamwera Orthobunyavirus Nucleocapsid Protein Gene." Journal of Virology 83, no. 21 (August 26, 2009): 11307–17. http://dx.doi.org/10.1128/jvi.01460-09.
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ABSTRACT The bunyavirus nucleocapsid protein, N, is a multifunctional protein that encapsidates each of the three negative-sense genome segments to form ribonucleoprotein complexes that are the functional templates for viral transcription and replication. In addition, N protein molecules interact with themselves to form oligomers, with the viral L (RNA polymerase) protein, with the carboxy-terminal regions of either or both of the virion glycoproteins, and probably also with host cell proteins. Bunyamwera virus (BUNV), the prototype bunyavirus, encodes an N protein of 233 amino acids in length. To learn more about the roles of individual amino acids in the different interactions of N, we performed a wide-scale mutagenic analysis of the protein, and 110 single-point mutants were obtained. When the mutants were employed in a minireplicon assay to examine their effects on viral RNA synthesis, a wide range of activities compared to those of wild-type N protein were observed; changes at nine amino acid positions resulted in severely impaired RNA synthesis. Seventy-seven mutant clones were selected for use in the bunyavirus reverse genetics system, and 57 viable recombinant viruses were recovered. The recombinant viruses displayed a range of plaque sizes and titers in cell culture (from approximately 103 to 108 PFU/ml), and a number of viruses were shown to be temperature sensitive. Different assays were applied to determine why 20 mutant N proteins could not be recovered into infectious virus. Based on these results, a preliminary domain map of the BUNV N protein is proposed.
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Qiu, Wenping, and James W. Moyer. "Tomato Spotted Wilt Tospovirus Adapts to the TSWV N Gene-Derived Resistance by Genome Reassortment." Phytopathology® 89, no. 7 (July 1999): 575–82. http://dx.doi.org/10.1094/phyto.1999.89.7.575.
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Pathogen- and host-derived resistance have been shown to suppress infection by many plant viruses. Tomato spotted wilt tospovirus (TSWV) is among these systems; however, it has easily overcome nearly all host resistance genes and has recently been shown to overcome resistance mediated by the TSWV N gene. To better understand the resistance-breaking mechanisms, we have chosen TSWV N gene-derived resistance (TNDR) as a model to study how plant viruses defeat resistance genes. A defined viral population of isolates TSWV-D and TSWV-10, both suppressed by TNDR, was subjected to TNDR selection by serial passage in an N-gene transgenic plant. The genotype analysis demonstrated that the mixed viral population was driven to form a specific reassortant, L10M10SD, in the presence of TNDR selection, but remained as a heterogeneous mixture in the absence of the selection. A genotype assay of 120 local lesion isolates from the first, fourth, and seventh transfers confirmed the shift of genomic composition. Further analysis demonstrated that the individual L10, M10, and SD RNA segments were each selected independently in response to TNDR selection rather than to a mutation or recombination event. Following the seventh transfer on the N-gene transgenic plants, TSWV S RNA remained essentially identical to the S RNA from TSWV-D, indicating that no intermolecular recombination occurred between the two S RNAs from TSWV-10 and TSWV-D nor with the transferred N gene. These results support the hypothesis that TSWV utilizes genome reassortment to adapt to new host genotypes rapidly and that elements from two or more segments of the genome are involved in suppression of the resistance reaction.
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Shelton, Holly, Matt Smith, Lorian Hartgroves, Peter Stilwell, Kim Roberts, Ben Johnson, and Wendy Barclay. "An influenza reassortant with polymerase of pH1N1 and NS gene of H3N2 influenza A virus is attenuated in vivo." Journal of General Virology 93, no. 5 (May 1, 2012): 998–1006. http://dx.doi.org/10.1099/vir.0.039701-0.
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Influenza viruses readily mutate by accumulating point mutations and also by reassortment in which they acquire whole gene segments from another virus in a co-infected host. The NS1 gene is a major virulence factor of influenza A virus. The effects of changes in NS1 sequence depend on the influenza polymerase constellation. Here, we investigated the consequences of a virus with the polymerase of pandemic H1N1 2009 acquiring an NS gene segment derived from a seasonal influenza A H3N2 virus, a combination that might arise during natural reassortment of viruses that currently circulate in humans. We generated recombinant influenza viruses with surface HA and NA genes and matrix M gene segment from A/PR/8/34 virus, but different combinations of polymerase and NS genes. Thus, any changes in phenotype were not due to differences in receptor use, entry, uncoating or virus release. In Madin–Darby canine kidney (MDCK) cells, the virus with the NS gene from the H3N2 parent showed enhanced replication, probably a result of increased control of the interferon response. However, in mice the same virus was attenuated in comparison with the virus containing homologous pH1N1 polymerase and NS genes. Levels of viral RNA during single-cycles of replication were lower for the virus with H3N2 NS, and this virus reached lower titres in the lungs of infected mice. Thus, virus with pH1N1 polymerase genes did not increase its virulence by acquiring the H3N2 NS gene segment, and MDCK cells were a poor predictor of the outcome of infection in vivo.
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Hatta, Masato, Hideo Goto, and Yoshihiro Kawaoka. "Influenza B Virus Requires BM2 Protein for Replication." Journal of Virology 78, no. 11 (June 1, 2004): 5576–83. http://dx.doi.org/10.1128/jvi.78.11.5576-5583.2004.
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ABSTRACT The BM2 protein of influenza B virus functions as an ion channel, which is suggested to be important for virus uncoating in endosomes of virus-infected cells. Because direct support for this function is lacking, whether BM2 plays an essential role in the viral life cycle remains unknown. We therefore attempted to generate BM2 knockout viruses by reverse genetics. Mutant viruses possessing M segments with the mutated initiation codon of BM2 protein at the stop-start pentanucleotide were viable and still expressed BM2. The introduction of multiple stop codons and a one-nucleotide deletion downstream of the stop-start pentanucleotide, in addition to disablement of the BM2 initiation codon, failed to generate viable mutant viruses, but the mutant M segments still expressed proteins that reacted with the BM2 peptide antiserum. To completely abolish BM2 expression, we generated a mutant M gene whose BM2 open reading frame was deleted. Although this mutant was not able to replicate in normal MDCK cells, it did replicate in a cell line that we established which constitutively expresses BM2. Furthermore, a virus possessing the mutant M gene lacking the BM2 open reading frame and a mutant NA gene containing the BM2 open reading frame instead of the NA open reading frame underwent multiple cycles of replication in MDCK cells, with exogenous sialidase used to supplement the deleted viral sialidase activity. These findings demonstrate that the BM2 protein is essential for influenza B virus replication.
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Blakqori, Gjon, and Friedemann Weber. "Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs." Journal of Virology 79, no. 16 (August 15, 2005): 10420–28. http://dx.doi.org/10.1128/jvi.79.16.10420-10428.2005.
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ABSTRACT La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3′ ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 108 infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.