The effect of RNA binding proteins on the regulation of Human papillomavirus type 16 late gene expression

Abstract

In this paper, we addressed whether cellular RNA binding proteins TIAR, hnRNP C1 and HuR induce HPV 16 late gene expression by interacting with elements of the viral genome that regulate splicing and polyadenylation. Pathogenesis and progression to HPV infected lesion is regulated by the viral gene expression program and it has been previously shown that several RNA binding proteins have an effect on this regulation at the level of RNA processing. The effect of RNA binding proteins was studied by RNA analysis of HeLa cells co-transfected with reporter subgenomic HPV 16 plasmids and RNA binding protein-encoding plasmid. In vitro binding assays showed that the factors studied here are interacting with key regulatory elements such as the early 3’ untranslated region (early 3'UTR) and splice silencer element upstream of major splice sites for viral late mRNAs. These results conclude that TIAR and HuR are inducing late gene expression through interaction with 3’ UTR and splice site SD3632.

Popular Abstract

RNA binding proteins that control HPV 16 late gene expression

Human Papillomavirus (HPV) is the most commonly sexually transmitted virus in humans. In most cases the infection is cleared by the immune system. However, a subset of HPVs such as HPV16 can cause persistent infections which can ultimately generate precancerous high-grade lesions and cervical cancer. An important feature of HPV 16 is its ability to evade the host immune system by restricting expression of the proteins to the very late stage of the infection. Thus, regulation of late gene expression is of paramount importance.

The life cycle of HPV 16 and gene expression is solely dependent on the host cells differentiation program. Viral particles first reach the basal cells. The viral genome is then released and enters the host-cell nucleus and early genes E1 and E2 are transcribed. The infected cells multiply and HPV 16 starts expressing proteins E6 and E7 which cause the host to cells to continue proliferate and become matured keratinocytes. Once the keratinocytes die the virus encodes L1 and L2 proteins, also the most immunogenic, that package its genome and virions are released from the cell.

In this project, I investigated whether RNA binding proteins could induce HPV 16 late gene expression. This is tested by transfecting human cells with HPV 16 subgenomic plasmids representing part of the genome. Furthermore, RNA extracted from the transfected cells was analysed to study the expression of specific viral mRNAs induced by the RNA binding proteins in question. Finally, in vitro experiments were performed to investigate whether the proteins in question interact with specific segments of the viral genome that affect the expression of HPV 16 genes.

Our results indicate that the RNA binding proteins TIAR and HuR do in fact cause the expression of HPV 16 late genes by interacting with regulatory sequences of the viral genome. Elucidating the mechanisms by which RNA binding proteins regulate HPV 16 gene expression program may lead to the identification of novel targets for antiviral drugs.


Master’s Degree Project in Molecular Biology 30 credits 2017
Department of Biology, Lund University

Advisors: Stefan Schwartz, Naoko Kajitani
Section of Medical Microbiology /Department of Laboratory medicine, BMC.

• author: Tsimtsirakis, Efthymios
• supervisor: Stefan Schwartz; Naoko Kajitani
• organization: Degree Projects in Molecular Biology
• course: MOBM01 20162
• year: 2017
• type: H1 - Master's Degree (One Year)
• subject: Biology and Life Sciences
• language: English
• id: 8904182
• date added to LUP: 2017-03-06 14:31:18
• date last changed: 2017-03-06 14:31:18