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The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication. Author summary: The influenza A virus (IAV) protein, NS2, plays a critical role in virus replication, as the abundance of NS2 influences viral transcription, viral RNA (vRNA) synthesis, and viral ribonucleoprotein (vRNP) export from the nucleus. Interestingly, high levels of NS2 are known to inhibit IAV transcription yet promote vRNA synthesis. In this study, we found that ubiquitination destabilizes NS2, and a host deubiquitinase, OTUB1, was shown to deubiquitinate and stabilize NS2, thus contributing to NS2 accumulation. OTUB1 has previously been reported to be a negative regulator of host cellular antiviral defense, and its upregulation enhances virion production. Our study sheds additional light on the role of OTUB1 during IAV infection which can now be considered for the development of anti-influenza strategies. [ABSTRACT FROM AUTHOR]

The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, which are involved in the transcriptional activation of EBV lytic genes. This study sought to elucidate the mechanism by which Rta activates transcription of the Zta-encoding gene, BZLF1, through the ZII element in the gene promoter. In a DNA affinity precipitation assay, ATF2 was found to associate with an Rta-interacting protein, MCAF1, at the ZII element. The interaction between Rta, MCAF1, and ATF2 at the same site in the ZII region was further verified in vivo by chromatin immunoprecipitation assay. The complex appears to be crucial for the activation of BZLF1 transcription, as the overexpression of two ATF2-dominant negative mutants, or the introduction of MCAF1 siRNA into 293T cells, were both found to substantially reduce Rta-mediated transcription levels of BZLF1. Moreover, this study also found that the Rta-MCAF1-ATF2 complex binds to a typical AP-1 binding sequence on the promoter of BMRF2, a key viral gene for EBV infection. Mutation of this sequence decreased Rta-mediated promoter activity significantly. Taken together, these results indicate a critical role for MCAF1 in AP-1-dependent Rta activation of BZLF1 transcription. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes. This protein usually binds to Rta-response elements or interacts with Sp1 or Zta via a mediator protein, MCAF1, to activate transcription. Rta is also known to interact with TBP and TFIIB to activate transcription. This study finds that Rta interacts with TAF4, a component of TFIID complex, in vitro and in vivo, and on the TATA sequence in the BcLF1 promoter. Rta also interacts with TAF4 and Sp1 on Sp1-binding sequences on TATA-less promoters, including those of BNLF1, BALF5, and the human androgen receptor. These interactions are important to the transcriptional activation of these genes by Rta since introducing TAF4 shRNA substantially reduces the ability of Rta to activate these promoters. This investigation reveals how Rta interacts with TFIID to stimulate transcription.