Download or read book Investigations of Vaccinia Virus DNA Replication The Roles of Three Viral Proteins A50 I3 and G5 written by Maciej W. Czarnecki and published by . This book was released on 2017 with total page 276 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Vaccinia Virus and Poxvirology written by Stuart N. Isaacs and published by Humana Press. This book was released on 2004-04-19 with total page 416 pages. Available in PDF, EPUB and Kindle. Book excerpt: A comprehensive collection of cutting-edge methods to study and work with the vaccinia virus and other poxvirus gene transcriptions. These readily reproducible techniques can be used for the construction and characterization of recombinant viruses; for the study of poxvirus gene transcription and DNA replication; for the investigation of the binding, entry, and movement of the virus in host cells; and for the use of the virus in immunologic and cell-cell fusion assays. Other methods cover poxvirus bioinformatics, aspects of viral pathogenesis at both a protein and an animal model level, and the study of immune responses to poxviruses-the latter a critical ability given the important role of vaccinia virus in smallpox vaccination and their potential role as vaccine vectors directed against infectious agents and cancer.

Download or read book Determination of Oligomeric State and Role of the Acidic C Terminal Tail of Vaccinia Virus I3 Single Stranded DNA Binding Protein written by Melissa L. Harrison and published by . This book was released on 2013 with total page 167 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-stranded DNA (ssDNA) binding proteins (SSB) play a major role in DNA replication, recombination, and repair, by protecting ssDNA from nuclease attack and removing inhibitory secondary structure. Poxviruses are large double stranded DNA viruses that replicate within the cytoplasm of cells, and must encode their own DNA replication proteins. Previous work has identified the Vaccinia I3 protein as the virally encoded SSB. The structure of I3 has not been solved and the amino acid sequence lacks conservation to other SSB proteins, excepting an acidic C terminus. Our work has shown that I3 can form dimeric and tetrameric complexes. Removal of the C terminus prevents tetramer formation and increases the affinity for ssDNA. We have demonstrated that the C terminus is surface exposed and can compete with ssDNA for SSB binding. The C terminus plays a role in virus infection, as its obstruction decreases the amount of viral DNA replication.

Download or read book Viral Genome Replication written by Craig E. Cameron and published by Springer Science & Business Media. This book was released on 2009-05-28 with total page 636 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides the first comprehensive review of viral genome replication strategies, emphasizing not only pathways and regulation but also the structure-function, mechanism, and inhibition of proteins and enzymes required for this process.

Download or read book Replication of Viral and Cellular Genomes written by Yechiel Becker and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 379 pages. Available in PDF, EPUB and Kindle. Book excerpt: Biosynthesis of cellular and viral DNA and RNA has been a major topic in molecular biology and biochemistry. The studies by Arthur Kornberg and his colleagues on the in-vitro synthesis of DNA have opened new avenues to understanding the processes controlling the duplication of the genetic information encoded in the DNA and RNA of bacterial and mammalian cells. Viral nucleic acids are replicated in infected cells (bacterial, plant, and animal) by virus coded enzymes with or without the involvement of proteins and enzymes coded by the host cells. The ability of the virus to replicate its genome within a relatively short period in the infected cell makes it an excellent biological tool for studying the molecular events in nucleic acid replication. Indeed, the identification of a number of virus-coded proteins that participate in the biosynthesis of X174 and SV40 DNA has led to the construction of in-vitro systems for the study of nucleic acid biosynthesis. Similarly, studies on the replication of other phage, animal and plant viruses have provided an insight into the nucleic acid sequences from which DNA synthesis is initiated, as well as the proteins and enzymes that regulate the catalyse biosynthetic processes. Investi gation of the molecular processes involved in the replication of cellular and mitochondrial genomes has gained momentum from the rapid developments in the analyses of viral nucleic acid biosynthesis.

Download or read book Viral Innovations to Fine Tune Reproduction written by Rong Sun (Ph. D. in plant pathology) and published by . This book was released on 2021 with total page 127 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viruses are the simplest organisms comprising only DNA or RNA genomes wrapped in protective shells of proteins or proteins and lipids. Their reproduction is only possible after they successfully enter a host cell, where they depend on the cellular apparatus for a variety of vital steps in their life cycles. Depending on the nature of viral genomes, these steps can include transcription of viral genes to synthesize viral mRNA, translation of viral proteins, and ultimately replication (multiplication) of viral genomes. The replicated viral genomes are then reassembled into virus particles, completing the life cycle of a typical virus. Throughout the entire process viruses interact intimately with the cellular environment, and must not only coerce the host cells for collaboration, but also counteract the hazard of host defense. As a result, viruses, despite their simple genomes, evolved to encode a vast array of regulative capacities that fine tune their gene expression and genome replication based on their surroundings and the progression of their life cycles. My thesis research focused on examining some of the virus-encoded regulative strategies. This dissertation contains four chapters. The first chapter reviewed the relevant literature, providing the background information and the current status of related research. The second chapter examined the impact of several cis-acting RNA elements on the replication of a virus with a single-stranded (ss), positive sense (+) RNA genome. The third chapter adopted a virus model with a genome of circular ssDNA, and investigated how a transcription factor encoded by this virus activates the transcription of late genes of the same virus. Finally, the last chapter sought to determine how a virus-encoded replication protein executes two opposite functions in the same infected cell. I used Turnip crinkle virus (TCV) as a small (+) RNA virus model for the research presented in Chapters two and four. Chapter two investigated how cis-acting RNA elements that are located in the coding region of TCV RNA-dependent-RNA polymerase (RdRp) influence the replication of this virus. Previous studies of these structures were limited to synonymous mutations that did not alter the amino acid sequence of RdRp, because the impact of nonsynonymous changes would be compounded by possible interference with the RdRp activity. Furthermore, those previous studies also could not determine whether certain cis-acting elements play multiple roles. For example, mutations within cis-acting elements important for RdRp translation could not be further tested to see if they are also important for the replication itself, because the latter step requires RdRp translation to be optimal. I have developed a new approach that decouples the translation and replication steps. This new approach allowed me to introduce a variety of mutations, including deletions of up to 1,000 nucleotides (nt), in the RdRp coding region. Using this approach, I was able to identify several new cis-acting elements that are important for the replication of TCV. In Chapter three, I studied a common phenomenon in a group of ssDNA viruses in the family Geminiviridae, known as bipartite begomoviruses. I used Mungbean yellow mosaic virus (MYMV) as a model to study the transcriptional activation of the late gene BV1 by AC2, an early expressing transcriptional activator protein of this virus. Previous research by others showed that AC2 bound to DNA but nonspecifically. I hence hypothesized that the transcriptional activation of BV1 gene required interactions between AC2 and transcription factors of the host plants, the latter binding to the promoter DNA of BV1 gene (PBV1) through specific promoter motifs. Therefore, my goal was to identify the specific motifs located in PBV1 that could be bound by potential AC2-interacting transcription factors of host plants. I was able to identify three ABA-responsive elements (ABREs) within the first 73 nt of PBV1 that collaboratively mediated the transcriptional activation of this promoter by the AC2 protein. Therefore, plant transcription factors involved in the ABA signaling pathways are likely candidates recruited by MYMV AC2 to mediate the activation of BV1 expression. In Chapter four, I investigated how p28, a TCV-encoded auxiliary replication protein (ARP), exerts two opposite functions in the same infected cell. My colleagues and I have established earlier that p28 plays two opposite roles in TCV replication: it supports TCV replication as an ARP, and represses TCV replication by eliciting superinfection exclusion (SIE). A p28 derivative with a C-terminal green fluorescent protein (GFP) tag, designated p28-GFP, exerted strong repression on the replication of a TCV replicon. However, p28-GFP failed to recapitulate the replication function of p28 because it was unable to complement the replication of a p28-defective TCV replicon. The focus of the Chapter four was to resolve why the p28-GFP, and other C-terminally tagged p28 variants, exhibited a strong repressive activity but lacked the replication activity. I initially hypothesized that the C-terminal tags stabilized the p28 protein, resulting in a higher p28 concentration in host cells, favoring p28 polymerization to form repressive protein aggregates. I first tested if the tendency of GFP to dimerize enhanced polymerization of p28-GFP, by replacing GFP with the non-dimerizing mNeonGreen (mNG). I found that the resulting p28-mNG still formed intracellular aggregates and exerted strong repressive activity to TCV replication. Simultaneously, I tested if the TCV RNA sequence encoding p28 was important for the formation of p28 protein aggregates using a codon shuffled (CS) p28, and found that the RNA sequence of p28 was not important for protein aggregation. Subsequently, I tested if I could abolish the repressive activity of C-terminally tagged p28 variants, and restore the replication-complementation activity to them, by diminishing their expression levels with weaker promoters. I found that very low expression of p28-mNG, as well as another p28 variant with a C-terminal duplicated HA tag (p28-2XHA), indeed abrogated their repressive activity. Surprisingly, even at very low expression levels, these C-terminally tagged p28 variants remained incapable of complementing the replication of a TCV mutant that did not encode its own p28. These results prompted the alternative hypothesis that an intact C-terminus free of any modifications is needed for the replication function of p28. Since our previous studies showed that HA-p28, a p28 derivative with an N-terminal, single-copy HA tag, did not significantly change the replication function of p28, I used it as the template to make small modifications at the C terminus of p28. I found that while deleting or mutating the last two amino acid (aa) residues substantially weakened the replication function of p28, a two-aa addition at this end had a relatively minor effect. These findings thus rejected my alternative hypothesis, suggesting that fusion of a C-terminal tag does not necessarily abolish the replication function of p28. Rather, there appears to be an upper limit on the size of the C-terminal tags. In summary, this dissertation examined three types of virus-encoded mechanisms that bolster the reproduction of viruses in infected cells. My research findings are expected to lay the foundation for additional investigations by fellow plant virologists, and contribute to the knowledge-based control and management of plant virus diseases.

Download or read book Vaccinia Virus Requires Heat shock Proteins for Genome Replication and Virion Assembly written by Harriet G. W. Mok and published by . This book was released on 2019 with total page 165 pages. Available in PDF, EPUB and Kindle. Book excerpt: The large double-stranded DNA virus, vaccinia virus (VACV), is closely related to variola virus, the causative agent of smallpox. While the poxvirus lifecycle has been studied for over 50 years, much is still unknown about the complex multi-step process of genome uncoating. Viral and cellular factors involved in poxvirus uncoating have been identified, including host cell proteasomes, ubiquitin, and heat-shock proteins (HSPs), which play a critical role in protein folding and the prevention of protein aggregation. Although HSPs have been linked to the lifecycle of many viruses including VACV, the role of these proteins in poxvirus infection has not been defined. Using various small compound inhibitors and RNAi, in combination with a battery of virus-specific assays, I show that heat-shock proteins are required for multiple stages of the VACV lifecycle. I demonstrate that Hsp90 is required for two stages of infection: release of the genome from the viral core and assembly of new virions. Following the finding that HSF1, the transcription factor for inducible HSPs, is required for infection, I also identify the Hsp105 as the inducible HSP required for post-replication formation of nascent virions. Given the clinical relevance of HSP inhibitors these results highlight HSPs as potential anti-poxviral drug targets. Collectively, through this work I define new cell factors required for poxvirus infection and demonstrate that subjugation of cellular HSPs during the poxvirus lifecycle is far more complex than previously appreciated.

Download or read book Biology of a Constitutively expressed Vaccinia Virus Gene Required for DNA Replication written by Nancy A. Roseman and published by . This book was released on 1987 with total page 200 pages. Available in PDF, EPUB and Kindle. Book excerpt: Replication of vaccinia virus in the cytoplasm of the infected cell occurs under the direction of virally encoded gene products. Expression of approximately 200 viral genes follows a highly regulated temporal scheme which can be followed biochemically and morphologically during the assembly process. In order to dissect this complex genetic program conditionally lethal mutants of vaccinia have previously been generated. This report describes the utilization of a collection of temperature sensitive (ts) mutants for the study of a required vaccinia virus gene. Previous biochemical analysis defined four biochemical phenotypes within this collection of is mutants: DNA negative, defective-late, abortive-late, and wild-type. One mutant from each phenotype was examined at the non-permissive temperature by electron microscopy. Each exhibited distinct morphological aberrations, but it was difficult to relate aberrant morphology to the biochemical phenotype. Therefore, one mutant, ts17, was chosen for more detailed study. Ts17 is a member of the DNA negative biochemical class. At the non-permissive temperature no viral DNA was produced in ts17 infected cells. Early viral proteins were synthesized for up to 24 hours post-infection, and late viral proteins were not expressed. The ts17 gene was then mapped by marker rescue techniques, and the nucleotide sequence of 3.6 kilobases of DNA form that region was determined. The nucleotide sequence of a fragment from tsi7 viral DNA, and from two ts17 revertants was also determined, and the nature of the tsi7 mutation was identified. Analysis of the wild-type sequence revealed three tightly spaced tandemly-oriented open reading frames. The predicted proteins encoded for by these open reading frames were confirmed by hybrid-selection in vitro translation of selected mRNAs. S1 mapping of the 5' and 3' ends of the encoded transcripts, in conjunction with a northern analysis, determined that two of the open reading frames terminate coincidentally. S1 analysis utilizing RNAs isolated over time demonstrated that the ts17 gene was transcribed throughout infection and is therefore a constitutive viral gene. Precise mapping showed the transcriptional start site of this gene to be in a proposed late regulatory element.

Download or read book Evidence for the Modification of Vaccinia Virus Core Proteins by ADP ribosylation written by Stephanie J. Child and published by . This book was released on 1992 with total page 230 pages. Available in PDF, EPUB and Kindle. Book excerpt: Vaccinia virus (VV), the prototype member of the orthopoxvirus family, is a large virus of complex morphology which contains a 191 Kbp double-stranded DNA genome whose expression is tightly regulated in a temporal fashion during viral replication. The regulation of gene expression can be exerted at various of levels, including transcriptional, translational, and post-translational points of control. In addition to transcriptional regulatory mechanisms, the occurrence of a variety of post-translational modifications in VV has been demonstrated. In an effort to better understand the role played by post-translational modifications during the viral replication cycle, we chose to focus on one specific modification event, ADP-ribosylation. Experiments were designed to determine whether any VV proteins might be subject to ADP-ribosylation. The ability to metabolically label a subset of viral proteins by growth of the virus in the presence of [3H]adenosine, in addition to the effects of the ADP-ribosylation inhibitor nicotinamide on viral core protein precursor processing and replication, provided evidence that this or some similar modification is an obligatory event during VV replication. Immunological reagents were used to identify several of the modified proteins. Biochemical evidence obtained via labeling with various precursor compounds, boronate affinity chromatography, and reverse phase HPLC analysis confirmed that the proteins were modified by ADP-ribose or a closely related compound. Additional ADP-ribosylation inhibitor studies provided further support for the initial finding that the viral proteins are subject to ADP-ribosylation or some related modification, and the evidence obtained from these experiments supports a model where this modification event might serve a function in either the proteolytic processing of the core protein precursors, or in localization of the mature core proteins to sites of VV replication within infected cells.

Download or read book DNA Replication and Telomere Resolution in Vaccinia Virus written by Xuekun Xing and published by . This book was released on 1997 with total page 240 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book DNA Replication and Telomere Resolution in Vaccinia Virus written by and published by . This book was released on 1997 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Characterization of Vaccinia Virus A12L Protein Its Proteolysis and Functional Analyses in Virus Replication written by Su Jung Yang and published by . This book was released on 2007 with total page 138 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteolytic processing of A12L produces multiple peptides, which do not appear to utilize AG/K sites, but rather cleavages occur at both the N- and C-terminus. Of the three AG/X motifs in A12L, cleavage has only been demonstrated at the AG/A site. The enzyme responsible for this cleavage has been shown to be I7L. Immunoprecipitation studies have shown that A12L associates with VV core and membrane proteins. A conditional mutant virus of A12L was constructed and determined the essentiality of A12L in virus replication and helped to elucidate its functions in the assembly of virus particles. An AG/A site mutation abrogated the ability of the transfected A12L gene to rescue the conditional mutant under non-permissive conditions, indicating that its proteolysis at the AG/A site is required during viral replication. Next, we compared the protein expression of A12L with D13L, an internal scaffolding protein, to investigate the role of A12L in virus assembly. We showed that A12L is stably synthesized only in the presence of D13L. Consequently, we established that A12L protein and its proteolysis participate in viral assembly subsequent to D13L involvement.

Download or read book Investigation of the Roles of Vaccinia Virus Protein F13 and Its Molluscum Contagiosum Virus Homolog MC021 at Steps Post Envelopment written by Peter J. Bryk and published by . This book was released on 2018 with total page 126 pages. Available in PDF, EPUB and Kindle. Book excerpt: The family Poxviridae encompasses a vast number of complex large, double-stranded DNA viruses. Among these are vaccinia virus (VACV), the virus used as the vaccine for smallpox, and molluscum contagiosum virus (MOCV), the only extant poxvirus that is human-specific and currently clinically relevant. VACV is the prototypic poxvirus and the protein F13 is conserved across the subfamily Chordopoxvirinae. It has been shown to be required to produce the wrapped forms of virus that are required for cell-to-cell spread. F13 is included on wrapped forms of virus, however what role it may play after virion envelopment remains unknown. During our investigations, two F13 temperature-sensitive VACV mutants were found to produce similar levels of extracellular virions (EV) compared to a virus with normal F13, yet displayed a small plaque phenotype similar to a F13L deletion mutant. Analysis of virions produced at the non-permissive temperature showed F13 was not incorporated, and that EV displayed delayed entry kinetics compared to virions containing F13. Furthermore, these virions were resistant to acid-induced dissolution of the EV envelope, suggesting F13 is involved in the modification or dissolution of this membrane. Little is known about MOCV virion morphogenesis, or if virion envelopment even occurs. Despite not having been shown to produce EV, MOCV encodes putative homologs of wrapped virion-specific proteins in VACV, of which the homolog of F13, MC021, shares the greatest amino acid sequence identity. A recombinant virus that expresses MC021 in place of F13 was capable of partially complementing EV production compared to an F13L deletion mutant, yet displayed markedly different localization from F13. We have identified interactions between MC021 and VACV proteins, including F13 and B5. Moreover, MC021 is incorporated into the EV envelope, and these EV enter cells at a rate similar to the parental strain, suggesting MC021 functionally replaces F13 on the EV membrane. Despite this, the virus has a plaque phenotype considerably smaller than that of the parental strain. Taken together, these results demonstrate a role for MC021 in the production of EV during infection, and suggest MOCV may produce wrapped forms of virus during its lifecycle.

Download or read book Selective Protein Synthesis During Vaccinia Virus induced Host Shutoff written by Pragyesh Dhungel and published by . This book was released on 2019 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: During infection, poxvirus makes host cells conducive for viral replication by causing host shutoff that is marked by global inhibition of host protein synthesis. Host shutoff facilitates the reallocation of cellular resources for viral replication and evasion of host antiviral immune responses. However, it poses a challenge for continuous synthesis of crucial cellular proteins and viral proteins that are important for viral replication. It is unclear whether and how viral and specific cellular proteins are selectively synthesized during poxvirus-induced host shutoff. In this dissertation, we elucidated that vaccinia virus boosts viral post-replicative protein synthesis by using the 5'-poly(A) leader at the 5'-UTR. Vaccinia virus has evolutionarily optimized the length of the poly(A) leader, and uninterrupted poly(A) leader is required for promoting poxvirus protein production. During vaccinia virus-induced shutoff, poly(A) leader stipulates viral post-replicative mRNAs an adaptive mechanism to translate efficiently. The poly(A) leader translation was not mediated by an internal ribosome entry site (IRES) mode, albeit poly(A) leader mediates cap-independent mode of translation. Through further investigation, we uncovered a cellular RNA-binding protein La-Related Protein 4 (LARP4) that was repurposed to augment vaccinia virus post-replicative mRNA translation. During VACV infection, LARP4 is enriched in the virus factory where VACV post-replicative mRNAs are translated. A decrease of LARP4 protein level reduces VACV replication, blocks post-replicative protein synthesis, and decreases 5'-poly(A) leader mediated translational advantage. Further studies showed that LARP4 is vital for the cap-independent mode of translation from poly(A) leader. We also showed that infection of vaccinia virus, the prototypic poxvirus, induced selective synthesis of cellular proteins involved in oxidative phosphorylation. Using simultaneous RNA-seq and ribosome profiling, we determined the mRNAs encoding proteins for oxidative phosphorylation complexes had increased relative translation efficiency. Indeed, vaccinia virus infection increased the activity of oxidative phosphorylation. Inhibition of oxidative phosphorylation function suppressed vaccinia virus replication significantly. Moreover, the mRNAs of oxidative phosphorylation have short 5'-UTRs with a less complex secondary structure that could confer oxidative phosphorylation mRNAs a translational advantage in vaccinia virus-infected cells during host shutoff. Together, these studies advanced our understanding of how vaccinia virus selectively synthesizes viral and cellular proteins for efficient viral replication during host shutoff. The findings may facilitate the development of novel anti-poxvirus strategies and the improvement of poxviruses as vaccine vectors and anti-cancer agents.

Download or read book Mesenchymal Epithelial Interactions in Neural Development written by Joachim R. Wolff and published by Springer Science & Business Media. This book was released on 2013-06-29 with total page 424 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book is based on the NATO Advanced Research Workshop on "Mesenchymal-Epithelial Interactions in Neural Development" which was held in Berlin during March 1986. The idea that it may be the time for this workshop arose from a discussion among the organizers who met at a conference in Innsbruck. During the twenties, Spemann demonstrated that, in vertebrates, the formation of neuroepithelium depends on the induction by specific mesodermal derivatives. This was about the first time that mesenchymal-epithelial interactions had been recognized as an important mechanism in the development of an epithelial organ. Since then two things had become clear: 1) The identity and role of molecules, elaborated at mesenchymal-neuroepithelial interfaces had been partially elucidated in spinal cord and peripheral nerve ontogeny but, until recently, had been ignored in studies on the development of the brain. 2) In many-non-neural epithelia, the surrounding mesenchyme regulates such fundamental developmental steps as, for example, cell proliferation, morphogenesis and cell differentia tion, and there is no reason to exclude similar influences and interactions from brain development. It seemed to us that the importance and implications of many of these findings had not been appreciated by many developmental neurobiologists and that an attempt should be made to initiate discussions and exchange of ideas among different groups of scientists involved in extracellular matrix (ECM) research. This turned out to be a formidable task because of the great diversity of disciplines contributing findings to this active field.

Download or read book Virus Taxonomy written by Frederik A Murphy and published by . This book was released on 1995-05-03 with total page 600 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book RNA Binding Proteins written by Kathryn Sandberg and published by Springer. This book was released on 2001-12-31 with total page 308 pages. Available in PDF, EPUB and Kindle. Book excerpt: RNA binding proteins are an exciting area of research in gene regulation. A multitude of RNA-protein interactions are used to regulate gene expression including pre-mRNA splicing, polyadenylation, editing, transport, cytoplasmic targeting, translation and mRNA turnover. In addition to these post-transcriptional processes, RNA-protein interactions play a key role in transcription as illustrated by the life cycle of retroviruses. Unlike DNA, the structure of RNA is highly variable and conformationally flexible, thus creating a number of unique binding sites and the potential for complex regulation by RNA binding proteins. Although there is a wide range of topics included in this volume, general themes have been repeated, highlighting the overall integrative nature of RNA binding proteins. The chapters have been separated into three different sections: Translational Control; mRNA Metabolism; and Hormonal and Homeostatic Regulation. The chapters of this volume were written with the seasoned investigator and student in mind. Summaries of key concepts are reviewed within each chapter as well as guiding questions that can be used to stimulate class discussions. The Editors of this volume hope that this compendium educates, enthralls, and stimulates the readers to look to the future possibilities in this rapidly evolving field.