A screen for gene regulators encoded by Kaposi’s sarcoma-associated herpesvirus uncovers a novel role for the ORF42 protein in regulating the production of cellular and viral proteins

Abstract

17The tight control of viral and host gene expression is critical to the replication of herpesviruses, 18 including the gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV). While 19 some of the KSHV proteins that contribute to viral and host gene regulation are known, it is clear 20 that there are additional uncharacterized contributing viral factors. Identifying these proteins and 21 their role in gene regulation is important to determine the mechanistic underpinnings of the 22 complex replication cycle of KSHV. Through a reporter-based screen, we have identified several 23 new potential KSHV-encoded gene regulators, including the previously uncharacterized protein 24 ORF42, which we find stimulates global protein production upon overexpression. We have 25 generated an ORF42-null virus, which revealed that ORF42 is required for wild-type levels of 26 virus production. Moreover, global protein synthesis and the accumulation of viral proteins are 27 reduced in infected cells in the absence of ORF42, suggesting that ORF42 regulates protein 28 synthesis during infection. A comparison of the effects of ORF42 on the levels of RNA and 29 protein suggests that ORF42 acts post-transcriptionally to control protein levels. In addition to 30 gene regulation, ORF42 may have other functions in virion formation, as it is found in viral 31 particles, which is consistent with the described roles of the ORF42 homologs in alpha-and beta-32 herpesviruses. 33 3 Importance 34 Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, an AIDS-associated 35 malignancy that remains one of the leading causes of cancer deaths in sub-Saharan Africa. 36 Replication of the virus is important for tumor formation and inhibition of viral replication may 37 be used for treatment. The correct levels and temporal expression of viral and host genes during 38 KSHV replication are key to viral replication, but the mechanisms that control this regulation 39 remain enigmatic. Here we identify several new KSHV proteins involved in viral and cellular 40 gene regulation and characterize the previously unstudied KSHV ORF42 protein in regulation of 41 viral and host protein levels and efficient formation of viral progeny. 42 4 Introduction 43