RNA-Seq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus 16 infection, including loss of epithelial barrier function

Klymenko, T. et al. (2017) RNA-Seq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus 16 infection, including loss of epithelial barrier function. Journal of Virology, 91(24), e01001-17. (doi: 10.1128/JVI.01001-17) (PMID:29021401) (PMCID:PMC5709591)

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The 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 immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq 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 up-regulated. HPV down-regulated many genes involved in epithelial barrier function that 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, IL1A and IL1B. However, IRF1, IFNκ and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR 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: Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNASeq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3000 genes were differentially expressed in keratinocyte 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 virus-host interaction crucial for production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Graham, Professor Sheila and Herbert, Miss Imogen and Stevenson, Mr Andrew and Iliev, Mr Victor and Gatherer, Dr Derek and Klymenko, Dr Tetyana and Herzyk, Dr Pawel and Gu, Dr Quan and Cuschieri, Dr Kate
Authors: Klymenko, T., Gu, Q., Herbert, I., Stevenson, A., Iliev, V., Watkins, G., Pollock, C., Bhatia, R., Cuschieri, K., Herzyk, P., Gatherer, D., and Graham, S.V.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Virology
Publisher:American Society for Microbiology
ISSN (Online):1098-5514
Published Online:11 October 2017

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
507841Human papillomavirus E2 regulation of splicing factors: consequences for the virus life cycleSheila GrahamWellcome Trust (WELLCOTR)088848/Z/09/ZMVLS III - CENTRE FOR VIRUS RESEARCH