• An interaction between KSHV ORF57 and UIF provides mRNA-adaptor redundancy in herpesvirus intronless mRNA export

      Jackson, B.R.; Boyne, James R.; Noerenberg, M.; Taylor, A.; Hautbergue, G.M.; Walsh, M.J.; Wheat, R.; Blackbourn, D.J.; Wilson, S.A.; Whitehouse, A. (2011)
      The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs.
    • ARID3B: A novel regulator of the Kaposi's sarcoma-associated herpesvirus lytic cycle

      Wood, J.J.; Boyne, James R.; Paulus, C.; Jackson, B.R.; Nevels, M.M.; Whitehouse, A.; Hughes, D.J. (2016-08-10)
      KSHV is the causative agent of commonly fatal malignancies of immuno-compromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic lifecycle – viral latency and the productive lytic cycle, and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein, replication and transcription activator (RTA), is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts on cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied SILAC-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B, ARID3B) whose expression was enhanced by RTA and that relocalised to replication compartments upon lytic reactivation. We also show that siRNA knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation.