Interaction of Human Cytomegalovirus protein UL84 with viral and cellular RNA transcripts
AuthorFelix, Jennifer N.
AdvisorPari, Gregory S.
Biochemistry and Molecular Biology
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Human Cytomegalovirus (HCMV), a member of the beta herpesvirus family, is a significant cause of life-threatening illness in immune compromised individuals and is the leading cause of congenital viral infection in neonates. As a member of the herpesvirus family, the HCMV life cycle occurs in two stages: lytic infection followed by latency. Lytic DNA replication requires 6 core proteins common to all herpesviruses. HCMV also requires the viral transactivators encoded by IRS1, UL112/113, UL84 and IE2 for origin-dependent DNA replication. Due to its requirement for lytic DNA replication and virus growth, HCMV UL84 has been identified as a protein of interest and potential candidate for the development of antiviral therapy. UL84 is a multifunctional phosphoprotein that interacts with several other required viral factors, including viral RNA and the HCMV origin of lytic DNA replication. UL84 also contains nuclear localization signals, giving it the ability to shuttle bi-directionally between the nucleus and the cytoplasm. This purpose of this shuttling function has not yet been ascertained.The finding that UL84 interacts with at least one known viral encoded mRNA suggests that one function of the bi-directional shuttling of UL84 is to increase the concentration of specific mRNA transcripts in the cytoplasm. We propose that UL84 interacts with several RNA transcripts and that this interaction plays a regulatory role in the expression of viral and cellular encoded genes.To study the interaction of UL84 with other RNA transcripts, a recombinant HCMV AD169 GFP BACmid expressing a flag epitope tagged UL84 protein was generated. This BACmid was utilized to reconstitute infectious virus containing a Flag tagged UL84 protein. Human Foreskin Fibroblasts (HFFs) were infected with HCMV AD169 UL84-Flag and allowed to incubate for 3 or 5 days post infection. Infected cell lysate was collected and RNA cross-linking and immunoprecipitation was performed (RNA CliP). The resulting RNA was used to prepare a library for next generation (deep) sequencing utilizing the Illumina MiSeq. Several viral and cellular transcripts were identified. Some transcripts of interest have been confirmed through RT-PCR.In identifying RNA transcripts with which UL84 interacts, we will have a better understanding how this key viral protein works to regulate lytic viral replication in the host cell.