The Role of ORF59 and PAN ENE in Viral Replication of KSHV
Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus and the etiological agent for a number of oncogenic diseases including Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). Similarly to other herpesviruses, KSHV can establish life-long infections. After a primary infection, the virus remains within the host as a persistent infection with periodic episodes of reactivation due to a variety of factors including environmental stimuli or waning immunity. Particularly, in immune-compromised individuals, reactivation can result in the production of new infectious virions which can in turn drive the progression of KSHV-associated malignancies. During reactivation, multiple viral and cellular factors are required to ensure progression through the KSHV viral lytic cycle. One of these factors is the viral polymerase processivity factor ORF59. The main functions of ORF59 are to localize the viral polymerase to the nucleus and to aid in polymerase processivity during viral DNA synthesis. Recently, ORF59 has been described to be a multifunctional protein. These functions extend beyond direct involvement in viral DNA synthesis to include regulation of viral gene transcription and modulation of the cellular environment to facilitate viral replication. Among the identified ORF59 binding partners are histones. Our work identified the domains of ORF59 that are required for interaction with histones and also determined that the carboxy-terminus is not required for viral replication. However, while we ascertained the domains required for ORF59 to interact with histones and found that these are also required for viral replication, the functional role for ORF59 and histone interaction remains to be determined. ORF59 is known to interact with a highly abundant viral long non-coding RNA (lnc RNA), polyadenylated nuclear (PAN) RNA. Previous work has suggested that PAN plays a crucial role during replication and also functions in regulation of gene expression and immune response. PAN possess two cis-acting elements, the MRE (Mta-response element) and the ENE (expression and nuclear retention element). While PAN has been shown to be required for viral replication, the function of these elements within PAN remains elusive. In our studies, we focused on determining a functional role of the ENE of PAN during viral replication. Although we found that the ENE is not required for replication or interaction with ORF59, we did confirm that the ENE functions to some extent in retaining PAN to the nucleus. Our work suggests that the cis element that may confer PAN its function is not within the ENE but may in fact be within the MRE of PAN or possibly extend outside the cis elements. In conclusion, these studies demonstrate the complexity in the regulation of KSHV replication and the viral and cellular factors that are crucial to support viral replication which ultimately leads to production of infectious virus and KSHV-associated disease pathologies. Furthermore, characterization of crucial viral factors will contribute to a better understanding of viral replication and the development of novel therapeutics to combat KSHV malignancies.
ENE, KSHV, long non-coding RNA, ORF59, PAN, Processivity Factor
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