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1
Upadhyay, Mohita. "Dinucleotide frequencies in DNA virus genomes: implications on virus evolution." Thesis, IIT Delhi, 2016. http://localhost:8080/xmlui/handle/12345678/6989.
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Altan, Eda, Valle Mendoza Juana Mercedes Del, Xutao Deng, Tung G. Phan, Mohammadreza Sadeghi, and Eric L. Delwart. "Small Circular Rep-Encoding Single-Stranded DNA Genomes in Peruvian Diarrhea Virome." American Society for Microbiology, 2017. http://hdl.handle.net/10757/622307.
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Metagenomic analysis of diarrhea samples revealed the presence of numerous human enteric viruses and small circular Rep-encoding single-stranded DNA (CRESS-DNA) genomes. One such genome was related to smacoviruses, while eight others were related to genomes reported in the feces of different mammals. The tropism of these CRESS-DNA viruses remains unknown.
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Karan, Mirko. "Sequence diversity of DNA components associated with banana bunchy top virus." Thesis, Queensland University of Technology, 1995.
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Jackson, Ronald James. "Analysis of hepatitis B virus DNA integrated into the genomes of rodent cells." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/15098.
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Tuthill, Tobias J. "Construction expression and preliminary biological analysis of HCV and HCV-dengue chimeric virus genomes." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368893.
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Yoosuf, Niyaz. "Genomes of mimiviruses of amoeba." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM5072/document.
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Les membres des familles Mimiviridae et Marseilleviridae, qui infectent et se répliquent dans Acanthamoeba spp. et d’autres protistes phagocytaires, ont été découverts au cours de la dernière décennie et rattachés à un groupe monophylétique de virus nommés les grands virus à ADN nucléocytoplasmiques (NCLDVs), qui infectent un large éventail d’eukaryotes y compris différents organismes unicellulaires. Récemment, il a été proposé de reclasser les NCLDVs dans un nouvel ordre viral nommé les Megavirales. Plusieurs dizaines de virus géants des amibes ont été isolés, mais le génome de peu d’entre eux a été étudié de façon approfondie. Nous avons étudié les génomes de ces virus géants d'amibe afin d’acquérir une meilleure compréhension de leur répertoire de gènes et leur importance évolutionnaire. L'analyse phylogénétique des virus géants d'amibe distingue clairement trois lignées, nommées A, B et C. Nous avons étudié en détail le génome de Acanthamoeba polyphaga moumouvirus, le membre fondateur de la lignée B et avons déchiffré son contenu en gènes et sa relation évolutive avec d'autres organismes. Nous avons également étudié les génomes de Terra1 virus et Terra2 virus, qui appartiennent respectivement aux lignées C et A, et ont été isolés à partir d'échantillons de sol alors que les mimivirus décrits aupravant ont été isolés à partir d'eau douce ou de mer. En outre, nous avons décrit le génome du virus Courdo11, qui appartient à la lignée C, et est étroitement lié au premier Mimivirus isolé d'un humain, qui présentait une pneumonie inexpliquéeThe members of families Mimiviridae and Marseilleviridae, which infect and replicate in Acanthamoeba spp. and other phagocytic protists, were discovered during the past decade and linked to a monophyletic group of viruses named the Nucleocytoplasmic Large DNA viruses (NCLDVs), which infect a broad variety of eukaryotes including diverse unicellular organisms. Recently, it has been proposed to reclassify the NCLDVs into a new viral order named the Megavirales. Several dozens of giant viruses of amoeba have been isolated but the genome of very few has been extensively studied. We studied the genomes of these giant viruses of amoeba to gain a better understanding of their gene repertoire and evolutionary importance. The phylogenetic analysis of giant viruses of amoeba clearly distinguished three lineages, named lineages A, B and C. We studied in detail the genome of Acanthamoeba polyphaga moumouvirus, the leader member of lineage B to decipher its gene content and its evolutionary relationship with other organisms. We further studied the genomes of Terra1 virus and Terra2 virus, which belong to lineages C and A, respectively, and were isolated from soil samples whereas previously described mimiviruses of amoeba were isolated from fresh or marine water. Furthermore, we described the genome of Courdo11 virus, which belongs to lineage C, and is closely related to the first mimivirus isolated from a human, who exhibited unexplained pneumonia
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Pereira, Bruna Tibirica. "Sequenciamento do baculovírus que infecta a broca-da-cana-de-açúcar Diatraea saccharalis." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-16052014-082057/.
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Baculovírus são vírus específicos de inseto que infectam principalmente membros da ordem Lepidoptera. Diatraea saccharalis granulovírus (DsGV) foi isolado de larvas de Diatraea saccharalis (Lepidoptera: Crambidae), a broca-da-cana-de-açúcar, um dos insetos-praga de maior importância na cultura de cana-de-açúcar no Brasil. O genoma completo de DsGV foi obtido através do sequenciamento 454 (Roche). O genoma de DsGV apresentou 98.463 pb e potencialmente codifica 116 genes. Foram identificados os 37 genes conservados em todos os baculovírus, 19 genes específicos de betabaculovírus e 17 genes únicos. DsGV é o primeiro betabaculovírus que possui o gene gp64, que codifica uma proteína de fusão, originalmente encontrado apenas em alfabaculovírus do grupo I. A análise filogenética utilizando a concatenação das sequências deduzidas de aminoácidos de 30 genes conservados em 61 baculovírus totalmente sequenciados sugere que DsGV está inserido no clado b do grupo dos betabaculovírus e parece estar mais estritamente relacionado a 5 GVs (ChocGV, PiraGV, ClanGV, CpGV e CrleGV).Baculoviruses are insect specific viruses that infect mainly members of the Order Lepidoptera. Diatraea saccharalis granulovirus (DsGV) was isolated from Diatraea saccharalis (Lepidoptera: Crambidae), one of the most important insect pest of the sugar cane culture in Brazil. The genome of DsGV was obtained by the 454 sequencing system (Roche). Our results showed that the nucleotide sequence of the DsGV genome is 98.463 bp in length and potentially encodes 116 putative genes. It contains the 37 baculovirus core genes, a set of 19 betabaculovirus-specific genes and 17 putative DsGV genes were not found in any genome of the baculoviruses sequenced up to the present. DsGV is the first betabaculovirus sequenced so far that has the gp64 envelope fusion protein gene, originally found only in alphabaculovirus group I. Phylogenetic analysis performed with concatamers of 30 conserved proteins from 61 fully sequenced baculovirus genomes suggests that DsGV is a member of clade b of the betabaculovirus and seems to be closer to 5 GVs (ChocGV, PiraGV, ClanGV, CpGV e CrleGV).
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Anderson, Marie June. "Complementary DNA cloning, sequencing, and analysis of the Pelargonium flower-break virus genome /." The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487842372897839.
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Akahata, Wataru. "DNA vaccination of macaques by full genome plasmids which produce noninfectious virus particles." Kyoto University, 2002. http://hdl.handle.net/2433/149869.
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Kyoto University (京都大学)0048
新制・課程博士
博士(人間・環境学)
甲第9661号
人博第145号
13||130(吉田南総合図書館)
新制||人||35(附属図書館)
UT51-2002-G419
京都大学大学院人間・環境学研究科人間・環境学専攻
(主査)教授 速水 正憲, 教授 津田 謹輔, 教授 松村 道一, 助教授 三浦 智行
学位規則第4条第1項該当
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Fick, Wilhelmina Christina. "Characterisation of promoter sequences in a Capripoxvirus genome." Master's thesis, University of Cape Town, 1992. http://hdl.handle.net/11427/25623.
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Capripoxviruses are of particular interest as live recombinant vectors for use in the veterinary field, since their host-range is restricted to cattle, goats and sheep. The work presented in this thesis is a preliminary study undertaken on the South African Neethling vaccine strain of lumpy skin disease virus (LSDV). As a departure point towards the eventual identification of strong promoter areas in the 143 kb genome of LSDV, a portion of its genome was cloned. Three methods for purification of LSDV DNA were compared, to determine which yielded the best quality DNA for cloning. DNA extracted directly from infected cells was excessively contaminated with bovine host-DNA, complicating the cloning of LSDV DNA. The use of pulsed field gel electrophoresis solved the contamination problem, by separating viral DNA from bovine DNA. However, insufficient amounts of viral DNA for cloning purposes, could be recovered from the gel. Sufficient amounts of good quality LSDV DNA was obtained by extraction from purified virions. Purified LSDV DNA was digested with various restriction enzymes to identify those which yielded several 4-1 0 kb fragments, for cloning into the Bluescribe plasmid transcription vector. Enrichment for large fragments (8-1 0 kb) was achieved by sucrose density centrifugation. Cloned fragments were analysed by Southern blot hybridisation to verify their viral origin. Hybridisation studies indicated that several unique regions of the LSDV genome were cloned as Pst I and Bam HI fragments respectively, i.e. the cloned fragments contained no overlapping regions. In total, 71.25 kb of the DNA of the LSDV Neethling vaccine strain has been cloned, representing approximately 50% of the viral genome. The availability of these clones now paves the way for further molecular investigations of the LSDV Neethling genome, including identification of promoter regions. A trial gene, which will be cloned and expressed in LSDV, namely the cloned VPS-gene of bluetongue virus serotype 4, was prepared and its nucleotide sequence determined. Homopolymer sequences present at the terminal ends of the gene as a result of the original cloning strategy, are known to interfere with expression and were removed by means of the polymerase chain reaction (PCR). The nucleotide sequence of the resulting PCR-tailored BTV4 VPS-genewas determined and used to deduce the amino acid sequence of the protein. The gene is 1638 bp in length and encodes a protein of 526 aa. Conserved sequences, 6 bp in length and unique to the 5'- and 3'terminal ends of all BTV genes, were detected at the termini of the tailored gene, confirming that the original clone was a full-length copy of the gene. Amplification by PCR did not mutate the open reading frame (OAF) of the gene, since it was of similar length to that reported for 5 other BTV serotypes. With a view to future investigations, including the identification of promoter sequences in the LSDV genome, a preliminary investigation of LSDV protein synthesis was undertaken, to acquire some knowledge of the growth cycle of the virus. Eighteen putative virus-specific proteins were identified by radio-labelling infected cells with [³⁵S]-methionine. By pulse-labelling infected cells with [³⁵S]methionine at various times post infection (p.i.), viral proteins were first detected at 16 hr p.i. It is, however, unlikely that the early phase of viral replication commences as late as 16 hr p.i. and these results might be attributed to various problems, such as the low multiplicity of infection used and that host protein shut-down was inefficient, thus masking the presence viral proteins. In conclusion, this investigation resulted in the cloning of 71,25 kb of the LSDV genome, the tailoring and sequencing of the BTV4 VPS gene and the identification of 18 putative LSDV proteins. This now paves the way for further research to develop LSDV as a vaccine vector.
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Horiuchi, Reii. "Development of DNA vaccine using full genome plasmids which produce non-infectious virus particles." Kyoto University, 2006. http://hdl.handle.net/2433/144059.
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Kyoto University (京都大学)0048
新制・課程博士
博士(人間・環境学)
甲第12403号
人博第321号
新制||人||79(附属図書館)
17||D||180(吉田南総合図書館)
24239
UT51-2006-J395
京都大学大学院人間・環境学研究科相関環境学専攻
(主査)教授 速水 正憲, 教授 津田 謹輔, 教授 小松 賢志
学位規則第4条第1項該当
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Anderson, Jon Paul. "Molecular diversity and evolution of human immunodeficiency virus type 1 /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8049.
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Papadopoulou, Eugenia. "Studies on tobacco yellow dwarf virus, a geminivirus of the genus Mastrevirus adapted to dicots : infectivity determinants, virion sense gene expression and ribosome inactivatin protein-based resistance." Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365008.
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Backovic, Ana. "Capsid Assembly and Single Stranded DNA Genome Formation of Adeno-Associated Virus Type2 in Yeast Cells." Doctoral thesis, Scuola Normale Superiore, 2011. http://hdl.handle.net/11384/85957.
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Saccharomyces cerevisiae has provided an array of genetic tools to study unknown aspects
of viral life cycles, supporting replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses). It also provides means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus) and as such represents a useful tool for vaccine development. To extend the utility of the S. cerevisiae expression system, we expressed AAV2 structural and nonstructural proteins in yeast cells, using both authentic AAV2 and heterologous yeast promoters. For the first time, we described the assembly of AAV2 virus-like particles from yeast-expressed AAV2 structural proteins. To do this we used AAV p40 promoter, whose activity in yeast cells resembled the one of yeast glycolytic promoters, resulting in the synthesis of the most abundant capsid protein VP3 when transformed yeast cells were
grown on glucose as a carbon source. The expression of other two VPs was induced from yeast, galactose inducible pGal1 promoter. Simultaneous production of all three VPs was achieved by growing the yeast cells in the medium containing both glucose and galactose, while their relative production levels were further optimized by varying amounts of each
carbon source in the induction medium, followed by the fine tuning of the induction time.
Moreover, we investigated the ability of the yeast Saccharomyces cerevisiae to carry out
the replication of a recombinant rAAV2. When a plasmid harboring the rAAV2 genome in
which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in
yeast with a plasmid expressing Rep68 from constitutive yeast promoter pADH, a
significant number of URA3+ clones were scored (more than 30-fold over controls).
Molecular analysis of low molecular weight DNA revealed that the single stranded DNA is
formed, in Rep68 and ITR dependent manner, and that the plasmid is entirely replicated.
The ss DNA contained the ITRs, URA3 gene and also vector sequences suggesting that ss
rAAV genomes were not obtained by the canonical AAV replication mechanism.
These results could open new prospects for using yeast cell in two ways: (i) as a model
system for studying viral and cellular factors involved in AAV2 capsid assembly and
packaging of rAAV ss genomes; and (ii) as a novel cell factory for developing superior
recombinant rAAV production technologies.
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Wade-Martins, Richard. "Developing Epstein-Barr virus-based stable episomes for gene expression from large genomic inserts to complement cell phenotypes." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301648.
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Chingandu, Nomatter, and Nomatter Chingandu. "Genomic Characterization of the Cacao Swollen Shoot Virus Complex and other Theobroma Cacao-Infecting Badnaviruses." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/621859.
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The cacao swollen shoot disease of Theobroma cacao L. (cacao) is caused by Cacao swollen shoot virus (CSSV; genus, Badnavirus, family, Caulimoviridae). The virus is endemic to West Africa, where it poses a serious threat to cocoa production. Despite efforts to control CSSV spread by replacement of infected trees with tolerant cultivars and mealybug vector management, the disease is widespread in West Africa. In Trinidad, leaf mosaic and vein-banding symptoms have been observed in cacao plants in the field since the 1940s, and recently at the International Cocoa Genebank (ICGT), a custodian of cacao germplasm resources. The strains A and B of the suspect Cacao Trinidad virus (CTV) caused the symptoms, and were thought to be related to CSSV, however, viral causality was not demonstrated, until now. To develop molecular detection methods for CSSV in infected plants, polymerase chain reaction (PCR) amplification of eight regions of the CSSV genome was implemented. The PCR results showed variable amplification frequencies of 19 - 42% at each region, for 124 isolates collected in Cote d'Ivoire and Ghana. Pairwise nucleotide (nt) analyses of the eight regions showed 66-99% shared identities, indicating that CSSV isolates exhibit extensive variability with respect to primer design. The results provided preliminary evidence for the existence of a CSSV complex consisting of four divergent species. The full length genome of 14 CSSV isolates from cacao determined using the Illumina HiSeq platform showed 70-99% shared nt identities. The pairwise nt identities placed CSSV sequences into a group of four distinct species, one of which represented a previously undescribed species. Moreover, the full-length genomes grouped phylogenetically with other badnaviruses and revealed two CSSV subclades with three types of genome arrangements; four, five or six open reading frames (ORFs). Predicted functional protein domains were conserved on each ORF. Two distinct, full-length genome sequences were determined using the Illumina HiSeq platform, from DNA isolated from cacao leaves exhibiting distinct symptoms in Trinidad. The sequences were validated by PCR-amplification and sequencing of overlapping viral genome fragments. Pairwise nt analysis indicated that each genome shared 52-62% nt identities with CSSV and other badnaviruses, suggesting that the two are distinct species. Phylogenetic analysis indicated that the two sequences are not strains of the same virus, as supposed, but they represent two previously undescribed species in the genus, Badnavirus, and they have been named Cacao mild mosaic virus (CaMMV) and Cacao yellow-vein-banding virus (CYVBV). Despite sharing the same host and causing similar symptoms in cacao, CSSV, CaMMV, and CYVBV are phylogenetically-distinct species. The discovery of a CSSV species complex and the identification of three new cacao-infecting badnavirus species will support the development of molecular detection tools using the partial and complete genome sequences determined in this study. The ability to develop validated molecular tools for the detection of CSSV and related viruses, CaMMV and CYVBV, in cacao will aid quarantine efforts and safe movement of germplasm from the ICGT in Trinidad to cacao-growing countries, worldwide. Also, molecular diagnostics tools are expected to be useful in efforts underway to develop CSSV-resistant planting material for countries in West Africa, which are currently experiencing continued or new disease outbreaks.
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Hauck, Markus Rüdiger. "Untersuchungen zur Integration von Nukleotidsequenzen des Retikuloendotheliose-Provirus in das Genom des Hühnerpocken-Virus." Berlin : Mensch-und-Buch-Verl, 2006. http://www.diss.fu-berlin.de/2006/389/index.html.
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NAM, THAM TO. "Organisation des transcrits et sequence nucleotidique de la region sali-f du genome du fv 3 (frog virus 3)." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13060.
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Le virus 3 grenouille (fv3), famille des iridoviridae, a un genome d'adn double brin de 160 kb hautement methyle, a redondances terminales et circulairement permute. Une arn polymerase ii de la cellule hote est impliquee dans la transcription precoce, mais l'adn viral purifie n'est pas infectieux, il est reactive par des proteines du virion. L'expression des genes est sequentielle. L'ordre des genes est determinee par selection des arns par hybridation aux recombinants suivi de leur traduction in vitro. L'organisation de transcripts est obtenue par cartographie a la nuclease s1. La comparaison des extremites 3'des arns revele la presence d'une sequence repetee inversee. Les sequences regulant l'initiation et la terminaison de la transcription sont voisines, sinon communes
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Shen, Ni 1969. "Role of the leader sequence of human immunodeficiency virus type 1 in viral replication, genome dimerization, encapsidation, and proviral DNA synthesis." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84434.
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Human immunodeficiency virus type 1 (HIV-1) genome consists of two identical RNAs that appear noncovalently linked near their 5' ends. The 5' untranslated region is called leader region. The 3' part of leader, i.e. nucleotides U200 to G335 in HIV-1 genomic RNA, between the primer binding site and the gag gene, can fold into 3 stem-loops: the kissing-loop domain (KLD) or stem-loop 1 (SL1), the 5' splicing junction hairpin (SD) or SL2 and SL3. The KLD, from nucleotide (nt) 243 to 277, forms a stem-loop (kissing loop hairpin) seated on top of a small stem bulge (stem B and loop B). The kissing-loop hairpin, or dimerization initiation site (DIS) hairpin consists of stem C and loop C. Loop C contains the autocomplementary sequence (ACS) GCGCGC262 or GUGCAC262, also called DIS.In the kissing-loop model of HIV-1 genome dimerization, HIV-1 RNA dimerization is initiated by base pairing between the ACS of one RNA monomer and that of an adjacent monomer.
To understand the role of the ACS in HIV-1 replication and HIV-1 genomic RNA dimerization, we replaced the central CGCG261 (or tetramer) of the HIV-1 Lai ACS by other tetramers. Genomic RNAs containing the UUAA tetramer (non-HIV-1 tetramer) were half dimeric, but UUAA genome packaging was unaffected. This was the first evidence that genomic RNA dimerization and packaging can be dissociated (Chapter 2). Destroying stem-loop C reduced genomic RNA dimerzation by ~50%, proviral DNA synthesis by ~85%, and reduced viral infectivity by ~3 logarithmic units. Destroying stem-loop B had similar effects on genome dimerization, reverse transcription, and viral infectivity. We also observed that mutations in stem-loop B and in the DIS hairpin were "non additive" (Chapter 2).
The existence of stem-loop C is supported by phylogenetic evidence, while that of stem-loop B is not, namely, its sequence is completely conserved. We investigated the role of stem B and loop B nucleotides in viral replication, and genomic RNA dimerization. The putative CUCG246/CGAG277 duplex was replaced by 9 alternative complementary sequences, 4 likely to base pair in long (~500 nts) RNAs, as assessed by the algorithm mfold. Among the 4 sequences, 3 preserved genome dimerzation, 1 did not significantly inhibit it, and 2 preserved viral replication. We also asked if 9 deletions or nucleotide substitutions within nucleotide 200 to 242 and/or 282 to 335 could influence genome dimerization. Delta200--226 and Delta236--242 genomic RNAs dimerized relatively poorly despite having neutral or positive influences on stem B, loop B and klh folding (Chapter 3).
Mutations within the Matrix, Capsid, p2 and nucleocapsid genes suppress several functional defects caused by KLD destruction. We tested the effect of these suppressor mutations on genome dimerization and infectiousness of viruses bearing moderate to crippling KLD mutations. Our conclusion is that these suppressor mutations can restore genomic RNA dimerization when DIS is weakened, but not when DIS is denatured or the KLD is destroyed (Chapter 4).
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Djacem, Karima. "Mécanisme moléculaire de reconnaissance et de clivage du génome chez le bactériophage SPP1, un virus à ADN double-brin." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS462/document.
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La reconnaissance spécifique du génome viral et son encapsidation est une étape cruciale pour l’assemblage de particules virales. Chez SPP1, comme chez d’autres bactériophages à queue, le moteur moléculaire qui encapside le génome viral est composé de la terminase, une enzyme hétéro-oligomérique qui possède une activité ATPasique et nucléasique, et de la protéine portale, un oligomère cyclique par lequel l’ADN viral est transloqué. Dans un grand nombre de ses virus, l’encapsidation de l’ADN est initiée par la reconnaissance et le clivage d’une séquence spécifique nommée « pac ». C’est un évènement qui se produit une seule fois au début d’une série de cycles d’encapsidation processive à partir d’un concatémère issu de la réplication du génome du phage. La région pac de SPP1 contient deux séquences (pacL et pacR) où TerS (gp1) se lie entourant la région (pacC) où TerL (gp2) coupe l’ADN de SPP1.Ici, nous montrons qu’une région de la séquence pacL et qu’un motif polyadénine de pacR agissent ensemble pour promouvoir le clivage en pacC. La dégénération de la région pacC n’a pas montré d’effet sur que le clivage endonucléolytique qui a lieu à une position bien définie de pacC avec une précision de ~6 pb. Des études avec des phages proches de SPP1 ont montré une conservation dans la position du clivage, malgré des variations dans pacC, pacR ou dans la distance entre pacL et pacC. Les données sont compatibles avec un modèle dans lequel TerS interagit spécifiquement avec la région pacL, sur laquelle le multimère cyclique TerS doit s’enrouler, et le motif polyadénine de la région pacR. Le complexe nucléoprotéique résultant va créer un contexte structural qui permet de recruter et positionner le domaine nucléase de TerL pour une coupure très précise sur pacC sans spécificité de séquenceThe specific recognition of the viral genome and its packaging is a critical step in viral particle assembly. In SPP1, as in many tailed bacteriophages, the macromolecular motor that encapsidates viral DNA is composed of terminase, a hetero-oligomeric enzyme possessing ATPase and nuclease activities, and of portal protein, a cyclic oligomer through which DNA is translocated. In a large number of these viruses, DNA packaging is initiated by recognition and cleavage of a specific sequence pac. This event occurs once at the beginning of a series of processive encapsidation events along a substrate concatemer of replicated phage genomes. The SPP1 pac region has two sequences where TerS (gp1) binds (pacL and pacR) flanking the segment where TerL (gp2) cleaves the SPP1 DNA (pacC). Here we show that a sequence segment of pacL and a poly-adenine motif in pacR act together to promote cleavage at pacC. Extensive degeneration of pacC sequence has no detectable effect in pac cleavage. The endonucleolytic cut occurs at a defined position with a precision of ~6 bp. Studies with SPP1-related phages show conservation of the cut position, irrespectively of sequence variation in pacC, in pacR or changes in pacL-pacC distance. The data is compatible with a model in which TerS interacts specifically with a region of pacL that probably wraps around the TerS cyclical multimer, and a poly-A tract in pacR. The resulting nucleoprotein complex architecture positions TerL for accurate cleavage at pacC without specific sequence requirement
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Jammeh, Saffie. "Study of the functional significance of mutations in the hepatitis B virus genome, following transfection of liver cell lines with cloned infectious DNA." Thesis, Imperial College London, 2006. http://hdl.handle.net/10044/1/11402.
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Mugnier, Marie-Ange. "Rna 3 du virus de la mosaique de la luzerne (almv) : obtention d'une copie cdna complete et etude conformationnelle de la region 5' du rna 3 de differentes souches." Université Louis Pasteur (Strasbourg) (1971-2008), 1986. http://www.theses.fr/1986STR13160.
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Synthese d'une copie complete d'adn complementaire qui est ensuite clone dans un vecteur d'expression pgemi. La comparaison de la sequence des cdna avec celle de l'arn 3 met en evidence une duplication dans la region 5' non codante, d'une sequence de 56 nucleotides qui constitue la difference majeure entre ces 2 sequences. La structure primaire de la region 5' non codante a ete examinee dans l'arn 3 de 3 souches du virus. Cette etude est completee par une analyse conformationnelle, en utilisant des sondes chimiques (dms) et enzymatique (v1 et s1)
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Cerva, Cristine. "Genomas de vírus de DNA de fita simples detectados em soros de suínos com síndrome multissistêmica do definhamento do suíno através de metagenômica." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/165145.
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As doenças associadas ao circovírus suíno (DACS) causam impacto econômico negativo nos sistemas de criação de suínos no mundo todo. As perdas incluem tratamento da doença, mortalidade, bem como diminuição no desempenho dos animais. Uma das manifestações mais relevantes das DACS é a síndrome multissistêmica do definhamento dos suínos (SMDS). O principal agente patogênico na causa da SMDS é o circovírus suíno tipo 2 (PCV2), no entanto, estudos observacionais e experimentais demonstraram que outros agentes estão envolvidos na patogênese e manifestação dos sinais clínicos. O sequenciamento de alto desempenho aliado a metagenômica são ferramentas que tornam possível a identificação da microbiota total de uma determinada amostra, independentemente de cultivo dos microrganismos. Visando contribuir para o conhecimento dos possíveis vírus envolvidos na SMDS, o presente trabalho realizou o sequenciamento genético de alto desempenho de soros de suínos e posterior análise do metagenoma resultante. Foram utilizadas amostras de soro coletadas em 2008, de 16 suínos com sinais clínicos da SMDS, entre 80 e 100 dias de idade, em uma granja no interior do Rio Grande do Sul. Os dados revelaram sequências virais de PCV2, parvovírus suíno tipo 1 a 6 (PPV1-6), torque teno vírus de suínos (TTSuV) tipo 1b, k2a e k2b e vírus de DNA circular de fita simples codificador de proteína associada a replicação (CRESS). A ocorrência de PCV2, PPV1-5 e TTSuV já foi descrita em suínos com SMDS, portanto este estudo reforça resultados anteriores. O PPV6 foi recentemente descrito na China, Europa e Estados Unidos, e os estudos não relacionaram o vírus com nenhuma doença específica de suínos. Os CRESS já foram identificados em todos os continentes, em vários tipos de amostras, incluindo fezes de suínos, mas sem nenhuma conexão com doenças de animais. Portanto, este é o primeiro relato de PPV6 e CRESS em suínos apresentando sinais de SMDS. Porém estudos posteriores são necessários para poder atribuir relação entre PPV6 e CRESS no desenvolvimento da SMDS.Porcine circovirus associated disease (PCVAD) is one of the causes of negative economic impact on pig farming systems described worldwide. Losses include expenditures with treatment, increased mortality rates, and decreased productivity. One of the most relevant manifestations of PCVAD is the post-weaning multisystemic wasting syndrome (PMWS). The main pathogen present in PMWS is porcine circovirus type 2 (PCV2). However, observational and experimental studies have shown that other agents may be involved in the pathogenesis and clinical manifestation. High-throughput sequencing combined with metagenomics analyses make it possible to identify the total microbiota in a given sample, regardless of microorganism culture. In order to contribute to the knowledge of the viruses involved in PMWS, the present study carried out the high-throughput sequencing of swine sera and subsequent analysis of the resulting metagenome. Sixteen serum samples collected in 2008 on a farm in Rio Grande do Sul, from 80 and 100 days old pigs with clinical signs of PMWS, were examined. Data revealed viral sequences of PCV2, porcine parvovirus type 1 through 6 (PPV1-6), torque teno sus virus (TTSuV) types 1b, k2a and k2b and circular replication-associated protein (Rep) encoding single-stranded (CRESS) DNA viruses. The occurrences of PCV2, PPV1-5 and TTSuV have already been described in pigs with PMWS, so this study reinforces previous results. PPV6 was recently described in China, Europe and the United States, and the studies did not correlate the virus to any specific disease. CRESS DNA viruses have been identified on all continents in various types of samples, including swine feces, but without any connection to animal diseases. The present study is the first report of PPV6 and CRESS in pigs presenting PMWS signals. However, further studies are necessary to be able to attribute the relationship between PPV6 and CRESS in the development of SMDS.
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Giri, Isabelle. "Le genome du papillomavirus de lapin cottontail : de la determination de la sequence aux proprietes biologiques des proteines." Paris 7, 1988. http://www.theses.fr/1988PA077060.
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Weiss, Etienne. "Caracterisation structurale et fonctionnelle de sous-populations de minichromosomes du virus sv40." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13019.
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Beaudenon, Sylvie. "Clonage moleculaire et caracterisation du genome de quatre papillomavirus humains associes a des lesions benignes ou a des neoplasies des muqueuses." Paris 6, 1988. http://www.theses.fr/1988PA066048.
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Ostler, Jeffery Brent Jr. "Characterization of Pol IV and Pol V-Dependent Non-Coding RNAs Derived from aGeminivirus Genome." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492698361649423.
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Johansson, Patrik. "Implications of Local Puumala Hantavirus Genetics and Epidemiology for Diagnostics and Vaccine Development." Doctoral thesis, Umeå : Klinisk mikrobiologi, Umeå universitet, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-532.
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Delmas, Véronique. "Structure et proprietes biologiques du papovavirus de hamster." Paris 6, 1986. http://www.theses.fr/1986PA066550.
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Le papovavirus de hamster (hapv) possede un tropisme restreint in vivo vis a vis des keratinocytes et des lymphocytes. Il se replique dans les tumeurs cutanes qui apparaissent chez des hamsters syriens, et induit egalement des lymphomes chez le hamster. L'organisation genetique du hapv deduite de sa sequence a montre qu'il appartient a la famille des polyomavirus. Le hapv est present dans les lymphomes sous forme de multiples copies libres possedant toujours une deletion localisee dans la meme region du genome. Les signaux de transcription precoce du hapv semblent etre actives par la region precoce de ce virus
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Albrecht, Huguette. "Les produits des genes i et iv du virus de la mosaique du chou-fleur (camv)." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13178.
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Bencherit, Djihad. "Etude des dommages à l'ADN induits par le virus de la maladie de Marek et de leur implication dans la pathogénèse virale." Thesis, Tours, 2016. http://www.theses.fr/2016TOUR4003/document.
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Le virus de la maladie de Marek (MDV) est un alphaherpesvirus à l’origine du développement de lymphomes chez les volailles. A ce jour, l’origine du développement des tumeurs induites par le MDV est encore peu connue malgré l’identification de plusieurs oncoprotéines virales. Au vu de l’implication des dommages à l’ADN dans la pathogenèse de plusieurs virus notamment les herpesvirus, mon projet de thèse avait pour objectif de déterminer l’impact de l’apparition de lésions d’ADN sur le cycle viral du MDV. Nous avons montré que l’infection cytolytique de MDV s’accompagne d’une accumulation de lésions dans l’ADN cellulaire de lymphocytes et cellules fibroblastiques de poulet. La phase de latente de l’infection MDV n’affecte pas l’intégrité de l’ADN des lymphocytes alors que la réactivation du virus conduit à l’apparition de lésions d’ADN. De plus, en utilisant une approche originale in vivo, nous avons confirmé le rôle essentiel de la protéine virale VP22 dans l’induction de ces dommages. Nous avons pu établir que l’induction des dommages à l’ADN au cours de l’infection et/ou la réponse cellulaire engendrée sont non seulement favorables à la réplication du virus mais également que l’apparition de ces lésions est étroitement liée au pouvoir oncogène du MDVMarek’s disease virus (MDV) is an alphaherpesvirus responsible of T lymphoma in chickens. Mechanisms leading to cellular transformation mediated by MDV are still incompletely understood. DNA damage and the associated cellular response participate actively in the life cycle of viruses, especially herpesviruses. Here, we aimed at deciphering the role of DNA damages in MDV pathogenesis. We show that MDV lytic infection leads to DNA lesions in lymphocytes and fibroblasts of chickens. Moreover, we demonstrated that MDV latently-infected lymphocytes exhibits undamaged DNA whereas MDV reactivation leads to an onset of DNA lesions. Also, using an original in vivo approach, we objectified the role of VP22 on DNA damages induction. Finally, we established that DNA damage and/or the associated DNA damage response are not only benefic to MDV replication but also that the DNA lesions onset might participate to MDV oncogenicity
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Magee, Wendy Colleen. "The mechanism of action of cidofovir and (S)-9-(3-hydroxy-2-phosphonomethoxypropyl) adenine against viral polymerases." 2009. http://hdl.handle.net/10048/534.
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Thesis (Ph.D.)--University of Alberta, 2009.Title from pdf file main screen (viewed on Sept. 18, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology, University of Alberta." Includes bibliographical references.
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Gammon, Donald Brad. "Vaccinia virus DNA polymerase and ribonucleotide reductase their role in replication, recombination and drug resistance /." 2010. http://hdl.handle.net/10048/860.
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Thesis (Ph.D.)--University of Alberta, 2010.A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology. Title from pdf file main screen (viewed on January 10, 2010). Includes bibliographical references.
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Huang, Jig-Fu, and 黃進福. "A Study of Virus Classification via Genomic DNA Sequences." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/57017642093554323280.
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碩士亞洲大學
生物資訊學系碩士班
99
Due to the availability of virus genome sequences nowadays, there provides a new approach to virus classification from the view point of molecular biology point of view, instead of from traditional morphol- ogy. To use the classifiers available in the vector space model, it is important to transfer the instances of virus into representative vectors. To transfer the instances of viruses (genomic sequences) into vectors as the input of experiments for virus classification, in this study, we adopted the k-mer(k) approach for pattern extraction and used the entropy of pattern distirbution for pattern weighting. To inspect the different effectiveness of coding/non-coding regions within one DNA nucleotide sequence, there were 4 types, ”ALL”, ”Coding”, ”NonCod- ing, and ”DirectedCoding”, of sequences extracted individually as the input for classification comparison. Experimental resources of viral genomes were downloaded from the NCBI and included 22 virus fami- lies consisting of ”1,601” virus species. Meanwhile, the values of the k ranged from 1 to 6 were evaluated for experiments. The results showed that the highest accuracy achieved by well known SVM classifier was 95.6%,by using the sequences of type ”ALL” when k = 5 . Further- more, the accuracy achieved via the ”DirectedCoding” was higher than that avhieved via the ”Coding”. It was out of our expectation that the accuracy achieved by using the sequence type of ”NonCoding” was as high as ”90%” when k = 6. This observation revealed that some information conserved in non-coding region (that)where worthy for further investigation for biologist.
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Huang, Ts-Hou, and 黃子厚. "The Study of Epstein-Barr Virus DNA Integration in Human Genome." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/43788662871549906069.
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碩士長庚大學
基礎醫學研究所
93
The Epstein-Barr virus (EBV) is a human herpes virus that is uaually carried lifelong as an asymptomatic infection. EBV is the causative agent of infectious mononucleosis, and has been linked to the development of several malignant tumors, including B-cell neoplasms such as Burkitt’s lymphoma. The hybrid cell line Clone 3 is a hybrid cell line from human cervical cancer cell line D98 with P3HR-1Burkitt’s lymphoma. This cell line was proved to be Epstein-Barr virus episomal form-free, and with three EBV DNA-integration sites in two human chromosomes by fluorescence in situ hybridization (FISH) with BamHI W probe. The study aimed to investigate the flanking human DNA sequences of the integration sites and integrated EBV DNA differentiation in the beginning. However, the deficiency of the background knowledge of the particular cell line, Clone3, disturbs us pursuing the exact DNA sequences locating at the integration sites at present. The alternative strategy is with Southern blotting technique by hybridizing the on hand probes cloned from the EBV genome to the blots with Clone 3 genome digested with restriction enzymes, mainly BamHI. The results indicate that there is neither C nor A fragments on both ends among three W-positive integration sites. However, the finding of the existence of G fragment, but no D fragment indicates that the possible breakpoint of one of the integrated EBV genomes may be between G and D fragments. The other finding concerning one of the hybridized fragments (~8-kb) of R probe is far larger than the regular site of R probe (at 3.4–kb). It implies that a possible breakpoint may be at R fragment. None of the Z-probe-hybridized fragments are at the 1.8-kb sites, but at ~6.6-kb, which indicates that there may be a translocation of the Z fragment in all integrated sites. The finding of this study helps us understanding the possible breakpoints of the integrated EBV genomes, and may be a model for further pursuing the possible fragile breakpoints in the human genome.
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Cheng, Hsien-Jen, and 鄭獻仁. "DNA Sequencing and Annotation of Genomic Segments of Insect Hz-1 Virus." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/10148435668508004493.
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碩士國立成功大學
生物學系
88
Hz-1 virus is an unclassified insect virus. It is the only insect virus that can infect insect cell lines persistently with temporal gene expressions between productive and persistent infections, and thus is a good model virus for studies of viral persistent infection. Previously, it was difficult to clone the HA, HD and HI genomic segments which came from Hz-1 viral genome digested by HindIII, and eventually theses fragments were cloned by using a low-copy number vector, pWSK29. With the Northern hybridization data, it was shown that there were several genes expressed in these three genomic segments. It was presumed that these genes encoded some transcription factors or toxin genes that influenced the viral infection and replication. In this study, the genomic segments of HA, HD and HI were sequenced and gene predicted and annotated. Finally, the predicted genes were assigned the corresponding bands on the previous Northern hybridization data to confirm the viral gene expressions. Three sequencing strategies were used to finished genomic segments sequencing in this study, including random shotgun cloning and sequencing, end sequencing of subclones and primer walking sequencing. These genomic segments contain 41,733 bp with a G+C content of 41.55 %. Total 108 open reading frames (ORFs) encoding at least 50 amino acids were predicted and patterns of transcriptional signals were searched. Then the full-length nucleotide sequences of these segments and peptide sequences of predicted ORFs were uploaded to Genbanks for searching alignments by BLASTP, BLASTX and FASTA programs to find potential genes and the similarity between Hz-1 virus and other viruses. The results of alignments showed that there were two significant genes contained in Hz-1 viral genome, they are the transposase (Tn) and the thymidilate synthease (Ts) respectively. The transposase was presumed that coming from contamination of Escherichia coli genomic DNA during experimental artifacts. The Hz-1 virus thymidilate synthease was aligned to herpesviruses ones with high similarities, and the gene was presumed to be obtained from its host, lepidopteran insects. In this study, the random shotgun sequencing strategy was used in genomic segment sequencing and can be considered to be used in genome project of Hz-1 virus in the future. The genomic sequences and alignment data can be afforded new data in Genbank and assist the studies in Hz-1 viral infection and classification.
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Huang, Yan-Hua, and 黃彥華. "The interaction between replication-associated protein (Rep) and viral genomic DNA of Tomato yellow leaf curl virus." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/70478466795679822881.
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碩士國立中興大學
生物科技學研究所
98
Tomato Yellow Leaf Curl Virus (TYLCV), a member of the family Geminiviridae, genus Begomovirus, harbors single-stranded circular DNA genome. The intergenic region of TYLCV genomic DNA contains bidirectional promoter elements interspersed within the origin of replication. The replication-associated protein (Rep) is essential for viral DNA replication, in which the Rep protein initiates rolling circle replication by binding to specific repeated sequences (iterons) within the origin of replication and acting as a topoisomerase. The aim of this study is to explore the underlying mechanism for the specific interaction between TYLCV Rep protein and the cognate viral genomic DNA by identifying the sequence and location of TYLCV iterons and DNA-binding domains of TYLCV Rep protein. The TYLCV genome was amplified from Bemisia tabacci (whiteflies) by Rolling Circle Amplification (RCA), and cloned into the pUC119 vector. The full length Rep open reading frame, N-terminus (Rep1-181) and C-terminus (Rep177-361) of Rep were cloned in pET21d and pET29a vectors and expressed in Escherichia coli. The target proteins were purified by electro-elution from polyacrylamide gels, and used to raise Rep-specific antibodies in rabbits. The interactions between Rep proteins and TYLCV genomic DNA were analyzed by Southwestern blot with Rep-specific antibodies. Preliminary results showed that Rep protein specifically interacted with double-strand circular geminivirus DNAs, instead of the linear form DNAs, suggesting that the binding specificity is partially determined by DNA topology. Both full-length Rep and Rep1-181 preferentially interact with IR and the coding regions of Rep and C4 protein, indicating that the main DNA-interacting domain locates at the N-terminus of Rep protein, while Rep177-361 might play the role of specificity determinant. The Type I topoisomerase function of Rep protein were analyzed by a mimicry RCA in vitro by using Φ29 DNA polymerase. The result confirmed that Rep protein possesses nicking / re-ligation activity, providing evidence that the native activity of the Rep protein expressed and purified from prokaryotic cells was still preserved. Through this study, it is expected that practical methods to disrupt the DNA replication cycles could be developed as the effective therapeutics for viral diseases caused by TYLCV and other geminiviruses.
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Lu, Chih-Chung. "Epstein-Barr Virus Genome-Wide Gene Expression Profile and the Function of BKRF3 Uracil-DNA Glycosylase in Viral Lytic Replication." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2811200620165600.
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Lu, Chih-Chung, and 呂志忠. "Epstein-Barr Virus Genome-Wide Gene Expression Profile and the Function of BKRF3 Uracil-DNA Glycosylase in Viral Lytic Replication." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/66203707137956188196.
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博士國立臺灣大學
微生物學研究所
95
Epstein-Barr virus (EBV) is a gamma herpesvirus which has been demonstrated to be associated with various human malignancies such as Burkitt’s lymphoma and nasopharynegeal carcinoma (NPC). Although EBV latent status was documented in most tumors, recent observations indicate that during the process of carcinogenesis in some malignancies EBV reactivation does occur. However, current knowledge regarding EBV lytic gene expression and function is far behind that of latent genes. In order to monitor simultaneously the genome-wide gene expression control, an EBV DNA array was generated to analyze the pattern of transcription of the entire EBV genome under various conditions. Firstly, a complete set of temporal expression clusters of EBV genes was displayed by analyzing the array data of anti-IgG induced Akata cells. A series of genes with unknown function were respectively assigned to various clusters, In addition, increasing expression of latent genes, including EBNA2, EBNA3A and EBNA 3C, was observed during virus replication. Secondly, gene expression independent of viral DNA replication was analyzed in PAA blocked Akata cells and in chemically induced Raji cells. Several genes with presumed late functions were unexepectedly found to be expressed with early kinetics and independent of viral DNA replication, suggesting possible novel functions for these genes. Finally, the EBV array was used to identify Rta responsive gene expression in Raji cells, and in the EBV positive epithelial cells NA, using a Zta siRNA strategy. The array data were confirmed by northern blotting, RT-PCR and reporter assays. All the information here thus provides a better understanding of the control of EBV lytic gene expression. According to microarray results, the virally encoded DNA replication associated enzymes were found to be catalogued into two clusters, suggesting their participation at early theta-form replication and late rolling-circle replication, respectively. Among the genes expressed in the second cluster, BKRF3 was previously demonstrated to enhance the oriLyt-dependent DNA replication in a co-transfection replication assay. However, the expression and function of BKRF3 have not yet been characterized. Based on its amino acid sequence, the putative Uracil-DNA glycosylase (UDG or UNG), BKRF3 belongs to the UNG family of proteins which are the primary DNA repair enzymes responsible for the removal of inappropriate uracil from DNA. Recent studies further suggested that the nuclear human UNG2, and the UDGs of large DNA viruses, may coordinate with their DNA polymerase accessory factors to enhance DNA replication. In the second part of this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the E. coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 UNG inhibitor, Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampicin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV positive lymphoid and epithelial cells using BKRF3 specific mouse antibodies. Expression of BKRF3 is mainly regulated by the immediate early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 siRNA, whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taken together, I demonstrated the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.
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Lai, Yi-Chin, and 賴儀瑾. "Molecular Interactions among the AV1 Protein, Coat Protein and Genomic DNA of Ageratum Yellow Vein Virus Ping-Tung Strain." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/35241287527650007360.
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碩士國立中興大學
農業生物科技學研究所
90
Most of the members of the family Geminiviridae belong to the genus Begomovirus, which are transmitted by whitefly to dicotyledonous plants and cause severe damage to crop plants. The begomoviruses are characterized by twinned isometric particles with a genome consisting of one or two circular single-stranded DNA. In the monopartite begomoviruses lacking DNA B, which encodes for movement related proteins, the AV1 protein is speculated to be involved in viral movement. The purpose of this study is to analyze the interactions between the AV1 protein, coat protein and genomic DNAs of a monopartite begomovirus, ageratum yellow vein virus Ping-Tung strain (AYVV-PT). The AV1 gene of AYVV-PT was cloned into plasmid pET21d and expressed in E. coli. The AV1 proteins were purified and subjected to the production of specific antiserum. In vitro, the AV1 protein could self-interact to form oligomers, as suggested by V8 protease digestion analysis. The existence of oligomeric forms of AV1 protein in plants was revealed by Western blot. The result of Western blot and protein overlay analyses demonstrated that AV1 protein could interact with the coat protein of AYVV-PT. The in vitro interaction between AV1 protein and both single- and double-stranded viral DNAs were detected by using nucleoprotein binding-enzyme linked immunosorbant assay (NB-ELISA) in different salt concentrations. However, the interactions appeared to be non-specific. The order of binding among the AV1 protein, coat protein, and viral DNA was also investigated by NB-ELISA, which showed that the AV1 protein might interact with the viral nucleic acids first. These results indicated that AV1 protein of monopartite begomoviruses might have the functions similar to BC1 and BV1 proteins of the bipartite begomoviruses, which may facilitate the movement of the monopartite begomoviruses by interacting with viral coat protein and DNAs.
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Wu, Chia-Ying, and 吳佳穎. "Molecular interactions among Rep protein and genomic DNA of ageratum yellow vein virus Ping-Tung strain and analyses of replication in prokaryotic system." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/47536085080349520695.
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碩士國立中興大學
農業生物科技學研究所
90
Abstract The replication ability is one of the key determinants for surviving of viruses. The Rep protein, the first expressed by geminiviruses during the early infection stage, is the only virus-encoded protein required for the replication process, including specific cleavage and ligation to initiate and terminate rolling-circle replication, self-interaction, and regulation of the host cell cycles. In this research, the monopartite begomovirus, ageratum yellow vein virus Ping-Tung strain (AYVV-PT) was used to investigate the interactions between Rep proteins and viral DNAs and to develop the prokaryotic replication system based on E.coli/phage M13 to facilitate the study of geminivirus replication. The Rep gene of AYVV-PT was cloned into plasmid pETBlue for expression, and gel-purified Rep proteins were used for producing specific polyclonal antibodies. The Rep gene was further sub-cloned into plasmid pGEX-4T-1 to obtain Rep proteins in native condition by affinity chromatography. The interactions between Rep proteins and viral genomic DNAs were investigated by Nucleoprotein Binding-ELISA (NB-ELISA) and Southwestern blot analyses. Rep protein has the highest affinity to nt. 1522-2731 of AYVV-PT genome under the optimized in vitro binding condition, 20 mM Tris-HCl buffer, pH 7.4 containing 0.4-0.6 % NaCl. The full-length AYVV-PT genome was cloned into phage M13 as a single unit, and specific Digoxygenin-labeled RNA and DNA probes for AYVV-PT were prepared by in vitro transcription and asymmetric PCR. The signals of small DNAs co-migrating with the single-stranded, circular DNAs encpasidated in virus particles were detected by Southern blot analysis, and the accumulation of the small DNAs was revealed by time course analysis. In addition, the small DNAs were further confirmed as single-stranded, circular DNA of AYVV-PT by RNase A, S1 nuclease digestions and PCR followed by nucleotide sequence analysis. The results suggested that the prokaryotic replication machinery of E.coli/phage M13 could support the generation of single-stranded, circular DNA of AYVV-PT in a construct containing only one copy of replication origin and a single unit viral genome. The E.coli/phage M13 system may facilitate the study of geminivirus replication and the development of convenient infection and foreign gene expression systems.