Abstract
ABSTRACTThe human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalized keratinocytes (NIKS) and NIKS stably transfected with HPV16 episomal genomes (NIKS16) were compared using next-generation sequencing (RNA-Seq). HPV16 infection altered expression of 2,862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNA-Seq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log2 = 1.8, 3.5-fold change), 670 genes were downregulated and 296 genes were upregulated. HPV downregulated many genes involved in epithelial barrier function, which involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant CCL20, and proinflammatory cytokines interleukin 1α (IL-1α) and IL-1β. However, the type I interferon regulator IRF1, kappa interferon (IFN-κ), and viral restriction factors (IFIT1, -2, -3, and -5, OASL, CD74, and RTP4) were upregulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions, and cell adhesion. Quantitative PCR (qRT-PCR) and Western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress.IMPORTANCE HPV genome amplification and capsid formation take place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNA-Seq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3,000 genes were differentially expressed in keratinocytes due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into the virus-host interaction that is crucial for the production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.
Highlights
The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation
In order to elucidate how cellular gene expression is altered during HPV infection, we examined changes in the keratinocyte transcriptome during differentiation and HPV16 infection using normal, spontaneously immortalized keratinocytes (NIKS) and the same cells stably transfected with HPV16 genomes (NIKS16)
We were interested in how differentiating keratinocytes respond to HPV infection during the late, productive phase of the viral life cycle
Summary
The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. The epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted These data provide new insights into the virus-host interaction that is crucial for the production of in-. The pathway of epithelial cell differentiation, from basal to granular layer, is tightly controlled by complex patterns of keratinocyte gene expression [8]. Viral gene expression required for vegetative viral genome amplification takes place in differentiating keratinocytes in the middle to upper epithelial layers [9]. At this stage, other viral regulatory proteins, E4 and E5, that can regulate the host cell are expressed [6]. Elucidating the interactions between HPV and the infected keratinocyte is key to understanding the HPV life cycle and how persistent infection may facilitate the development of cervical disease
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