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Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation.

ABSTRACT: Papillomaviruses are small DNA viruses that are associated with benign and malignant epithelial lesions, including >95% of cervical cancers and approximately 20% of head and neck cancers. Because papillomavirus replication and virion production are tied to epithelial cell differentiation, infectious papillomavirus virion production has been limited to cumbersome organotypic cultures and mouse xenografts. Consequent difficulties in obtaining useful amounts of wild-type or mutant human papillomavirus (HPV) virions have greatly limited studies on many aspects of papillomavirus biology. To overcome these limitations, we developed a system to encapsidate the full-length papillomaviral genome into infectious virions, independently of viral DNA replication and epithelial differentiation. This transient-transfection-based system produces >1,000 times more infectious virus per cell culture dish than the much more labor-intensive organotypic culture. Furthermore, we show that this method allows the facile generation of infectious particles containing wild-type, mutant, or chimeric papillomaviral genomes, overcoming barriers to studying many facets of replication, host interactions, and vaccine and drug development, which has been limited by the insufficient availability of infectious virions.

SUBMITTER: Pyeon D

PROVIDER: S-EPMC1166641 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation.

Pyeon Dohun D Lambert Paul F PF Ahlquist Paul P

Proceedings of the National Academy of Sciences of the United States of America 20050615 26

Papillomaviruses are small DNA viruses that are associated with benign and malignant epithelial lesions, including >95% of cervical cancers and approximately 20% of head and neck cancers. Because papillomavirus replication and virion production are tied to epithelial cell differentiation, infectious papillomavirus virion production has been limited to cumbersome organotypic cultures and mouse xenografts. Consequent difficulties in obtaining useful amounts of wild-type or mutant human papillomavi ...[more]

PMID: 15958530

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Project description:Human papillomaviruses induce a host of anogenital cancers, as well as oropharyngeal cancer (HPV+OPC); human papillomavirus 16 (HPV16) is causative in around 90% of HPV+OPC cases. Using telomerase reverse transcriptase (TERT) immortalized foreskin keratinocytes (N/Tert-1), we have identified significant host gene reprogramming by HPV16 (N/Tert-1+HPV16) and demonstrated that N/Tert-1+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is transcriptionally regulated by HPV16 in N/Tert-1. CRISPR/Cas9 removal of SAMHD1 from N/Tert-1 and N/Tert-1+HPV16 demonstrates that SAMHD1 controls cell proliferation of N/Tert-1 only in the presence of HPV16; the deletion of SAMHD1 promotes hyperproliferation of N/Tert-1+HPV16 cells in organotypic raft cultures but has no effect on N/Tert-1. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in nonimmortalized primary keratinocytes due to their limited life span. To confirm the relevance of our results, we repeated the analysis with human tonsil keratinocytes (HTK) immortalized by HPV16 (HTK+HPV16) and observed the same hyperproliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of N/Tert-1 with N/Tert-1+HPV16, combined with HTK+HPV16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in keratinocytes.IMPORTANCE HPVs are causative agents in human cancers and are responsible for around of 5% of all cancers. A better understanding of the viral life cycle in keratinocytes will facilitate the development of novel therapeutics to combat HPV-positive cancers. Here, we present a unique keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein levels in keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in keratinocytes and elevated viral replication but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.

Project description:Human papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here, we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions compared to normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression, there was enhanced basal cell proliferation, DNA damage, and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium, and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle.IMPORTANCE HPV16 is the major human viral carcinogen, responsible for around 3 to 4% of all cancers worldwide. Our understanding of how the viral replication machinery interacts with host factors to control/activate the DNA damage response to promote the viral life cycle remains incomplete. Recently, we demonstrated a SIRT1-WRN axis that controls HPV16 replication, and here we demonstrate that this axis persists in clinical cervical lesions induced by HPV16. Here, we describe the effects of WRN depletion on cellular differentiation with or without HPV16; WRN depletion results in enhanced proliferation and DNA damage irrespective of HPV16 status. Also, WRN is a restriction factor for the viral life cycle since replication is disrupted in the absence of WRN. Future studies will focus on enhancing our understanding of how WRN regulates viral replication. Our goal is to ultimately identify cellular factors essential for HPV16 replication that can be targeted for therapeutic gain.

Project description:Hepatitis C virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacement of the NS3 helicase and NS5B-to-3'X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications within these genomic regions needed to establish replication-competent J6CF. We previously identified 4 mutations in the NS5B-to-3'X region that could be used instead of replacement of this region to confer J6CF replication competence. Here, we induced cell culture-adaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3'X or individual parts of this but not the NS3 helicase region. After 2 months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of the coexpressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introduction of the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in the NS5B-to-3'X region. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3'X and established a replication-competent J6CF strain with minimum essential modifications in cultured cells. The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications needed for cell culture adaptation will be valuable as another representative of genotype 2a strains.

Project description:The association between partner human papillomavirus (HPV) viral load and incident HPV detection in heterosexual couples is unknown.HPV genotypes were detected in 632 human immunodeficiency virus (HIV)-negative couples followed for 2 years in a male circumcision trial in Rakai, Uganda, using the Roche HPV Linear Array. This assay detects 37 genotypes and provides a semiquantitative measure of viral load based on the intensity (graded 1-4) of the genotype-specific band; a band intensity of 1 indicates a low genotype-specific HPV load, whereas an intensity of 4 indicates a high load. Using Poisson regression with generalized estimating equations, we measured the association between partner's genotype-specific viral load and detection of that genotype in the HPV-discordant partner 1 year later.Incident detection of HPV genotypes was 10.6% among men (54 of 508 genotype-specific visit intervals) and 9.0% among women (55 of 611 genotype-specific visit intervals). Use of male partners with a baseline genotype-specific band intensity of 1 as a reference yielded adjusted relative risks (aRRs) of 1.14 (95% confidence interval [CI], .58-2.27]) for incident detection of that genotype among women whose male partner had a baseline band intensity of 2, 1.75 (95% CI, .97-3.17) among those whose partner had an intensity of 3, and 2.52 (95% CI, 1.40-4.54) among those whose partner had an intensity of 4. Use of female partners with a baseline genotype-specific band intensity of 1 as a reference yielded an aRR of 2.83 (95% CI, 1.50-5.33) for incident detection of that genotype among men whose female partner had a baseline band intensity of 4. These associations were similar for high-risk and low-risk genotypes. Male circumcision also was associated with significant reductions in incident HPV detection in men (aRR, 0.53 [95% CI, .30-.95]) and women (aRR, 0.42 [95% CI, .23-.76]).In heterosexual couples, the genotype-specific HPV load in one partner is associated with the risk of new detection of that genotype in the other partner. Interventions that reduce the HPV load may reduce the incidence of HPV transmission.

Project description:Human papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCE Human papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.

Dataset Information

Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation.

Publications

Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation.

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