scholarly journals Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 772
Author(s):  
Isao Murakami ◽  
Takashi Iwata ◽  
Tohru Morisada ◽  
Kyoko Tanaka ◽  
Daisuke Aoki
Keyword(s):  
Life Cycle ◽  
Cultured Cells ◽  
Basal Layer ◽  
Nucleosome Positioning ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Host Cells ◽  
Micrococcal Nuclease ◽  
Hpv Dna ◽  
Dna Replication And Repair

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

scholarly journals Nucleosome positioning on episomal human papillomavirus DNA in cultured cells

2021 ◽  
Author(s):  
Isao Murakami ◽  
Takashi Iwata ◽  
Tohru Morisada ◽  
Kyoko Tanaka ◽  
Daisuke Aoki
Keyword(s):  
Life Cycle ◽  
Cultured Cells ◽  
Nucleosome Position ◽  
Basal Layer ◽  
Nucleosome Positioning ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Host Cells ◽  
Micrococcal Nuclease ◽  
Hpv Dna

Abstract Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that change in nucleosome position on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

scholarly journals Pathogenesis of Human Papillomaviruses in Differentiating Epithelia

2004 ◽  
Vol 68 (2) ◽  
pp. 362-372 ◽  
Author(s):  
Michelle S. Longworth ◽  
Laimonis A. Laimins
Keyword(s):  
Life Cycle ◽  
Genital Tract ◽  
Basal Layer ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Basal Cells ◽  
Human Pathogens ◽  
Late Gene Expression ◽  
Differentiated Cells ◽  
Hpv Types

SUMMARY Human papillomaviruses (HPV) are the etiological agents of cervical and other anogenital malignancies. Over 100 different types of HPVs have been identified to date, and all target epithelial tissues for infection. One-third of HPV types specifically infect the genital tract, and a subset of these are the causative agents of anogenital cancers. Other HPV types that infect the genital tract induce benign hyperproliferative lesions or genital warts. The productive life cycle of HPVs is linked to epithelial differentiation. Papillomaviruses are thought to infect cells in the basal layer of stratified epithelia and establish their genomes as multicopy nuclear episomes. In these cells, viral DNA is replicated along with cellular chromosomes. Following cell division, one of the daughter cells migrates away from the basal layer and undergoes differentiation. In highly differentiated suprabasal cells, vegetative viral replication and late-gene expression are activated, resulting in the generation of progeny virions. Since virion production is restricted to differentiated cells, infected basal cells can persist for up to several decades or until the immune system clears the infection. The E6 and E7 genes encode viral oncoproteins that target Rb and p53, respectively. During the viral life cycle, these proteins facilitate stable maintenance of episomes and stimulate differentiated cells to reenter the S phase. The E1 and E2 proteins act as origin recognition factors as well as regulators of early viral transcription. The functions of the E5 and E1^E4 proteins are still largely unknown, but these proteins have been implicated in modulating late viral functions. The L1 and L2 proteins form icosahedral capsids for progeny virion generation. The characterization of the cellular targets of these viral proteins and the mechanisms regulating the differentiation-dependent viral life cycle remain active areas for the study of these important human pathogens.

scholarly journals Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

Viruses ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 82 ◽  
Author(s):  
Di Sun ◽  
Shun Chen ◽  
Anchun Cheng ◽  
Mingshu Wang
Keyword(s):  
Life Cycle ◽  
Viral Life Cycle ◽  
Host Cells

scholarly journals SAMHD1 Regulates Human Papillomavirus 16-Induced Cell Proliferation and Viral Replication during Differentiation of Keratinocytes

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Claire D. James ◽  
Apurva T. Prabhakar ◽  
Raymonde Otoa ◽  
Michael R. Evans ◽  
Xu Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Human Papillomavirus ◽  
Life Cycle ◽  
Viral Replication ◽  
Cellular Proliferation ◽  
Specific Interaction ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Cellular Growth ◽  
Human Papillomavirus 16

ABSTRACT 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.

scholarly journals Transactivation by the E2 Protein of Oncogenic Human Papillomavirus Type 31 Is Not Essential for Early and Late Viral Functions

1998 ◽  
Vol 72 (10) ◽  
pp. 8115-8123 ◽  
Author(s):  
Frank Stubenrauch ◽  
Angela M. E. Colbert ◽  
Laimonis A. Laimins
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Human Keratinocytes ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Late Gene ◽  
Late Gene Expression ◽  
Positive Role ◽  
E2 Protein ◽  
Biochemical Analyses

ABSTRACT The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN6GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.

scholarly journals The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Dipon Das ◽  
Nathan Smith ◽  
Xu Wang ◽  
Iain M. Morgan
Keyword(s):  
Life Cycle ◽  
Dna Replication ◽  
Viral Replication ◽  
Viral Proteins ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Dependent Manner ◽  
Viral Origin ◽  
Replication Complex

ABSTRACT Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle. IMPORTANCE HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.

scholarly journals SARS-CoV-2 mutations in Brazil: from genomics to putative clinical conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Luis Fernando Saraiva Macedo Timmers ◽  
Julia Vasconcellos Peixoto ◽  
Rodrigo Gay Ducati ◽  
José Fernando Ruggiero Bachega ◽  
Leandro de Mattos Pereira ◽  
...  
Keyword(s):  
Life Cycle ◽  
Drug Discovery ◽  
Structural Analysis ◽  
Vaccine Development ◽  
Protein Structures ◽  
Viral Life Cycle ◽  
Host Cells ◽  
High Rate ◽  
Clinical Conditions

AbstractDue to the high rate of transmissibility, Brazil became the new COVID-19 outbreak epicenter and, since then, is being monitored to understand how SARS-CoV-2 mutates and spreads. We combined genomic and structural analysis to evaluate genomes isolated from different regions of Brazil and show that the most prevalent mutations were located in the S, N, ORF3a and ORF6 genes, which are involved in different stages of viral life cycle and its interaction with the host cells. Structural analysis brought to light the positions of these mutations on protein structures, contributing towards studies of selective structure-based drug discovery and vaccine development.

scholarly journals Structural basis of anti-SARS-CoV-2 activity of hydroxychloroquine: specific binding to NTD/CTD and disruption of LLPS of N protein

2021 ◽  
Author(s):  
Mei Dang ◽  
Jianxing Song
Keyword(s):  
Life Cycle ◽  
Specific Binding ◽  
Viral Life Cycle ◽  
Host Cells ◽  
Structural Basis ◽  
N Protein ◽  
Terminal Domain ◽  
Underlying Mechanisms ◽  
Key Steps ◽  
Viral Target

SARS-CoV-2 is the coronavirus causing the catastrophic pandemic which already led to >120 millions of infections and >2.6 millions of deaths. Hydroxychloroquine (HCQ) has been shown to own promising potential in clinically combating SARS-CoV-2 but the underlying mechanisms still remain almost unknown. So far, all action sites are proposed on the host cells, and in particular no specific viral target protein has been experimentally identified. In this study, by use of DIC microscopy and NMR spectroscopy, for the first time we have decoded that HCQ specifically binds to both N-terminal domain (NTD) and C-terminal domain (CTD) of SARS-CoV-2 nucleocapsid (N) protein to inhibit their interactions with nucleic acids (NAs), as well as to disrupt its NA-induced liquid-liquid phase separation (LLPS) essential for the viral life cycle including the package of gRNA and N protein into new virions. These results suggest that HCQ may achieve its anti-SARS-CoV-2 activity by interfering in several key steps of the viral life cycle. The study not only provides a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also indicates that SARS-CoV-2 N protein and its LLPS represent key targets for further optimization and development of anti-SARS-CoV-2 drugs.

scholarly journals Quantitative Role of the Human Papillomavirus Type 16 E5 Gene during the Productive Stage of the Viral Life Cycle

2003 ◽  
Vol 77 (5) ◽  
pp. 2832-2842 ◽  
Author(s):  
Sybil M. Genther ◽  
Stephanie Sterling ◽  
Stefan Duensing ◽  
Karl Münger ◽  
Carol Sattler ◽  
...  
Keyword(s):  
Life Cycle ◽  
Epithelial Cells ◽  
Dna Synthesis ◽  
Viral Gene Expression ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Keratinocyte Differentiation ◽  
Viral Gene ◽  
Viral Dna

ABSTRACT Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle.

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