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      Genome-Wide Transcriptome Analysis of Human Papillomavirus 16-Infected Primary Keratinocytes Reveals Subtle Perturbations Mostly due to E7 Protein Expression

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          Abstract

          To establish infection and complete the viral life cycle, human papillomavirus (HPV) needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocyte-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an extracellular matrix (ECM)-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S-phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and, possibly, disease progression.

          ABSTRACT

          It is established that the host cell transcriptomes of natural lesions, organotypic rafts, and human papillomavirus (HPV)-immortalized keratinocytes are altered in the presence of HPV genomes. However, the establishment of HPV-harboring cell lines requires selection and immortalization, which makes it impossible to distinguish between alterations directly induced by HPV or indirectly by the need for immortalization or selection. To address direct effects of HPV infection on the host cell transcriptome, we have used our recently established infection model that allows efficient infection of primary keratinocytes with HPV16 virions. We observed only a small set of genes to be deregulated at the transcriptional level at 7 days postinfection (dpi), most of which fall into the category regulated by pocket proteins pRb, p107, and p130. Furthermore, cell cycle genes were not deregulated in cells infected with a virus lacking E7 despite the presence of episomal genome and viral transcripts. These findings imply that the majority of transcriptional changes are due to the E7 protein impairing pocket protein function. Additional pathways, such as the Fanconi anemia-BRCA pathway, became perturbed only after long-term culturing of infected cells. When grown as organotypic raft cultures, keratinocytes infected with wild-type but not E7 mutant virus had perturbed transcriptional regulation of pathways previously identified in natural lesions and in rafts derived from immortalized keratinocytes. We conclude that the HPV infection model provides a valuable tool to distinguish immediate transcriptional alterations from those induced by persistent infection and the need for selection and immortalization.

          IMPORTANCE To establish infection and complete the viral life cycle, human papillomavirus (HPV) needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocyte-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an extracellular matrix (ECM)-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S-phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and, possibly, disease progression.

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          Author and article information

          Contributors
          Role: Editor
          Journal
          J Virol
          J. Virol
          jvi
          jvi
          JVI
          Journal of Virology
          American Society for Microbiology (1752 N St., N.W., Washington, DC )
          0022-538X
          1098-5514
          20 November 2019
          17 January 2020
          February 2020
          : 94
          : 3
          : e01360-19
          Affiliations
          [a ] Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Feist Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, USA
          International Centre for Genetic Engineering and Biotechnology
          Author notes
          Address correspondence to Martin Sapp, msapp1@ 123456lsuhsc.edu .

          Citation Bienkowska-Haba M, Luszczek W, Zwolinska K, Scott RS, Sapp M. 2020. Genome-wide transcriptome analysis of human papillomavirus 16-infected primary keratinocytes reveals subtle perturbations mostly due to E7 protein expression. J Virol 94:e01360-19. https://doi.org/10.1128/JVI.01360-19.

          Author information
          https://orcid.org/0000-0003-3840-9189
          Article
          PMC7000963 PMC7000963 7000963 01360-19
          10.1128/JVI.01360-19
          7000963
          31748387
          00c5586b-f225-4789-bb3f-704a4033a6da
          Copyright © 2020 American Society for Microbiology.

          All Rights Reserved.

          History
          : 14 August 2019
          : 10 November 2019
          Page count
          Figures: 9, Tables: 2, Equations: 0, References: 48, Pages: 17, Words: 8922
          Funding
          Funded by: HHS | NIH | National Cancer Institute (NCI), https://doi.org/10.13039/100000054;
          Award ID: R01CA211576
          Award Recipient :
          Funded by: HHS | NIH | National Institute of General Medical Sciences (NIGMS), https://doi.org/10.13039/100000057;
          Award ID: P30GM110703
          Award Recipient :
          Funded by: HHS | NIH | National Institute of Dental and Craniofacial Research (NIDCR), https://doi.org/10.13039/100000072;
          Award ID: R01DE25565
          Award Recipient :
          Categories
          Cellular Response to Infection
          Spotlight
          Custom metadata
          February 2020

          pocket protein,RB,infection model,p53,organotypic raft culture,HPV16,E7,transcriptome,primary foreskin keratinocytes

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