PhD student, University of Wisconsin-Madison
Interactions between two human tumor viruses, HPV and EBV, in the oral epithelium
Epstein-Barr virus (EBV) and human papillomaviruses (HPVs) are two important human tumor viruses that cause 7% of all human cancers including a shared ability to cause head and neck cancers. Both viruses infect and replicate in upper aerodigestive tract epithelia from which these cancers arise. Some studies have detected co-infection in both oropharyngeal and nasopharyngeal cancers raising the possibility that these viruses could potentially affect each other’s life cycles and/or oncogenic potential. We established an in vitro model system using organotypic raft cultures to study the life cycles of EBV and HPV and to test the effects of EBV and HPV on each other in stratified squamous oral epithelial cells. We found that oral epithelial cells infected or transfected with EBV or HPV support the life cycles of these two viruses with clear parallels in that the production of progeny HPV and EBV is tied to the differentiation of the infected epithelial host cells. Interestingly we found that, in cells harboring both viruses, HPV dramatically augmented the EBV life cycle. Specifically, HPV led to EBV being more stably retained in epithelial cells that were grown under conditions that limit their differentiation, and, under conditions that stimulate cell differentiation, HPV enhanced the expression of an EBV protein, Z, that triggers EBV lytic reactivation, increased the level of synthesis of EBV genomic DNA, and dramatically increased yields of EBV progeny virus. We further discovered that HPV’s ability to promote EBV lytic reactivation is driven by the HPV oncoprotein E7, and correlates with E7’s ability to inactivate, pRb, a well-known tumor suppressor that controls key steps in the cell cycle. Studying the effects of HPV on EBV thus has illuminated mechanisms underlying EBV lytic reactivation and demonstrated potential consequences of co-infection on viral spread and head and neck carcinogenesis.
Abstract: Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.
Pub.: 15 Oct '14, Pinned: 29 Jun '17
Abstract: Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.
Pub.: 03 Oct '15, Pinned: 29 Jun '17
Abstract: Epstein-Barr virus and human papillomaviruses are human tumor viruses that infect and replicate in upper aerodigestive tract epithelia and cause head and neck cancers. The productive phases of both viruses are tied to stratified epithelia highlighting the possibility that these viruses may affect each other's life cycles. Our lab has established an in vitro model system to test the effects of EBV and HPV co-infection in stratified squamous oral epithelial cells. Our results indicate that HPV increases maintenance of the EBV genome in the co-infected cells and promotes lytic reactivation of EBV in upper layers of stratified epithelium. Expression of the HPV oncogenes E6 and E7 were found to be necessary and sufficient to account for HPV-mediated lytic reactivation of EBV. Our findings indicate that HPV increases the capacity of epithelial cells to support the EBV life cycle, which could in turn increase EBV-mediated pathogenesis in the oral cavity.
Pub.: 15 May '16, Pinned: 29 Jun '17
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