scholarly journals Human Papillomavirus 16 Capsids Mediate Nuclear Entry during Infection

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Patricia M. Day ◽  
Andrea S. Weisberg ◽  
Cynthia D. Thompson ◽  
Michelle M. Hughes ◽  
Yuk Ying Pang ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Viral Entry ◽  
Large Family ◽  
Viral Capsid ◽  
Mitotic Chromosomes ◽  
Nuclear Entry ◽  
Human Papillomavirus 16 ◽  
Transport Vesicle ◽  
Causative Agents

ABSTRACTInfectious human papillomavirus 16 (HPV16) L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA. We also demonstrate that L1 nuclear localization and mitotic chromosome association can occurin vivoin the murine cervicovaginal challenge model of HPV16 infection. These findings challenge the prevailing concepts of PV uncoating and disassembly. More generally, they document that a largely intact viral capsid can enter the nucleus within a transport vesicle, establishing a novel mechanism by which a virus accesses the nuclear cellular machinery.IMPORTANCEPapillomaviruses (PVs) comprise a large family of nonenveloped DNA viruses that include HPV16, among other oncogenic types, the causative agents of cervical cancer. Delivery of the viral DNA into the host cell nucleus is necessary for establishment of infection. This was thought to occur via a subviral complex following uncoating of the larger viral capsid. In this study, we demonstrate that little disassembly of the PV capsid occurs prior to nuclear delivery. These surprising data reveal a previously unrecognized viral strategy to access the nuclear replication machinery. Understanding viral entry mechanisms not only increases our appreciation of basic cell biological pathways but also may lead to more effective antiviral interventions.

scholarly journals CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

mBio ◽  
2021 ◽  
Author(s):  
Apurva T. Prabhakar ◽  
Claire D. James ◽  
Dipon Das ◽  
Raymonde Otoa ◽  
Matthew Day ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Cellular Protein ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Human Papillomavirus 16 ◽  
Causative Agents ◽  
Mitotic Chromatin

Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 in vitro and in vivo .

scholarly journals Virus–Host Protein–Protein Interactions between Human Papillomavirus 16 E6 A1 and D2/D3 Sub-Lineages: Variances and Similarities

2020 ◽  
Vol 21 (21) ◽  
pp. 7980
Author(s):  
Guillem Dayer ◽  
Mehran L. Masoom ◽  
Melissa Togtema ◽  
Ingeborg Zehbe
Keyword(s):  
Human Papillomavirus ◽  
Asian American ◽  
Protein Interactions ◽  
Host Protein ◽  
Cellular Proteins ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Human Papillomavirus 16 ◽  
Novel Approach ◽  
Causative Agents

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers, with type 16 being the most frequent. Compared to the European Prototype (EP; A1), the Asian-American (AA; D2/D3) sub-lineage seems to have increased abilities to promote carcinogenesis. Here, we studied protein–protein interactions (PPIs) between host proteins and sub-lineages of the key transforming E6 protein. We transduced human keratinocyte with EP or AA E6 genes and co-immunoprecipitated E6 proteins along with interacting cellular proteins to detect virus–host binding partners. AAE6 and EPE6 may have unique PPIs with host cellular proteins, conferring gain or loss of function and resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to AAE6 and EPE6, along with 13 E6 targets common to both. A novel approach based on pathway selection discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets. Interpretations were made using databases, such as UniProt, BioGRID, and Reactome. Detected E6 targets were differentially implicated in important hallmarks of cancer: deregulating Notch signaling, energetics and hypoxia, DNA replication and repair, and immune response.

scholarly journals In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by e6 siRNA

2003 ◽  
Vol 8 (5) ◽  
pp. 762-768 ◽  
Author(s):  
Mitsuo Yoshinouchi ◽  
Taketo Yamada ◽  
Masahiro Kizaki ◽  
Jin Fen ◽  
Takeyoshi Koseki ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Cancer Cells ◽  
Growth Suppression ◽  
Human Papillomavirus 16 ◽  
Cervical Cancer Cells ◽  
In Vivo Growth

scholarly journals Enhanced oncogenicity of Asian-American human papillomavirus 16 is associated with impaired E2 repression of E6/E7 oncogene transcription

2004 ◽  
Vol 85 (6) ◽  
pp. 1433-1444 ◽  
Author(s):  
Rosa M. Ordóñez ◽  
Ana María Espinosa ◽  
Dolores Javier Sánchez-González ◽  
Juan Armendáriz-Borunda ◽  
Jaime Berumen
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Asian American ◽  
Long Control Region ◽  
Hpv 16 ◽  
E2 Protein ◽  
Human Papillomavirus 16 ◽  
E2 Gene ◽  
E7 Oncogene

Asian-American (AA) variants of human papillomavirus 16 (HPV-16) are linked to a high incidence of cervical cancer in Mexico, with some evidence strongly suggesting that they are more oncogenic than European (E) variants, including their association with younger women and their higher associated risk of cervical cancer. Differences in the regulation of viral E6/E7 oncogene transcription by the E2 protein may be involved in the higher oncogenicity of AA variants. In E variants, E6/E7 oncogene transcription is repressed by the E2 protein and is frequently up-regulated by the destruction of the E2 gene during viral integration. In contrast, the E2 gene is retained in full in most AA-positive carcinomas, raising the possibility of alternative mechanisms for increasing viral oncogene transcription. The authors investigated whether the higher oncogenicity of AA variants is linked to differences in E6/E7 oncogene transcription and the mechanism of E2 deactivation. E6/E7 and E1/E2 transcripts were explored by RT-PCR in 53 HPV-16-positive cervical carcinomas, 39 retaining (20 European and 19 AA) and 14 having lost (12 European and 2 AA) the E1/E2 genes, and transcription repression activity of the AA E2 genes was tested in four cell lines that constitutively express the β-galactosidase reporter or E6/E7 genes driven by the viral long control region. E6/E7 oncogene transcripts were found in all carcinomas, but only those positive for AA variants with E1/E2 genes had complete E2 transcripts. E2 transcripts were down-regulated by splicing in E-positive carcinomas retaining E1/E2. AA E2 genes were impaired for repression of E6/E7 oncogene transcription in vivo. These results suggest that E6/E7 oncogene expression starts earlier in AA than E variant infections, since E variants need E2 to be destroyed or down-regulated.

scholarly journals Interferon Gamma Prevents Infectious Entry of Human Papillomavirus 16 via an L2-Dependent Mechanism

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Patricia M. Day ◽  
Cynthia D. Thompson ◽  
Douglas R. Lowy ◽  
John T. Schiller
Keyword(s):  
Human Papillomavirus ◽  
Viral Genome ◽  
Antiviral Effect ◽  
Late Endosome ◽  
Human Papillomavirus 16 ◽  
Causative Agents ◽  
Ifn Γ ◽  
Biochemical Analyses ◽  
Infectious Entry ◽  
L1 Protein

ABSTRACT In this study, we report that gamma interferon (IFN-γ) treatment, but not IFN-α, -β, or -λ treatment, dramatically decreased infection of human papillomavirus 16 (HPV16) pseudovirus (PsV). In a survey of 20 additional HPV and animal papillomavirus types, we found that many, but not all, PsV types were also inhibited by IFN-γ. Microscopic and biochemical analyses of HPV16 PsV determined that the antiviral effect was exerted at the level of endosomal processing of the incoming capsid and depended on the JAK2/STAT1 pathway. In contrast to infection in the absence of IFN-γ, where L1 proteolytic products are produced during endosomal capsid processing and L2/DNA complexes segregate from L1 in the late endosome and travel to the nucleus, IFN-γ treatment led to decreased L1 proteolysis and retention of L2 and the viral genome in the late endosome/lysosome. PsV sensitivity or resistance to IFN-γ treatment was mapped to the L2 protein, as determined with infectious hybrid PsV, in which the L1 protein was derived from an IFN-γ-sensitive HPV type and the L2 protein from an IFN-γ-insensitive type or vice versa. IMPORTANCE A subset of HPV are the causative agents of many human cancers, most notably cervical cancer. This work describes the inhibition of infection of multiple HPV types, including oncogenic types, by treatment with IFN-γ, an antiviral cytokine that is released from stimulated immune cells. Exposure of cells to IFN-γ has been shown to trigger the expression of proteins with broad antiviral effector functions, most of which act to prevent viral transcription or translation. Interestingly, in this study, we show that infection is blocked at the early step of virus entry into the host cell by retention of the minor capsid protein, L2, and the viral genome instead of trafficking into the nucleus. Thus, a novel antiviral mechanism for IFN-γ has been revealed.

scholarly journals CK2 phosphorylation of human papillomavirus 16 E2 on serine 23 promotes interaction with TopBP1 and is critical for E2 plasmid retention function

2021 ◽  
Author(s):  
Apurva T. Prabhakar ◽  
Claire D. James ◽  
Dipon Das ◽  
Raymonde Otoa ◽  
Matthew Day ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Life Cycle ◽  
Cellular Protein ◽  
Human Papillomaviruses ◽  
Retention Function ◽  
Critical Function ◽  
Human Papillomavirus 16 ◽  
Ck2 Inhibitors

AbstractDuring the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here, we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo, and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A activates and represses transcription identically to E2-WT (wild-type), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin when compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis when compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. We next tested whether this difference in E2-S23A levels during mitosis disrupts E2 plasmid retention function. We developed a novel plasmid retention assay and demonstrate that E2-S23A is deficient in plasmid retention when compared with E2-WT. siRNA targeted knockdown of TopBP1 abrogates E2-WT plasmid retention function. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, which is critical for E2 plasmid retention function and in HPV16 immortalization of keratinocytes.ImportanceHuman papillomaviruses are causative agents in around 5% of all cancers, with no specific anti-viral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex formation with the cellular protein TopBP1 in vitro and in vivo. This complex results in stabilization of E2 during mitosis and mediates plasmid retention by E2. This function promotes the partitioning of viral genomes into the nuclei of daughter cells following mitosis. We demonstrate that CK2 phosphorylates E2 on serine 23 in vivo, and that CK2 inhibitors disrupt the E2-TopBP1 complex. Mutation of E2 serine 23 to alanine disrupts the HPV16 life cycle, demonstrating a critical function for this residue. Together, our results suggest that CK2 inhibitors may disrupt the E2-TopBP1 dependent HPV16 life cycle and potentially kill HPV16 positive cancers, which lays a molecular foundation to develop novel therapeutic approaches for combating HPV16 disease.

scholarly journals Cellular factor YY1 downregulates the human papillomavirus 16 E6/E7 promoter, P97, in vivo and in vitro from a negative element overlapping the transcription-initiation site

2009 ◽  
Vol 90 (10) ◽  
pp. 2402-2412 ◽  
Author(s):  
Michael J. Lace ◽  
Yasushi Yamakawa ◽  
Masato Ushikai ◽  
James R. Anson ◽  
Thomas H. Haugen ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Transcription Initiation ◽  
Regulatory Region ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Wild Type ◽  
Hpv 16 ◽  
Human Papillomavirus 16

Cellular factors that bind to cis sequences in the human papillomavirus 16 (HPV-16) upstream regulatory region (URR) positively and negatively regulate the viral E6 and E7 oncogene promoter, P97. DNase I footprinting has revealed the binding of cellular proteins to two previously undetected cis elements overlapping and 3′ of the transcription-initiation site of the P97 promoter. Mutations within homologous motifs found in both of these cis elements abolished their negative function in vivo and the binding of the same cellular complex in vitro. This factor was identified as YY1 by complex mobility and binding specificity in comparison with vaccinia virus-expressed, purified recombinant YY1 protein and by antigenic reactivity with YY1 antisera. Cis mutations in the ‘initiator’ YY1 site activated the P97 promoter in vivo and in vitro. P97 was also activated threefold in vitro by depletion of endogenous YY1 with wild-type, but not mutant, YY1 oligonucleotides from the IgH kappa E3′ enhancer. Furthermore, increasing concentrations of exogenous, purified recombinant YY1 repressed wild-type P97 transcript levels by up to threefold, but did not influence the P97 promoter mutated in the ‘initiator’ YY1 site. Thus, the promoter-proximal YY1 site was not necessary for correct transcription initiation at the P97 promoter, but was found to be required for downregulation of P97 transcription in vivo and in vitro. In contrast to other viral and cellular promoters, where YY1 is thought to function as a positive transcription-‘initiator’ factor, HPV-16 P97 transcription is downregulated by YY1 from a critical motif overlapping the transcription start site.

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