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1
Tsai, Chi-Wei, Margaret G. Redinbaugh, Kristen J. Willie, Sharon Reed, Michael Goodin, and Saskia A. Hogenhout. "Complete Genome Sequence and In Planta Subcellular Localization of Maize Fine Streak Virus Proteins." Journal of Virology 79, no. 9 (May 1, 2005): 5304–14. http://dx.doi.org/10.1128/jvi.79.9.5304-5314.2005.
Анотація:
ABSTRACT The genome of the nucleorhabdovirus maize fine streak virus (MFSV) consists of 13,782 nucleotides of nonsegmented, negative-sense, single-stranded RNA. The antigenomic strand consisted of seven open reading frames (ORFs), and transcripts of all ORFs were detected in infected plants. ORF1, ORF6, and ORF7 had significant similarities to the nucleocapsid protein (N), glycoprotein (G), and polymerase (L) genes of other rhabdoviruses, respectively, whereas the ORF2, ORF3, ORF4, and ORF5 proteins had no significant similarities. The N (ORF1), ORF4, and ORF5 proteins localized to nuclei, consistent with the presence of nuclear localization signals (NLSs) in these proteins. ORF5 likely encodes the matrix protein (M), based on its size, the position of its NLS, and the localization of fluorescent protein fusions to the nucleus. ORF2 probably encodes the phosphoprotein (P) because, like the P protein of Sonchus yellow net virus (SYNV), it was spread throughout the cell when expressed alone but was relocalized to a subnuclear locus when coexpressed with the MFSV N protein. Unexpectedly, coexpression of the MFSV N and P proteins, but not the orthologous proteins of SYNV, resulted in accumulations of both proteins in the nucleolus. The N and P protein relocalization was specific to cognate proteins of each virus. The subcellular localizations of the MFSV ORF3 and ORF4 proteins were distinct from that of the SYNV sc4 protein, suggesting different functions. To our knowledge, this is the first comparative study of the cellular localizations of plant rhabdoviral proteins. This study indicated that plant rhabdoviruses are diverse in genome sequence and viral protein interactions.
2
Kondo, Hideki, Takanori Maeda, Yukio Shirako, and Tetsuo Tamada. "Orchid fleck virus is a rhabdovirus with an unusual bipartite genome." Journal of General Virology 87, no. 8 (August 1, 2006): 2413–21. http://dx.doi.org/10.1099/vir.0.81811-0.
Анотація:
Orchid fleck virus (OFV) has an unusual bipartite negative-sense RNA genome with clear sequence similarities to those of nucleorhabdoviruses. The OFV genome consists of two single-stranded RNA molecules, RNA1 and RNA2 that are 6413 and 6001 nt long, respectively, with open reading frame (ORF) information in the complementary sense. RNA1 encodes 49 (ORF1), 26 (ORF2), 38 (ORF3), 20 (ORF4) and 61 kDa (ORF5) proteins, and RNA2 encodes a single protein of 212 kDa (ORF6). ORF1, ORF5 and ORF6 proteins had significant similarities (21–38 % identity) to the nucleocapsid protein (N), glycoprotein (G) and polymerase (L) gene products, respectively, of other rhabdoviruses, especially nucleorhabdoviruses, whereas ORF2, ORF3 and ORF4 proteins had no significant similarities to other proteins in the international databases. Similarities between OFV and rhabdoviruses were also found in the sequence complementarity at both termini of each RNA segment (the common terminal sequences are 3′-UGUGUC---GACACA-5′), the conserved intergenic sequences and in being negative sense. It was proposed that a new genus Dichorhabdovirus in the family Rhabdoviridae of the order Mononegavirales should be established with OFV as its prototype member and type species.
3
Melzer, M. J., A. V. Karasev, D. M. Sether, and J. S. Hu. "Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2." Journal of General Virology 82, no. 1 (January 1, 2001): 1–7. http://dx.doi.org/10.1099/0022-1317-82-1-1.
Анотація:
The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3′-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5′ to 3′, potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3′ terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family Closteroviridae the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.
4
Li, Jiapeng, Xiaoyin Wu, Hui Liu, Xiaomei Wang, Shaokui Yi, Xueting Zhong, Yaqin Wang, and Zhanqi Wang. "Identification and Molecular Characterization of a Novel Carlavirus Infecting Chrysanthemum morifolium in China." Viruses 15, no. 4 (April 21, 2023): 1029. http://dx.doi.org/10.3390/v15041029.
Анотація:
Chrysanthemum (Chrysanthemum morifolium) is an important ornamental and medicinal plant suffering from many viruses and viroids worldwide. In this study, a new carlavirus, tentatively named Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN), was identified from chrysanthemum plants in Zhejiang Province, China. The genome sequence of CiCV1-CN was 8795 nucleotides (nt) in length, with a 68-nt 5′-untranslated region (UTR) and a 76-nt 3′-UTR, which contained six predicted open reading frames (ORFs) that encode six corresponding proteins of various sizes. Phylogenetic analyses based on full-length genome and coat protein sequences revealed that CiCV1-CN is in an evolutionary branch with chrysanthemum virus R (CVR) in the Carlavirus genus. Pairwise sequence identity analysis showed that, except for CiCV1, CiCV1-CN has the highest whole-genome sequence identity of 71.3% to CVR-X6. At the amino acid level, the highest identities of predicted proteins encoded by the ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6 of CiCV1-CN were 77.1% in the CVR-X21 ORF1, 80.3% in the CVR-X13 ORF2, 74.8% in the CVR-X21 ORF3, 60.9% in the CVR-BJ ORF4, 90.2% in the CVR-X6 and CVR-TX ORF5s, and 79.4% in the CVR-X21 ORF6. Furthermore, we also found a transient expression of the cysteine-rich protein (CRP) encoded by the ORF6 of CiCV1-CN in Nicotiana benthamiana plants using a potato virus X-based vector, which can result in a downward leaf curl and hypersensitive cell death over the time course. These results demonstrated that CiCV1-CN is a pathogenic virus and C. morifolium is a natural host of CiCV1.
5
Shafat, Zoya. "Sequence to structural analysis of ORF5 protein in Norway rat Hepatitis E Virus." Bioinformation 18, no. 1 (January 31, 2022): 19–25. http://dx.doi.org/10.6026/97320630018019.
Анотація:
Hepatitis E virus (HEV) is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF5 is attributed to life cycle of rat HEV. The ORFF5 protein’s function remains undetermined. Therefore, it is of interest to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. Analysis of the ORF5 protein revealed it as highly unstable, hydrophilic with basic pI. The ORF5 protein consisted mostly of Arg, Pro, Ser, Leu and Gly. The 3D structural homology model of the ORF5 protein generated showed mixed α/β structural fold with predominance of coils. Structural analysis revealed the presence of clefts, pores and a tunnel. This data will help in the sequence, structure and functional annotation of ORF5.
6
Shafat, Zoya. "Intrinsically disordered regions in the rodent hepevirus proteome." Bioinformation 18, no. 2 (February 28, 2022): 111–18. http://dx.doi.org/10.6026/97320630018111.
Анотація:
Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or intrinsically disordered proteins (IDPs) are regions or proteins that are characterized by lack of definite structure. These IDPRs or IDPs play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPR/IDP in proteins is associated with the virus’s pathogenicity and infectivity. The prevalence of IDPR/IDP in rat HEV proteome remains undetermined. Hence, we examined the unstructured/disordered regions of the open reading frame (ORF) encoded proteins of rat HEV by analyzing the prevalence of intrinsic disorder. The intrinsic disorder propensity analysis showed that the different ORF proteins consisted of varying fraction of intrinsic disorder. The protein ORF3 was identified with maximum propensity for intrinsic disorder while the ORF6 protein had the least fraction of intrinsic disorder. The analysis revealed ORF6 as a structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins (IDPs). The protein ORF2 was found to be moderately as well as highly disordered using different predictors, thus, was categorized into both IDPR and IDP. Such disordered regions have important roles in pathogenesis and replication of viruses.
7
Garcia, Miguel, Madalena Pimentel та José Moniz-Pereira. "Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA". Journal of Bacteriology 184, № 11 (1 червня 2002): 3034–43. http://dx.doi.org/10.1128/jb.184.11.3034-3043.2002.
Анотація:
ABSTRACT A mycobacteriophage Ms6 strong promoter region (Plys ) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem σ70-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region Plys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that β-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.
8
Kiatpapan, Pornpimon, Yoshiteru Hashimoto, Hisako Nakamura, Yong-Zhe Piao, Hisayo Ono, Mitsuo Yamashita, and Yoshikatsu Murooka. "Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici, and Its Use for Development of a Host-Vector System in Propionibacteria." Applied and Environmental Microbiology 66, no. 11 (November 1, 2000): 4688–95. http://dx.doi.org/10.1128/aem.66.11.4688-4695.2000.
Анотація:
ABSTRACT The complete nucleotide sequence of pRGO1, a cryptic plasmid fromPropionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4,orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containingorf1 (repA), orf2(repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 × 106 CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 104 to 107 CFU/μg of DNA. The vector was stably maintained in strains of P. freudenreichiisubsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp.freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.
9
Li, Tong, Massoud Kheir Khah, Snjezana Slavnic, Ingegerd Johansson, and Nicklas Strömberg. "Different Type 1 Fimbrial Genes and Tropisms of Commensal and Potentially Pathogenic Actinomyces spp. with Different Salivary Acidic Proline-Rich Protein and Statherin Ligand Specificities." Infection and Immunity 69, no. 12 (December 1, 2001): 7224–33. http://dx.doi.org/10.1128/iai.69.12.7224-7233.2001.
Анотація:
ABSTRACT Actinomyces spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands. Actinomyces spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of Actinomyces naeslundiigenospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (orf1- to -6 andfimP) in genospecies 1 and 2 strains, except thatorf5 and orf3 were divergent in genospecies 1. (ii) In preferential statherin binding, oralActinomyces viscosus strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (orfA to -C andfimP). Bioinformatics suggested sortase (orfB, orf4, and part oforf5), prepilin peptidase (orfC andorf6), fimbria subunit (fimP), and usher- and autotransporter-like (orfA and orf1to -3) functions. Those gene regions corresponding toorf3 and orf5 were divergent, those corresponding to orf2, orf1, andfimP were moderately conserved, and those corresponding to orf4 and orf6 were highly conserved. Restriction fragment length polymorphism analyses using a fimP probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of A. naeslundii genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only theorf4 and orf6 gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both types of fimbria on the investigated Actinomyces strains.
10
Shimizu, Takeo, Hiroshi Kinoshita, and Takuya Nihira. "Identification and In Vivo Functional Analysis by Gene Disruption of ctnA, an Activator Gene Involved in Citrinin Biosynthesis in Monascus purpureus." Applied and Environmental Microbiology 73, no. 16 (June 22, 2007): 5097–103. http://dx.doi.org/10.1128/aem.01979-06.
Анотація:
ABSTRACT Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5′-flanking region and one ORF (orf5) in the 3′-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis.
11
Lymanska, О. Yu. "Comparative analysis of the open reading frames protein genes of genotype 4 Hepatitis E virus in swine and wild boar." Journal for Veterinary Medicine, Biotechnology and Biosafety 9, no. 1-2 (June 7, 2023): 12–19. http://dx.doi.org/10.36016/jvmbbs-2023-9-1-2-3.
Анотація:
The goal of this study was to determine the molecular diversity of the open reading frames (ORFs) ORF1, ORF2, ORF3 protein genes from full-length genomes of genotype 4 hepatitis E virus (HEV) from pigs and wild boars at protein and gene level. Statistical technique Shannon entropy was used for mutational analysis of ORF1–ORF3 protein genes to identify amino acid substitutions in the HEV 4 sequences isolated from pigs and wild boars that were most susceptible to mutations. Gene selective pressure for genes was estimated using Tajima’s neutrality test. The ORF regions of 11 swine and 11 wild boar genotype 4 HEV isolates with complete genomes from the GenBank database were analyzed comparatively. The total number of polymorphic sites was determined. Nonsynonymous (amino acid changing) and synonymous (amino acid preserving) substitutions were identified in ORF1, ORF2, ORF3 in swine and wild boar HEV 4 isolates. No evidence of recombination was found for ORFs in 11 swine HEV 4 isolates, ORF2, ORF3 in 8 wild boar HEV 4 isolates. However, a recombination fragment with a length of 430 nucleotides was detected in the ORF1 gene of 3 wild boar HEV 4 isolates. Positive D Tajima factors were determined for ORF1, ORF2, ORF3 genes of swine HEV 4 and ORF1, ORF2 genes of wild boar HEV 4. While a negative value of D Tajima’s factor was determined for ORF3 gene of wild boar HEV 4. Molecular characteristics showing principal distinctions between the open-reading frames of swine and wild boar genotype 4 hepatitis E virus were obtained. Wild boar ORF1 is characterized by lower nucleotide diversity π value (0.144) and higher number of segregated sites S value (1,688) comparing with higher π value (0.159) and lower S value (1,602) of swine ORF1. Positive values of D Tajima’s factor for ORF1, ORF2 ORF3 genes of swine HEV 4 and ORF1, ORF2 genes of wild boar HEV 4 show on positive selection of these genes. Negative value of D Tajima’s factor for ORF3 gene of wild boar HEV 4 indicates onto purifying selection decreasing variability in ORF3 gene of wild boar HEV 4. The largest number of amino acid variation sites (19.2%) was found for wild boar HEV 4 ORF3 followed by swine HEV 4 ORF3 (15.7%) comparing with other swine and wild boars HEV 4 ORFs
12
Thomas, Darby L., Jerome Schaack, Hannes Vogel, and Ronald Javier. "Several E4 Region Functions Influence Mammary Tumorigenesis by Human Adenovirus Type 9." Journal of Virology 75, no. 2 (January 15, 2001): 557–68. http://dx.doi.org/10.1128/jvi.75.2.557-568.2001.
Анотація:
ABSTRACT Among oncogenic adenoviruses, human adenovirus type 9 (Ad9) is unique in eliciting exclusively estrogen-dependent mammary tumors in rats and in not requiring viral E1 region transforming genes for tumorigenicity. Instead, studies with hybrid viruses generated between Ad9 and the closely related nontumorigenic virus Ad26 have roughly localized an Ad9 oncogenic determinant(s) to a segment of the viral E4 region containing open reading frame 1 (E4-ORF1), E4-ORF2, and part of E4-ORF3. Although subsequent findings have shown that E4-ORF1 codes for an oncoprotein essential for tumorigenesis by Ad9, it is not known whether other E4 region functions may similarly play a role in this process. We report here that new results with Ad9/Ad26 hybrid viruses demonstrated that the minimal essential Ad9 E4-region DNA sequences include portions of both E4-ORF1 and E4-ORF2. Investigations with Ad9 mutant viruses additionally showed that the E4-ORF1 protein and certain E4-ORF2 DNA sequences are necessary for Ad9-induced tumorigenesis, whereas the E4-ORF2 and E4-ORF3 proteins are not. In fact, the E4-ORF3 protein was found to antagonize this process. Also pertinent was that certain crucial nucleotide differences between Ad9 and Ad26 within E4-ORF1 and E4-ORF2 were found to be silent with respect to the amino acid sequences of the corresponding proteins. Furthermore, supporting a prominent role for the E4-ORF1 oncoprotein in Ad9-induced tumorigenesis, an E1 region-deficient Ad5 vector that expresses the Ad9 but not the Ad26 E4-ORF1 protein was tumorigenic in rats and, like Ad9, promoted solely mammary tumors. These findings argue that the E4-ORF1 oncoprotein is the major oncogenic determinant of Ad9 and that an undefined regulatory element(s) within the E4 region represents a previously unidentified second function likewise necessary for tumorigenesis by this virus.
13
Surjit, Milan, Shahid Jameel, and Sunil K. Lal. "The ORF2 Protein of Hepatitis E Virus Binds the 5′ Region of Viral RNA." Journal of Virology 78, no. 1 (January 1, 2004): 320–28. http://dx.doi.org/10.1128/jvi.78.1.320-328.2004.
Анотація:
ABSTRACT Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3′ structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5′ and 3′ ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5′ end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5′ end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.
14
Alves-Freitas, Dione M. T., Bruna Pinheiro-Lima, Josias C. Faria, Cristiano Lacorte, Simone G. Ribeiro, and Fernando L. Melo. "Double-Stranded RNA High-Throughput Sequencing Reveals a New Cytorhabdovirus in a Bean Golden Mosaic Virus-Resistant Common Bean Transgenic Line." Viruses 11, no. 1 (January 21, 2019): 90. http://dx.doi.org/10.3390/v11010090.
Анотація:
Using double-strand RNA (dsRNA) high-throughput sequencing, we identified five RNA viruses in a bean golden mosaic virus (BGMV)-resistant common bean transgenic line with symptoms of viral infection. Four of the identified viruses had already been described as infecting common bean (cowpea mild mottle virus, bean rugose mosaic virus, Phaseolus vulgaris alphaendornavirus 1, and Phaseolus vulgaris alphaendornavirus 2) and one is a putative new plant rhabdovirus (genus Cytorhabdovirus), tentatively named bean-associated cytorhabdovirus (BaCV). The BaCV genome presented all five open reading frames (ORFs) found in most rhabdoviruses: nucleoprotein (N) (ORF1) (451 amino acids, aa), phosphoprotein (P) (ORF2) (445 aa), matrix (M) (ORF4) (287 aa), glycoprotein (G) (ORF5) (520 aa), and an RNA-dependent RNA polymerase (L) (ORF6) (114 aa), as well as a putative movement protein (P3) (ORF3) (189 aa) and the hypothetical small protein P4. The predicted BaCV proteins were compared to homologous proteins from the closest cytorhabdoviruses, and a low level of sequence identity (15–39%) was observed. The phylogenetic analysis shows that BaCV clustered with yerba mate chlorosis-associated virus (YmCaV) and rice stripe mosaic virus (RSMV). Overall, our results provide strong evidence that BaCV is indeed a new virus species in the genus Cytorhabdovirus (family Rhabdoviridae), the first rhabdovirus to be identified infecting common bean.
15
Gaur, Arti, and William R. Green. "Role of a Cytotoxic-T-Lymphocyte Epitope-Defined, Alternative gag Open Reading Frame in the Pathogenesis of a Murine Retrovirus-Induced Immunodeficiency Syndrome." Journal of Virology 79, no. 7 (April 1, 2005): 4308–15. http://dx.doi.org/10.1128/jvi.79.7.4308-4315.2005.
Анотація:
ABSTRACT LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60 gag ). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.
16
Zhang, Yun-Ping, Jerry K. Uyemoto, Deborah A. Golino, and Adib Rowhani. "Nucleotide Sequence and RT-PCR Detection of a Virus Associated with Grapevine Rupestris Stem-Pitting Disease." Phytopathology® 88, no. 11 (November 1998): 1231–37. http://dx.doi.org/10.1094/phyto.1998.88.11.1231.
Анотація:
Grapevine rupestris stem pitting (RSP) is a graft-transmissible disease of unknown etiology. We have characterized a virus associated with this disease. The entire genomic sequence (GenBank accession number AF026278) consisted of 8,725 nucleotides excluding a poly(A) tail. Six open reading frames (ORF) were found. ORF1 potentially encodes a polypeptide with a methyltransferase domain, a papain-like proteinase domain, a helicase domain, and a RNA-dependent RNA polymerase domain; ORF2, ORF3, and ORF4 compose a triple-gene block; ORF5 encodes a coat protein; and ORF6 is located near the 3′ end with unknown function. Sequence analysis indicated that the virus is most similar to apple stem-pitting virus and may be allied with the carla- and potexviruses and grouped with other viruses that infect woody hosts. A specific reverse-transcription polymerase chain reaction (RT-PCR)-based detection method was developed. Among 62 grapevine sources known to be infected with rupestris stem-pitting disease, 60 sources tested positive by RT-PCR. Among 43 healthy vines tested, all were negative. The name grapevine rupestris stem-pitting-associated virus is proposed.
17
Basim, Huseyin, Gerald V. Minsavage, Robert E. Stall, Jaw-Fen Wang, Savita Shanker, and Jeffrey B. Jones. "Characterization of a Unique Chromosomal Copper Resistance Gene Cluster from Xanthomonas campestris pv. vesicatoria." Applied and Environmental Microbiology 71, no. 12 (December 2005): 8284–91. http://dx.doi.org/10.1128/aem.71.12.8284-8291.2005.
Анотація:
ABSTRACT We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.
18
Mahé, Dominique, Philippe Blanchard, Catherine Truong, Claire Arnauld, Pierre Le Cann, Roland Cariolet, François Madec, Emmanuel Albina, and André Jestin. "Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes." Microbiology 81, no. 7 (July 1, 2000): 1815–24. http://dx.doi.org/10.1099/0022-1317-81-7-1815.
Анотація:
Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated.
19
Göbel, Markus, Kerstin Kassel-Cati, Eberhard Schmidt, and Walter Reineke. "Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Cloning, Characterization, and Analysis of Sequences Encoding 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase." Journal of Bacteriology 184, no. 1 (January 1, 2002): 216–23. http://dx.doi.org/10.1128/jb.184.1.216-223.2002.
Анотація:
ABSTRACT 3-Oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF. Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.
20
Graff, Judith, Hanh Nguyen, Claro Yu, William R. Elkins, Marisa St. Claire, Robert H. Purcell, and Suzanne U. Emerson. "The Open Reading Frame 3 Gene of Hepatitis E Virus Contains a cis-Reactive Element and Encodes a Protein Required for Infection of Macaques." Journal of Virology 79, no. 11 (June 1, 2005): 6680–89. http://dx.doi.org/10.1128/jvi.79.11.6680-6689.2005.
Анотація:
ABSTRACT An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.
21
Martínez-Guinó, Laura, Maria Ballester, Joaquim Segalés, and Tuija Kekarainen. "Expression profile and subcellular localization of Torque teno sus virus proteins." Journal of General Virology 92, no. 10 (October 1, 2011): 2446–57. http://dx.doi.org/10.1099/vir.0.033134-0.
Анотація:
In the present study, the expression, generation and subcellular localization of Torque teno sus virus (TTSuV) proteins were characterized into two genetically distinct TTSuV species (TTSuV1 and TTSuV2). Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative splice variants encoding new TTSuV protein isoforms were identified for the first time. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 in the cytoplasm and nucleus excluding the nucleoli. The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus (TTV). Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms probably occurs in vivo. Obtained data provide new information on molecular biology of TTSuV and anelloviruses, which until now has been solely based on results obtained from human TTV.
22
Ralfs, Philipp, Eduardo Salinas, Charuta Ambardekar, Bill Bremer, Heather Blasczyk, Jingting Zhu, Brantley Holland, Christopher M. Walker, Zongdi Feng, and Arash Grakoui. "Contribution of soluble ORF2 viral protein to viral replication and host immune response during acute Hepatitis E virus infection." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 20.34. http://dx.doi.org/10.4049/jimmunol.206.supp.20.34.
Анотація:
Abstract The hepatitis E virus (HEV) is a small, positive-stranded RNA virus that is a major cause of acute viral hepatitis globally. Acute HEV infection is typically asymptomatic and resolves within 8–10 weeks. HEV encodes 2 forms of capsid protein. A cytoplasmic form (ORF2c) is essential for virion structure. A secreted glycosylated form (ORF2s) accumulates at high titer in serum and can mask anti-ORF2 neutralizing antibodies. Here, we explored the contribution of ORF2s to HEV replicative fitness in vivo, and its role in generating anti-ORF2 antibodies (Abs). Rhesus Macaques (RM) were challenged by direct hepatic injection of infectious ORF2s+ and ORF2s− RNA. The replication of an HEV mutant lacking ORF2s expression was delayed by ~2 weeks when compared with wildtype virus and peak titers were nearly 10-fold lower for ORF2s−. No reversion of the 3 ORF2s silencing mutations was detected in the ORF2s− genomes, indicating genetic stability. The delay in replication and lower peak titer was unexpected as the viruses replicate similarly in cell culture. In addition, our data demonstrated that ORF2s has a significant and unexpected impact on generation of antibodies. Specifically, serum anti-ORF2 antibodies were only transiently detected in ORF2s− infected RM. As expected, anti-ORF2 titers were high and sustained in ORF2s+ infected RM. Furthermore, anti-ORF2 Ab response primed by ORF2s− infection differed in protection against reinfection when compared to ORF2s+. The ORF2s− challenged animals were re-infected upon second exposure to HEV infection. These findings indicate ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody response to mediate protection against re-exposure.
23
Hervouet, Kévin, Martin Ferrié, Maliki Ankavay, Claire Montpellier, Charline Camuzet, Virginie Alexandre, Aïcha Dembélé, et al. "An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell." PLOS Pathogens 18, no. 8 (August 25, 2022): e1010798. http://dx.doi.org/10.1371/journal.ppat.1010798.
Анотація:
Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.
24
Xu, Zhi, Mahmoud E. Khalifa, Rebekah A. Frampton, Grant R. Smith, Rebecca L. McDougal, Robin M. MacDiarmid, and Falk Kalamorz. "Characterization of a Novel Double-Stranded RNA Virus from Phytophthora pluvialis in New Zealand." Viruses 14, no. 2 (January 26, 2022): 247. http://dx.doi.org/10.3390/v14020247.
Анотація:
A new dsRNA virus from the oomycete Phytophthora pluvialis has been characterized and designated as Phytophthora pluvialis RNA virus 1 (PplRV1). The genome of the PplRV1 reference genome is 6742 bp that encodes two predicted open reading frames (ORFs). ORF1 and ORF2 overlap by a 47 nt “slippery” frameshift sequence. ORF1 encodes a putative protein of unknown function. ORF2 shows high similarity to the RNA-dependent RNA polymerase (RdRp) of other dsRNA viruses. Phylogenetic analysis of the putative PplRV1 RdRp and its most closely related viruses showed PplRV1 is distinct from other known viruses (below 33% amino acid similarity), which indicates this virus may belong to a new virus family. Analyses of the geographical distribution of PplRV1 in relation to two genetically distinct classes of its host revealed two corresponding genotypes of the PplRV1 (termed a and b), which share 92.3% nt identity. The reference genome for the second genotype is 6760 bp long and a prediction of its genetic organization shows three ORFs, with ORF2 being split into two ORFs, ORF2a and ORF2b, that is conserved in seven of eleven genotype b isolates. Additionally, a quick and simple diagnostic method using qPCR has been developed, which is suitable for large scale screens to identify PplRV1 in Phytophthora.
25
Bertolotti-Ciarlet, Andrea, Sue E. Crawford, Anne M. Hutson, and Mary K. Estes. "The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein." Journal of Virology 77, no. 21 (November 1, 2003): 11603–15. http://dx.doi.org/10.1128/jvi.77.21.11603-11615.2003.
Анотація:
ABSTRACT Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3′ untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3′ terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3′ elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3′UTR (ORF2+3+3′UTR). In contrast, expression of VP1 from constructs that lacked the 3′UTR (ORF2+3), ORF3 (ORF2+3′UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3′UTR revealed that the 3′UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.
26
Balak, O. K., and O. Yu Lymanska. "Structural analysis of open reading frames of bovine immunodeficiency virus proteins." Veterinary Medicine: inter-departmental subject scientific collection, no. 109 (September 27, 2023): 26–34. http://dx.doi.org/10.36016/vm-2023-109-5.
Анотація:
The goal of this study was determining the structural organization peculiarities of the ORF2 and ORF3 proteins of the bovine immunodeficiency virus (BIV). Five ORFs were determined for two BIV isolates with complete genome using the ATGpr software, which permits effective prediction of translation initiation codons with nucleotide accuracy. Phyre2 software was used to predict, analyze the secondary structure and function of proteins. PONDR-FIT software was used to search for protein fragments in a disordered or natively unfolded state. Analysis of the amino acid composition of ORF2 and ORF3 proteins of BIVisolates regarding the presence of nonpolar, polar, aromatic, and hydrophobic amino acid residues was carried out using PSIPRED software. Models of the 3D-structure of proteins were obtained by I-TASSER server. 14% of α helices, 17% of β strands and 43% of disordered structure are predicted for the ORF3 protein. 37% of α helices, 0% of β strands, and 41% of disordered structure were determined for Gag polyprotein, which is translated from ORF2. The distribution of charged amino acid residues characterizes the surface properties of proteins. Their number reaches 23.9% for ORF2 protein. The amount of Arg is 5.2%, Lys — 8.0%, Glu — 7.3%, Asp — 3.4%. The total number of charged amino acid residues of ORF3 is 23.3%. The number of Arg is 12.6%, Lys — 4.9%, Glu — 1.9%, Asp — 3.9%. Only two ORFs of five ones coincide in nucleotide length (and, therefore, in length of corresponding proteins) for the two BIV isolates. The ORF3 protein belongs to the intrinsically disordered proteins that cannot be stably folded into a unique three-dimensional structure under physiological conditions, and the Gag polyprotein, which is translated from ORF2, belongs to the class of fully structured proteins. The secondary structure of both proteins shows the presence of α-helices
27
Vakulenko, Yulia A., Artem V. Orlov, and Alexander N. Lukashev. "Patterns and Temporal Dynamics of Natural Recombination in Noroviruses." Viruses 15, no. 2 (January 28, 2023): 372. http://dx.doi.org/10.3390/v15020372.
Анотація:
Noroviruses infect a wide range of mammals and are the major cause of gastroenteritis in humans. Recombination at the junction of ORF1 encoding nonstructural proteins and ORF2 encoding major capsid protein VP1 is a well-known feature of noroviruses. Using all available complete norovirus sequences, we systematically analyzed patterns of natural recombination in the genus Norovirus both throughout the genome and across the genogroups. Recombination events between nonstructural (ORF1) and structural genomic regions (ORF2 and ORF3) were found in all analyzed genogroups of noroviruses, although recombination was most prominent between members of GII, the most common genogroup that infects humans. The half-life times of recombinant forms (clades without evidence of recombination) of human GI and GII noroviruses were 10.4 and 8.4–11.3 years, respectively. There was evidence of many recent recombination events, and most noroviruses that differed by more than 18% of nucleotide sequence were recombinant relative to each other. However, there were no distinct recombination events between viruses that differed by over 42% in ORF2/3, consistent with the absence of systematic recombination between different genogroups. The few inter-genogroup recombination events most likely occurred between ancient viruses before they diverged into contemporary genogroups. The recombination events within ORF1 or between ORF2/3 were generally rare. Thus, noroviruses routinely exchange full structural and nonstructural blocks of the genome, providing a modular evolution.
28
Sadowy, Ewa, Anna Maasen, Marek Juszczuk, Chantal David, Wlodzimierz Zagórski-Ostoja, Bruno Gronenborn, and M. Danuta Hulanicka. "The ORF0 product of Potato leafroll virus is indispensable for virus accumulation." Journal of General Virology 82, no. 6 (June 1, 2001): 1529–32. http://dx.doi.org/10.1099/0022-1317-82-6-1529.
Анотація:
Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.
29
Silva, Gonçalo, Moritz Bömer, Ajith I. Rathnayake, Steven O. Sewe, Paul Visendi, Joshua O. Oyekanmi, Marian D. Quain, Belinda Akomeah, P. Lava Kumar, and Susan E. Seal. "Molecular Characterization of a New Virus Species Identified in Yam (Dioscorea spp.) by High-Throughput Sequencing." Plants 8, no. 6 (June 11, 2019): 167. http://dx.doi.org/10.3390/plants8060167.
Анотація:
To date, several viruses of different genera have been reported to infect yam (Dioscorea spp.). The full diversity of viruses infecting yam, however, remains to be explored. High-throughput sequencing (HTS) methods are increasingly being used in the discovery of new plant viral genomes. In this study, we employed HTS on yam to determine whether any undiscovered viruses were present that would restrict the international distribution of yam germplasm. We discovered a new virus sequence present in 31 yam samples tested and have tentatively named this virus “yam virus Y” (YVY). Twenty-three of the samples in which YVY was detected showed mosaic and chlorotic leaf symptoms, but Yam mosaic virus was also detected in these samples. Complete genome sequences of two YVY viral isolates were assembled and found to contain five open reading frames (ORFs). ORF1 encodes a large replication-associated protein, ORF2, ORF3 and ORF4 constitute the putative triple gene block proteins, and ORF5 encodes a putative coat protein. Considering the species demarcation criteria of the family Betaflexiviridae, YVY should be considered as a novel virus species in the family Betaflexiviridae. Further work is needed to understand the association of this new virus with any symptoms and yield loss and its implication on virus-free seed yam production.
30
Salaipeth, Lakha, Sotaro Chiba, Ana Eusebio-Cope, Satoko Kanematsu, and Nobuhiro Suzuki. "Biological properties and expression strategy of rosellinia necatrix megabirnavirus 1 analysed in an experimental host, Cryphonectria parasitica." Journal of General Virology 95, no. 3 (March 1, 2014): 740–50. http://dx.doi.org/10.1099/vir.0.058164-0.
Анотація:
Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNA-silencing-defective strain (Δdcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being ~20-fold higher in Δdcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in Δdcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a −1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of Δdcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virus–host interactions and virus gene expression strategies.
31
Mazalovska, Milena, and J. Calvin Kouokam. "Progress in the Production of Virus-Like Particles for Vaccination against Hepatitis E Virus." Viruses 12, no. 8 (July 30, 2020): 826. http://dx.doi.org/10.3390/v12080826.
Анотація:
Hepatitis E virus (HEV), a pathogen that causes acute viral hepatitis, is a small icosahedral, quasi-enveloped, positive ssRNA virus. Its genome has three open reading frames (ORFs), with ORF1 and ORF3 encoding for nonstructural and regulatory proteins, respectively, while ORF2 is translated into the structural, capsid protein. ORF2 is most widely used for vaccine development in viral hepatitis. Hepatitis E virus-like particles (VLPs) are potential vaccine candidates against HEV infection. VLPs are composed of capsid subunits mimicking the natural configuration of the native virus but lack the genetic material needed for replication. As a result, VLPs are unable to replicate and cause disease, constituting safe vaccine platforms. Currently, the recombinant VLP-based vaccine Hecolin® against HEV is only licensed in China. Herein, systematic information about the expression of various HEV ORF2 sequences and their ability to form VLPs in different systems is provided.
32
Kojima, Kenji K., Takumi Matsumoto, and Haruhiko Fujiwara. "Eukaryotic Translational Coupling in UAAUG Stop-Start Codons for the Bicistronic RNA Translation of the Non-Long Terminal Repeat Retrotransposon SART1." Molecular and Cellular Biology 25, no. 17 (September 1, 2005): 7675–86. http://dx.doi.org/10.1128/mcb.25.17.7675-7686.2005.
Анотація:
ABSTRACT Most eukaryotic cellular mRNAs are monocistronic; however, many retroviruses and long terminal repeat (LTR) retrotransposons encode multiple proteins on a single RNA transcript using ribosomal frameshifting. Non-long terminal repeat (non-LTR) retrotransposons are considered the ancestor of LTR retrotransposons and retroviruses, but their translational mechanism of bicistronic RNA remains unknown. We used a baculovirus expression system to produce a large amount of the bicistronic RNA of SART1, a non-LTR retrotransposon of the silkworm, and were able to detect the second open reading frame protein (ORF2) by Western blotting. The ORF2 protein was translated as an independent protein, not as an ORF1-ORF2 fusion protein. We revealed by mutagenesis that the UAAUG overlapping stop-start codon and the downstream RNA secondary structure are necessary for efficient ORF2 translation. Increasing the distance between the ORF1 stop codon and the ORF2 start codon decreased translation efficiency. These results are different from the eukaryotic translation reinitiation mechanism represented by the yeast GCN4 gene, in which the probability of reinitiation increases as the distance between the two ORFs increases. The translational mechanism of SART1 ORF2 is analogous to translational coupling observed in prokaryotes and viruses. Our results indicate that translational coupling is a general mechanism for bicistronic RNA translation.
33
Nawagitgul, Porntippa, Igor Morozov, Steven R. Bolin, Perry A. Harms, Steven D. Sorden, and Prem S. Paul. "Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein." Journal of General Virology 81, no. 9 (September 1, 2000): 2281–87. http://dx.doi.org/10.1099/0022-1317-81-9-2281.
Анотація:
Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa.
34
Glass, Pamela J., Carl Q. Zeng, and Mary K. Estes. "Two Nonoverlapping Domains on the Norwalk Virus Open Reading Frame 3 (ORF3) Protein Are Involved in the Formation of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions." Journal of Virology 77, no. 6 (March 15, 2003): 3569–77. http://dx.doi.org/10.1128/jvi.77.6.3569-3577.2003.
Анотація:
ABSTRACT Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF31-179, ORF31-152, and ORF31-107) no longer produced the 35K form. An N-terminal truncation mutant (ORF351-212) and a site-directed mutant (ORF3T201V) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3FL), ORF31-179, ORF31-152, and ORF351-212 interacted with the ORF2 protein, while an ORF31-107 protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.
35
Hardy, Richard W., and Gail W. Wertz. "The Product of the Respiratory Syncytial Virus M2 Gene ORF1 Enhances Readthrough of Intergenic Junctions during Viral Transcription." Journal of Virology 72, no. 1 (January 1, 1998): 520–26. http://dx.doi.org/10.1128/jvi.72.1.520-526.1998.
Анотація:
ABSTRACT The mRNA encoding the M2 protein of respiratory syncytial (RS) virus contains two open reading frames (ORFs). ORF1 encodes the 22-kDa structural protein, M2, and ORF2 has the potential to encode a 10-kDa protein (90 amino acids). Using a vaccinia virus T7 expression system, we examined the RNA synthetic activities of mono- and dicistronic subgenomic replicons of RS virus by direct metabolic labeling of RNA in the presence and absence of the products of ORF1 and ORF2. In the absence of ORF1 and ORF2, the negative- and positive-sense products of genomic RNA replication and positive-sense polyadenylated mRNA(s) were synthesized. Expression of the whole M2 transcription unit (containing ORF1 and ORF2) or ORF1 alone caused an increase in the synthesis of polyadenylated mRNA, the majority of which was due to a substantial increase in the quantity of polycistronic mRNAs generated by the polymerase failing to terminate at gene end signals. In agreement with previous reports, the ORF2 product was found to inhibit viral RNA replication and mRNA transcription. These data show that the M2 protein functions as a transcriptional antiterminator that enhances the ability of the viral RNA polymerase to read through intergenic junctions. The role of such a function during the viral life cycle is discussed.
36
Elsner, Andrea, Bernd Kreikemeyer, Andrea Braun-Kiewnick, Barbara Spellerberg, Bettina A. Buttaro, and Andreas Podbielski. "Involvement of Lsp, a Member of the LraI-Lipoprotein Family in Streptococcus pyogenes, in Eukaryotic Cell Adhesion and Internalization." Infection and Immunity 70, no. 9 (September 2002): 4859–69. http://dx.doi.org/10.1128/iai.70.9.4859-4869.2002.
Анотація:
ABSTRACT Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between the mga core regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein of Streptococcus pyogenes) and ORF2 exhibited a high degree of homology to the lmb/ORF2 genes of S. agalactiae (B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistronic lsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase. lsp and ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn2+ and Cu2+ ion concentrations decreased lsp operon expression, Lsp did not transport divalent cations as described for other LraI-type operons. The lsp mutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, the lsp mutant showed decreased transcription of prtF2 encoding the fibronectin-binding protein F2. Both the lsp and ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in the lsp mutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription of speB (cysteine protease) and/or the global regulators mga, csrRS, and nra.
37
Chuang, Duen-Yau, Ampaabeng G. Kyeremeh, Yuichi Gunji, Yoshiyuki Takahara, Yoshio Ehara, and Toshio Kikumoto. "Identification and Cloning of an Erwinia carotovorasubsp. carotovora Bacteriocin Regulator Gene by Insertional Mutagenesis." Journal of Bacteriology 181, no. 6 (March 15, 1999): 1953–57. http://dx.doi.org/10.1128/jb.181.6.1953-1957.1999.
Анотація:
ABSTRACT Avirulent Erwinia carotovora subsp.carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3′ end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5′ end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qβ-replicase; andAzorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coliDH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.
38
Yin, Xin, Dong Ying, Sébastien Lhomme, Zimin Tang, Christopher M. Walker, Ningshao Xia, Zizheng Zheng, and Zongdi Feng. "Origin, antigenicity, and function of a secreted form of ORF2 in hepatitis E virus infection." Proceedings of the National Academy of Sciences 115, no. 18 (April 18, 2018): 4773–78. http://dx.doi.org/10.1073/pnas.1721345115.
Анотація:
The enterically transmitted hepatitis E virus (HEV) adopts a unique strategy to exit cells by cloaking its capsid (encoded by the viral ORF2 gene) and circulating in the blood as “quasi-enveloped” particles. However, recent evidence suggests that the majority of the ORF2 protein present in the patient serum and supernatants of HEV-infected cell culture exists in a free form and is not associated with virus particles. The origin and biological functions of this secreted form of ORF2 (ORF2S) are unknown. Here we show that production of ORF2Sresults from translation initiated at the previously presumed AUG start codon for the capsid protein, whereas translation of the actual capsid protein (ORF2C) is initiated at a previously unrecognized internal AUG codon (15 codons downstream of the first AUG). The addition of 15 amino acids to the N terminus of the capsid protein creates a signal sequence that drives ORF2Ssecretion via the secretory pathway. Unlike ORF2C, ORF2Sis glycosylated and exists as a dimer. Nonetheless, ORF2Sexhibits substantial antigenic overlap with the capsid, but the epitopes predicted to bind the putative cell receptor are lost. Consistent with this, ORF2Sdoes not block HEV cell entry but inhibits antibody-mediated neutralization. These results reveal a previously unrecognized aspect in HEV biology and shed new light on the immune evasion mechanisms and pathogenesis of this virus.
39
Sasaya, Takahide, Shinnosuke Kusaba, Koichi Ishikawa, and Hiroki Koganezawa. "Nucleotide sequence of RNA2 of Lettuce big-vein virus and evidence for a possible transcription termination/initiation strategy similar to that of rhabdoviruses." Journal of General Virology 85, no. 9 (September 1, 2004): 2709–17. http://dx.doi.org/10.1099/vir.0.80061-0.
Анотація:
Lettuce big-vein virus (LBVV) is the type species of the genus Varicosavirus and is a two-segmented negative-sense single-stranded RNA virus. The larger LBVV genome segment (RNA1) consists of 6797 nt and encodes an L polymerase that resembles that of rhabdoviruses. Here, the nucleotide sequence of the second LBVV genome segment (RNA2) is reported. LBVV RNA2 consisted of 6081 nt and contained antisense information for five major ORFs: ORF1 (nt 210–1403 on the viral RNA), ORF2 (nt 1493–2494), ORF3 (nt 2617–3489), ORF4 (nt 3843–4337) and ORF5 (nt 4530–5636), which had coding capacities of 44, 36, 32, 19 and 41 kDa, respectively. The gene at the 3′ end of the viral RNA encoded a coat protein, while the other four genes encoded proteins of unknown functions. The 3′-terminal 11 nt of LBVV RNA2 were identical to those of LBVV RNA1, and the 5′-terminal regions of LBVV RNA1 and RNA2 contained a long common nucleotide stretch of about 100 nt. Northern blot analysis using probes specific to the individual ORFs revealed that LBVV transcribes monocistronic RNAs. Analysis of the terminal sequences, and primer extension and RNase H digestion analysis of LBVV mRNAs, suggested that LBVV utilizes a transcription termination/initiation strategy comparable with that of rhabdoviruses.
40
Oleksiewicz, M. B., A. Bøtner, and P. Normann. "Porcine B-cells recognize epitopes that are conserved between the structural proteins of American- and European-type porcine reproductive and respiratory syndrome virus." Journal of General Virology 83, no. 6 (June 1, 2002): 1407–18. http://dx.doi.org/10.1099/0022-1317-83-6-1407.
Анотація:
By selecting phage display libraries with immune sera from experimentally infected pigs, porcine B-cell epitopes in the open reading frame (ORF) 2, 3, 5 and 6 proteins of European-type porcine reproductive and respiratory syndrome virus (PRRSV) were identified. The sequences of all the epitopes were well conserved in European-type PRRSV and even between European- and American-type PRRSV. Accordingly, sera from pigs infected with American-type PRRSV cross-reacted with the European-type epitopes. Thus, this study showed, for the first time, the presence of highly conserved epitopes in the matrix protein and envelope glycoproteins of PRRSV. ORF5 and 6 epitopes localized to protein parts that are predicted to be hidden in PRRSV virions. In contrast, ORF2 and 3 epitopes localized to putative protein ectodomains. Due to the interesting localization, the sequence surrounding the ORF2 and 3 epitopes was subjected to closer scrutiny. A heptad motif, VSRRIYQ, which is present in a single copy in ORF2 and 3 proteins, was identified; this arrangement is completely conserved in all European-type PRRSV sequences available. The VSRRIYQ repeat motif colocalized closely with one of the ORF2 epitopes and secondary structure modelling showed that this segment of the ORF2 protein could form an amphipathic helix. Intriguingly, a mutation associated with virulence/attenuation of an American vaccine strain of PRRSV also localized to this ORF2 protein segment and affected the hydrophobic face of the predicted amphipathic helix. Further work is needed to determine whether these findings delineate a functional domain in the PRRSV ORF2 protein.
41
Ilves, H., O. Kahre, and M. Speek. "Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting." Molecular and Cellular Biology 12, no. 9 (September 1992): 4242–48. http://dx.doi.org/10.1128/mcb.12.9.4242-4248.1992.
Анотація:
The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.
42
Ilves, H., O. Kahre, and M. Speek. "Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting." Molecular and Cellular Biology 12, no. 9 (September 1992): 4242–48. http://dx.doi.org/10.1128/mcb.12.9.4242.
Анотація:
The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.
43
Cowley, Jeff A., Lee C. Cadogan, Kirsten M. Spann, Nusra Sittidilokratna, and Peter J. Walker. "The Gene Encoding the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeus monodon Prawns Is Located Upstream of the Glycoprotein Gene." Journal of Virology 78, no. 16 (August 15, 2004): 8935–41. http://dx.doi.org/10.1128/jvi.78.16.8935-8941.2004.
Анотація:
ABSTRACT The ORF2 gene of Gill-associated virus (GAV) of Penaeus monodon prawns resides 93 nucleotides downstream of the ORF1a-ORF1b gene and encodes a 144-amino-acid hydrophilic polypeptide (15,998 Da; pI, 9.75) containing 20 basic (14%) and 13 acidic (9%) residues and 19 prolines (13%). Antiserum to a synthetic ORF2 peptide or an Escherichia coli-expressed glutathione S-transferase-ORF2 fusion protein detected a 20-kDa protein in infected lymphoid organ and gill tissues in Western blots. The GAV ORF2 fusion protein antiserum also cross-reacted with the p20 nucleoprotein in virions of the closely related Yellow head virus. By immuno-gold electron microscopy, it was observed that the ORF2 peptide antibody localized to tubular GAV nucleocapsids, often at the ends or at lateral cross sections. As GAV appears to contain only two structural protein genes (ORF2 and ORF3), these data indicate that GAV differs from vertebrate nidoviruses in that the gene encoding the nucleocapsid protein is located upstream of the gene encoding the virion glycoproteins.
44
Llamas, María A., Juan L. Ramos, and José J. Rodríguez-Herva. "Transcriptional Organization of the Pseudomonas putida tol-oprL Genes." Journal of Bacteriology 185, no. 1 (January 1, 2003): 184–95. http://dx.doi.org/10.1128/jb.185.1.184-195.2003.
Анотація:
ABSTRACT Proteins of the Tol system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. In Pseudomonas putida, the seven genes, orf1, tolQ, tolR, tolA, tolB, oprL, and orf2, which encode the proteins of this complex, are clustered in a 5.8-kb region of chromosomal DNA. Analysis of polar mutations, reverse transcriptase PCR assays, and transcriptional fusion constructs with a promoterless lacZ gene revealed that the genes are arranged in two operons: orf1 tolQ tolR tolA tolB and oprL orf2. We were also able to find a transcript that was initiated at the orf1 promoter and covered the two operons in a single mRNA. On the basis of the OprL protein level, we surmised that this transcript contributed only about 10 to 15% of the total OprL protein. Primer extension analysis identified the oprL orf2 operon promoter within the tolB gene, and the −10 and −35 regions exhibited some similarity to those of σ70-recognized promoters. The transcription start point of orf1 was located 91 bp upstream of the orf1 start codon, and the −10/−35 region also exhibited σ70 −10/−35 recognition sequences. The expression from both promoters in rich and minimal media was constitutive and was very little influenced by the growth phase or iron-deficient conditions. In addition, analyses of the β-galactosidase activities of different translational fusion constructs revealed that translation of tolA and orf2 genes was dependent on the translation of their corresponding upstream genes (tolR and oprL, respectively).
45
Robinson, D. N., and L. Cooley. "Examination of the function of two kelch proteins generated by stop codon suppression." Development 124, no. 7 (April 1, 1997): 1405–17. http://dx.doi.org/10.1242/dev.124.7.1405.
Анотація:
The Drosophila kelch gene produces a single transcript with a UGA stop codon separating two open reading frames (ORF1 and ORF2). From the transcript, 76 kDa ORF1 and 160 kDa full-length (ORF1 + ORF2) proteins are made. The expression of these two proteins is regulated in a tissue-specific manner causing the ratio of full-length to ORF1 protein to vary in different tissues. The only detected defect for kelch mutants is female sterility, and kelch protein is localized to the ovarian ring canals. kelch mutant ring canals are disorganized and have partly occluded lumens, causing a failure to transport cytoplasm. ORF1 and full-length kelch proteins co-sediment with ring canals suggesting that both proteins are found in the ring canals. Transgenetic analysis reveals that ORF1 kelch protein is sufficient to rescue ring canal morphology and fertility. In addition, we have mutated the UGA stop codon to a UAA stop codon and to three sense codons that allow constitutive readthrough. Analysis of these mutants reveals that a full-length kelch protein can partially compensate for the loss of endogenous kelch, but the residue included at the stop codon is critical for function. Finally, these studies suggest that the mechanism of stop codon suppression of kelch is by tRNA suppression.
46
Ichinose, Koji, Makoto Ozawa, Keiko Itou, Kanako Kunieda, and Yutaka Ebizuka. "Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters." Microbiology 149, no. 7 (July 1, 2003): 1633–45. http://dx.doi.org/10.1099/mic.0.26310-0.
Анотація:
Medermycin is a Streptomyces aromatic C-glycoside antibiotic classified in the benzoisochromanequinones (BIQs), which presents several interesting biosynthetic problems concerning polyketide synthase (PKS), post-PKS tailoring and deoxysugar pathways. The biosynthetic gene cluster for medermycin (the med cluster) was cloned from Streptomyces sp. AM-7161. Completeness of the clone was proved by the heterologous expression of a cosmid carrying the entire med cluster in Streptomyces coelicolor CH999 to produce medermycin. The DNA sequence of the cosmid (36 202 bp) revealed 34 complete ORFs, with an incomplete ORF at either end. Functional assignment of the deduced products was made for PKS and biosynthetically related enzymes, tailoring steps including strereochemical control, oxidation, angolosamine pathway, C-glycosylation, and regulation. The med cluster was estimated to be about 30 kb long, covering 29 ORFs. An unusual characteristic of the cluster is the disconnected organization of the minimal PKS genes: med-ORF23 encoding the acyl carrier protein is 20 kb apart from med-ORF1 and med-ORF2 for the two ketosynthase components. Secondly, the six genes (med-ORF14, 15, 16, 17, 18 and 20) for the biosynthesis of the deoxysugar, angolosamine, are all contiguous. Finally, the finding of a glycosyltransferase gene, med-ORF8, suggests a possible involvement of conventional C-glycosylation in medermycin biosynthesis. Comparison among the three complete BIQ gene clusters – med and those for actinorhodin (act) and granaticin (gra) – revealed some common genes whose deduced functions are unavailable from database searches (the ‘unknowns’). An example is med-ORF5, a homologue of actVI-ORF3 and gra-ORF18, which was highlighted by a recent proteomic analysis of S. coelicolor A3(2).
47
Emerson, Suzanne U., Hanh T. Nguyen, Udana Torian, Danielle Burke, Ronald Engle, and Robert H. Purcell. "Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif." Journal of Virology 84, no. 18 (July 7, 2010): 9059–69. http://dx.doi.org/10.1128/jvi.00593-10.
Анотація:
ABSTRACT Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.
48
Sunna, Anwar, Moreland D. Gibbs, Charles W. J. Chin, Peter J. Nelson та Peter L. Bergquist. "A Gene Encoding a Novel Multidomain β-1,4-Mannanase from Caldibacillus cellulovorans and Action of the Recombinant Enzyme on Kraft Pulp". Applied and Environmental Microbiology 66, № 2 (1 лютого 2000): 664–70. http://dx.doi.org/10.1128/aem.66.2.664-670.2000.
Анотація:
ABSTRACT Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a β-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85°C and pH 6.0 and was extremely thermostable at 70°C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable β-xylanase on oxygen-delignifiedPinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.
49
Kaiser, William J., Yasmin Chaudhry, Stanislav V. Sosnovtsev, and Ian G. Goodfellow. "Analysis of protein–protein interactions in the feline calicivirus replication complex." Journal of General Virology 87, no. 2 (February 1, 2006): 363–68. http://dx.doi.org/10.1099/vir.0.81456-0.
Анотація:
Caliciviruses are a major cause of gastroenteritis in humans and cause a wide variety of other diseases in animals. Here, the characterization of protein–protein interactions between the individual proteins of Feline calicivirus (FCV), a model system for other members of the family Caliciviridae, is reported. Using the yeast two-hybrid system combined with a number of other approaches, it is demonstrated that the p32 protein (the picornavirus 2B analogue) of FCV interacts with p39 (2C), p30 (3A) and p76 (3CD). The FCV protease/RNA polymerase (ProPol) p76 was found to form homo-oligomers, as well as to interact with VPg and ORF2, the region encoding the major capsid protein VP1. A weak interaction was also observed between p76 and the minor capsid protein encoded by ORF3 (VP2). ORF2 protein was found to interact with VPg, p76 and VP2. The potential roles of the interactions in calicivirus replication are discussed.
50
Taguchi, Fumiko, Yujiro Ogawa, Kasumi Takeuchi, Tomoko Suzuki, Kazuhiro Toyoda, Tomonori Shiraishi, and Yuki Ichinose. "A Homologue of the 3-Oxoacyl-(Acyl Carrier Protein) Synthase III Gene Located in the Glycosylation Island of Pseudomonas syringae pv. tabaci Regulates Virulence Factors via N-Acyl Homoserine Lactone and Fatty Acid Synthesis." Journal of Bacteriology 188, no. 24 (October 6, 2006): 8376–84. http://dx.doi.org/10.1128/jb.00763-06.
Анотація:
ABSTRACT Pseudomonas syringae pv. tabaci 6605 possesses a genetic region involved in flagellin glycosylation. This region is composed of three open reading frames: orf1, orf2, and orf3. Our previous study revealed that orf1 and orf2 encode glycosyltransferases; on the other hand, orf3 has no role in posttranslational modification of flagellin. Although the function of Orf3 remained unclear, an orf3 deletion mutant (Δorf3 mutant) had reduced virulence on tobacco plants. Orf3 shows significant homology to a 3-oxoacyl-(acyl carrier protein) synthase III in the fatty acid elongation cycle. The Δorf3 mutant had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules, suggesting that Orf3 is required for AHL synthesis. In comparison with the wild-type strain, swarming motility, biosurfactant production, and tolerance to H2O2 and antibiotics were enhanced in the Δorf3 mutant. A scanning electron micrograph of inoculated bacteria on the tobacco leaf surface revealed that there is little extracellular polymeric substance matrix surrounding the cells in the Δorf3 mutant. The phenotypes of the Δorf3 mutant and an AHL synthesis (ΔpsyI) mutant were similar, although the mutant-specific characteristics were more extreme in the Δorf3 mutant. The swarming motility of the Δorf3 mutant was greater than that of the ΔpsyI mutant. This was attributed to the synergistic effects of the overproduction of biosurfactants and/or alternative fatty acid metabolism in the Δorf3 mutant. Furthermore, the amounts of iron and biosurfactant seem to be involved in biofilm development under quorum-sensing regulation in P. syringae pv. tabaci 6605.