Viral Life Cycle

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Craig Meyers - One of the best experts on this subject based on the ideXlab platform.

  • the role of the human papillomavirus type 18 e7 oncoprotein during the complete Viral Life Cycle
    Virology, 2005
    Co-Authors: Margaret E Mclaughlindrubin, Jennifer L Brombergwhite, Craig Meyers
    Abstract:

    The role of the human papillomavirus oncoprotein E7 in carcinogenesis has been extensively studied. While the role of HPV E7 in the Viral Life Cycle has also been studied, certain disparities exist, indicating that genotype differences may influence the role that E7 plays in the Viral Life Cycle. In this study, we investigated the role of HPV18 E7 in the Viral Life Cycle in order to gain a further understanding of this issue. To determine the role that HPV18 E7 plays in the Viral Life Cycle, a translation termination substitution mutant of E7 in the context of the full HPV18 genome was created. We introduced linearized HPV18 E7-deficient genomic DNA into primary keratinocytes, where it recircularized and was maintained episomally at a range of five to several hundred copies of HPV genomic DNA. The mutant genomes failed to amplify following epithelial stratification and differentiation in organotypic culture. Moreover, virion morphogenesis did not occur. We found that the expression of HPV16 or HPV18 E7 in trans was able to rescue the amplification defect but not the defect in virion morphogenesis. These studies indicate that HPV18 E7 plays a critical role in the productive stage of the Viral Life Cycle. In addition, these studies add further proof to the hypothesis that genotype differences exist for the role of E7 during the Viral Life Cycle.

  • genetic and biochemical analysis of cis regulatory elements within the keratinocyte enhancer region of the human papillomavirus type 31 upstream regulatory region during different stages of the Viral Life Cycle
    Journal of Virology, 2004
    Co-Authors: Samina Alam, Craig Meyers
    Abstract:

    Using linker scanning mutational analysis, we recently identified potential cis regulatory elements contained within the 5′ upstream regulatory region (URR) domain and auxiliary enhancer (AE) region of the human papillomavirus type 31 (HPV31) URR involved in the regulation of E6/E7 promoter activity at different stages of the Viral Life Cycle. For the present study, we extended the linker scanning mutational analysis to identify potential cis elements located in the keratinocyte enhancer (KE) region (nucleotides 7511 to 7762) of the HPV31 URR and to characterize cellular factors that bind to these elements under conditions representing different stages of the Viral Life Cycle. The linker scanning mutational analysis identified Viral cis elements located in the KE region that regulate transcription in the presence and absence of any Viral gene products or Viral DNA replication and determine the role of host tissue differentiation on Viral transcriptional regulation. Using electrophoretic mobility shift assays, we illustrated defined reorganization in the composition of cellular transcription factors binding to the same cis regulatory elements at different stages of the HPV differentiation-dependent Life Cycle. Our studies provide an extensive map of functional elements in the KE region of the HPV31 URR, identify cis regulatory elements that exhibit significant transcription regulatory potential, and illustrate changes in specific protein-DNA interactions at different stages of the Viral Life Cycle. The variable recruitment of transcription factors to the same cis element under different cellular conditions may represent a mechanism underlying the tight link between keratinocyte differentiation and E6/E7 expression.

  • genetic analysis of cis regulatory elements within the 5 region of the human papillomavirus type 31 upstream regulatory region during different stages of the Viral Life Cycle
    Journal of Virology, 2002
    Co-Authors: Jennifer L Brombergwhite, Craig Meyers
    Abstract:

    The function of the 5′ region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5′ region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the Viral Life Cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the Viral Life Cycle. This study identified (i) Viral cis elements that regulate transcription in the presence and absence of any Viral gene products or Viral DNA replication, (ii) the role of host tissue differentiation in Viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5′ region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the Viral Life Cycle.

  • Temporal and spatial expression of the E5a protein during the differentiation-dependent Life Cycle of human papillomavirus type 31b.
    Virology, 1998
    Co-Authors: Timothy J. Mayer, Craig Meyers
    Abstract:

    Abstract Human papillomaviruses (HPVs) are epitheliotropic viruses, and their Life Cycle is intimately linked to the stratification and differentiation state of the host epithelial tissues. Defining a role for the E5 gene product in the differentiation-dependent Viral Life Cycle has been difficult due to the lack of a suitable culture system. We used the organotypic (raft) culture system to investigate the spatial and temporal expression pattern of the E5 protein during the differentiation-dependent Life Cycle of HPV-31b. We report the generation of antisera specific to the HPV-31b E5a protein. The HPV-31b E5a protein was detected throughout the Viral Life Cycle in raft cultures as determined by immunostaining analyses, and the protein was localized predominantly to the basal and granular layers. Expression of epidermal growth factor receptor or platelet-derived growth factor receptors, two proteins with which E5 has been shown to interact in cell culture, did not specifically colocalize with E5a expression. However, HPV-31b E5a expression did colocalize with the epithelial differentiation-specific marker filaggrin. The kinetics of E5a protein expression during the complete Viral Life Cycle was analyzed by immunoblotting, and the highest level was found to be coincidental with the onset of virion morphogenesis.

Laimonis A Laimins - One of the best experts on this subject based on the ideXlab platform.

  • An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment
    Journal of Virology, 2012
    Co-Authors: Mary Donaldson, Laimonis A Laimins, Lorna J. Mackintosh, Jason M. Bodily, Edward S. Dornan, Iain M. Morgan
    Abstract:

    In human papillomavirus DNA replication, the Viral protein E2 forms homodimers and binds to 12-bp palindromic DNA sequences surrounding the origin of DNA replication. Via a protein-protein interaction, it then recruits the Viral helicase E1 to an A/T-rich origin of replication, whereupon a dihexamer forms, resulting in DNA replication initiation. In order to carry out DNA replication, the Viral proteins must interact with host factors that are currently not all known. An attractive cellular candidate for regulating Viral replication is TopBP1, a known interactor of the E2 protein. In mammalian DNA replication, TopBP1 loads DNA polymerases onto the replicative helicase after the G1-to-S transition, and this process is tightly cell Cycle controlled. The direct interaction between E2 and TopBP1 would allow E2 to bypass this cell Cycle control, resulting in DNA replication more than once per cell Cycle, which is a requirement for the Viral Life Cycle. We report here the generation of an HPV16 E2 mutant compromised in TopBP1 interaction in vivo and demonstrate that this mutant retains transcriptional activation and repression functions but has suboptimal DNA replication potential. Introduction of this mutant into a Viral Life Cycle model results in the failure to establish Viral episomes. The results present a potential new antiViral target, the E2-TopBP1 interaction, and increase our understanding of the Viral Life Cycle, suggesting that the E2-TopBP1 interaction is essential.

  • Human papillomaviruses activate caspases upon epithelial differentiation to induce Viral genome amplification
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Cary A. Moody, Amélie Fradet-turcotte, Jacques Archambault, Laimonis A Laimins
    Abstract:

    The Life Cycle of human papillomaviruses (HPVs) is linked to epithelial differentiation, with late Viral events restricted to the uppermost stratified layers. Our studies indicated that HPV activates capases-3, -7, and -9 upon differentiation, whereas minimal activation was observed in differentiating normal keratinocytes. Activation occurred in the absence of significant levels of apoptosis, suggesting a potential role for caspases in the Viral Life Cycle. In support of this, the addition of caspase inhibitors significantly impaired differentiation-dependent Viral genome amplification. A conserved caspase cleavage motif was identified in the replication protein E1 (46DxxD49) that was targeted in vitro by both recombinant caspase-3 and caspase-7. Mutation of this site inhibited amplification of Viral genomes, indicating that caspase cleavage is necessary for the productive Viral Life Cycle. Our study demonstrates that HPV activates caspases upon differentiation to facilitate productive Viral replication and represents a way by which HPV controls Viral gene function in differentiating cells.

  • Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 Life Cycle
    Virology, 2006
    Co-Authors: Tonia R. Wooldridge, Laimonis A Laimins
    Abstract:

    Abstract The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive Viral Life Cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the Viral Life Cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another α-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the Viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the Viral Life Cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the Life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the Viral Life Cycle and for extension of cell Life span.

  • human papillomavirus type 31 replication modes during the early phases of the Viral Life Cycle depend on transcriptional and posttranscriptional regulation of e1 and e2 expression
    Journal of Virology, 2002
    Co-Authors: Walter G Hubert, Laimonis A Laimins
    Abstract:

    The E1 and E2 proteins are both required for papillomavirus DNA replication, and replication efficiency is controlled by the abundance of these factors. In human papillomaviruses (HPVs), the regulation of E1 and E2 expression and its effect on Viral replication are not well understood. In particular, it is not known if E1 and E2 modulate their own expression and how posttranscriptional mechanisms may affect the levels of the replication proteins. Previous studies have implicated splicing within the E6 open reading frame (ORF) as being important for modulating replication of HPV type 31 (HPV31) through altered expression of E1 and E2. To analyze the function of the E6 intron in Viral replication more specifically, we examined the effects of E6 splicing mutations in the context of entire Viral genomes in transient assays. HPV31 genomes which had mutations in the splice donor site (E6SD) or the splice acceptor site (E6SA), a deletion of the intron (E6ID), or substituted heterologous intron sequences (E6IS) were constructed. Compared to wild-type (wt) HPV31, pHPV31-E6SD, -E6SA, and -E6IS replicated inefficiently while pHPV31-E6ID replicated at an intermediate level. Cotransfection of the E6 mutant genomes with an E1 expression vector strongly activated their replication levels, indicating that efficient expression of E1 requires E6 internal splicing. In contrast, replication was activated only moderately with an E2 expression vector. Replacing the wt E6 intron in HPV31 with a heterologous intron from simian virus 40 (E6SR2) resulted in replication levels similar to that of the wt in the absence of expression vectors, suggesting that mRNA splicing upstream of the E1 ORF is important for high-level replication. To examine the effects of E6 intron splicing on E1 and E2 expression directly, we constructed reporter DNAs in which the luciferase coding sequences were fused in frame to the E1 (E1Luc) or E2 (E2Luc) gene. Reporter activities were then analyzed in transient assays with cotransfected E1 or E2 expression vectors. Both reporters were moderately activated by E1 in a dose-dependent manner. In addition, E1Luc was activated by low doses of E2 but was repressed at high doses. In contrast, E2 had little effect on E2Luc activity. These data indicate that E1 expression and that of E2 are interdependent and regulated differentially. When the E6 splicing mutations were analyzed in both reporter backgrounds, only E1Luc activities correlated with splicing competence in the E6 ORF. These findings support the hypothesis that the E6 intron primarily regulates expression of E1. Finally, in long-term replication assays, none of the E6 mutant genomes could be stably maintained. However, cotransfection of the E6 splicing mutant genomes with pHPV31-E7NS, which contains a nonsense mutation in the E7 coding sequence, restored stable replication of some mutants. Our observations indicate that E1 expression and that of E2 are differentially regulated at multiple levels and that efficient expression of E1 is required for transient and stable Viral replication. These regulatory mechanisms likely act to control HPV copy number during the various phases of the Viral Life Cycle.

  • human papillomavirus type 31 oncoproteins e6 and e7 are required for the maintenance of episomes during the Viral Life Cycle in normal human keratinocytes
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Jennifer T Thomas, Walter G Hubert, Margaret N Ruesch, Laimonis A Laimins
    Abstract:

    The E6 and E7 oncoproteins of the high-risk human papillomavirus (HPV) types are able to immortalize human keratinocytes in vitro and likely contribute to the development of anogenital malignancies in vivo. The role of these oncoproteins in the productive Viral Life Cycle, however, is not known. To begin to examine these possible roles, mutations in E6 were introduced in the context of the complete HPV 31 genome. Although transfected wild-type HPV 31 genomes, as well as genomes containing an E6 translation termination linker, an E6 frameshift mutation, and a point mutation in the p53 interacting domain were able to replicate in transient assays, only the wild-type genome was stably maintained as an episome. Interestingly, mutant genomes in either the E6 splice-donor site or splice-acceptor site were reduced in replication ability in transient assays; however, cotransfection of E1 and E2 expression vectors restored this function. In a similar fashion, genomes containing mutant HPV 31 E7 genes, including a translation termination mutant, two Rb-binding site mutants, a casein kinase II phosphorylation site mutant, and a transformation deficient mutant, were constructed. Although transient replication was similar to wild type in all of the E7 mutants, only the casein kinase II mutant had the ability to maintain high copies of episomal genomes. These findings suggest a role for E6 and E7 in the Viral Life Cycle beyond their ability to extend the Life span of infected cells.

Jennifer L Brombergwhite - One of the best experts on this subject based on the ideXlab platform.

  • the role of the human papillomavirus type 18 e7 oncoprotein during the complete Viral Life Cycle
    Virology, 2005
    Co-Authors: Margaret E Mclaughlindrubin, Jennifer L Brombergwhite, Craig Meyers
    Abstract:

    The role of the human papillomavirus oncoprotein E7 in carcinogenesis has been extensively studied. While the role of HPV E7 in the Viral Life Cycle has also been studied, certain disparities exist, indicating that genotype differences may influence the role that E7 plays in the Viral Life Cycle. In this study, we investigated the role of HPV18 E7 in the Viral Life Cycle in order to gain a further understanding of this issue. To determine the role that HPV18 E7 plays in the Viral Life Cycle, a translation termination substitution mutant of E7 in the context of the full HPV18 genome was created. We introduced linearized HPV18 E7-deficient genomic DNA into primary keratinocytes, where it recircularized and was maintained episomally at a range of five to several hundred copies of HPV genomic DNA. The mutant genomes failed to amplify following epithelial stratification and differentiation in organotypic culture. Moreover, virion morphogenesis did not occur. We found that the expression of HPV16 or HPV18 E7 in trans was able to rescue the amplification defect but not the defect in virion morphogenesis. These studies indicate that HPV18 E7 plays a critical role in the productive stage of the Viral Life Cycle. In addition, these studies add further proof to the hypothesis that genotype differences exist for the role of E7 during the Viral Life Cycle.

  • genetic analysis of cis regulatory elements within the 5 region of the human papillomavirus type 31 upstream regulatory region during different stages of the Viral Life Cycle
    Journal of Virology, 2002
    Co-Authors: Jennifer L Brombergwhite, Craig Meyers
    Abstract:

    The function of the 5′ region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5′ region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the Viral Life Cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the Viral Life Cycle. This study identified (i) Viral cis elements that regulate transcription in the presence and absence of any Viral gene products or Viral DNA replication, (ii) the role of host tissue differentiation in Viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5′ region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the Viral Life Cycle.

Alison J Sinclair - One of the best experts on this subject based on the ideXlab platform.

  • epigenetic control of epstein barr virus transcription relevance to Viral Life Cycle
    Frontiers in Genetics, 2013
    Co-Authors: Alison J Sinclair
    Abstract:

    DNA methylation normally leads to silencing of gene expression but Epstein–Barr virus (EBV) provides an exception to the epigenetic paradigm. DNA methylation is absolutely required for the expression of many Viral genes. Although the Viral genome is initially un-methylated in newly infected cells, it becomes extensively methylated during the establishment of Viral latency. One of the major regulators of EBV gene expression is a Viral transcription factor called Zta (BZLF1, ZEBRA, Z) that resembles the cellular AP1 transcription factor. Zta recognizes at least 32 variants of a 7-nucleotide DNA sequence element, the Zta-response element (ZRE), some of which contain a CpG motif. Zta only binds to the latter class of ZREs in their DNA-methylated form, whether they occur in Viral or cellular promoters and is functionally relevant for the activity of these promoters. The ability of Zta to interpret the differential DNA methylation of the Viral genome is paramount for both the establishment of Viral latency and the release from latency to initiate Viral replication.

  • Epigenetic control of Epstein–Barr virus transcription – relevance to Viral Life Cycle?
    Frontiers in Genetics, 2013
    Co-Authors: Alison J Sinclair
    Abstract:

    DNA methylation normally leads to silencing of gene expression but Epstein–Barr virus (EBV) provides an exception to the epigenetic paradigm. DNA methylation is absolutely required for the expression of many Viral genes. Although the Viral genome is initially un-methylated in newly infected cells, it becomes extensively methylated during the establishment of Viral latency. One of the major regulators of EBV gene expression is a Viral transcription factor called Zta (BZLF1, ZEBRA, Z) that resembles the cellular AP1 transcription factor. Zta recognizes at least 32 variants of a 7-nucleotide DNA sequence element, the Zta-response element (ZRE), some of which contain a CpG motif. Zta only binds to the latter class of ZREs in their DNA-methylated form, whether they occur in Viral or cellular promoters and is functionally relevant for the activity of these promoters. The ability of Zta to interpret the differential DNA methylation of the Viral genome is paramount for both the establishment of Viral latency and the release from latency to initiate Viral replication.

  • epigenetic control of Viral Life Cycle by a dna methylation dependent transcription factor
    PLOS ONE, 2011
    Co-Authors: Kirsty Flower, David Thomas, James M Heather, Sharada Ramasubramanyan, Susan Jones, Alison J Sinclair
    Abstract:

    Epstein-Barr virus (EBV) encoded transcription factor Zta (BZLF1, ZEBRA, EB1) is the prototype of a class of transcription factor (including C/EBPalpha) that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation Cycle; it is extensively methylated during Viral latency but is reset to an unmethylated state following Viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs) has been proposed to regulate EBV replication, specifically it could aid the activation of Viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic Cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic Cycle genes at the majority of EBV promoters.

Samina Alam - One of the best experts on this subject based on the ideXlab platform.

  • genetic and biochemical analysis of cis regulatory elements within the keratinocyte enhancer region of the human papillomavirus type 31 upstream regulatory region during different stages of the Viral Life Cycle
    Journal of Virology, 2004
    Co-Authors: Samina Alam, Craig Meyers
    Abstract:

    Using linker scanning mutational analysis, we recently identified potential cis regulatory elements contained within the 5′ upstream regulatory region (URR) domain and auxiliary enhancer (AE) region of the human papillomavirus type 31 (HPV31) URR involved in the regulation of E6/E7 promoter activity at different stages of the Viral Life Cycle. For the present study, we extended the linker scanning mutational analysis to identify potential cis elements located in the keratinocyte enhancer (KE) region (nucleotides 7511 to 7762) of the HPV31 URR and to characterize cellular factors that bind to these elements under conditions representing different stages of the Viral Life Cycle. The linker scanning mutational analysis identified Viral cis elements located in the KE region that regulate transcription in the presence and absence of any Viral gene products or Viral DNA replication and determine the role of host tissue differentiation on Viral transcriptional regulation. Using electrophoretic mobility shift assays, we illustrated defined reorganization in the composition of cellular transcription factors binding to the same cis regulatory elements at different stages of the HPV differentiation-dependent Life Cycle. Our studies provide an extensive map of functional elements in the KE region of the HPV31 URR, identify cis regulatory elements that exhibit significant transcription regulatory potential, and illustrate changes in specific protein-DNA interactions at different stages of the Viral Life Cycle. The variable recruitment of transcription factors to the same cis element under different cellular conditions may represent a mechanism underlying the tight link between keratinocyte differentiation and E6/E7 expression.