Regulation of Vaccinia Virus E3 Protein by Small Ubiquitin-Like Modifier Proteins

Kaposi's sarcoma-associated herpesvirus (KSHV), which belongs to the Gammaherpesviridae, typically displays two different phases in its life cycle, the latent phase and the lytic phase. Latency-associated nuclear antigen (LANA), the primary viral product during latency, has been reported to bind to a series of cellular gene promoters to modulate gene transcription. To systemically elucidate the cellular genes regulated by LANA, we identified genome-wide LANA binding sites by chromatin immunoprecipitation coupled with sequencing (ChIP-seq). We stratified ChIP-seq data and found that LANA might be involved in the macromolecule catabolic process. Specifically, we found and verified that LANA could directly bind to the promoter of the SUMO/sentrin-specific peptidase 6 (SENP6) gene in vivo and in vitro. LANA could repress SENP6 promoter activity in a dose-dependent manner in a reporter gene assay. LANA expression was sufficient to inhibit endogenous SENP6 expression at both the RNA and protein levels. Moreover, SENP6 overexpression in KSHV-infected cells reduced LANA at the protein level. Mechanistically, we found that SENP6 could interact with LANA and reduce the formation of sumoylated LANA, which relies on the desumoylation ability of SENP6. During de novo infection, SENP6 overexpression would decrease the abundance of LANA and enhance viral gene expression, which would hamper the establishment of latency. Taken together, these data suggest that KSHV-encoded LANA could inhibit SENP6 expression to regulate the abundance of itself, which may play an important role in controlling the establishment of latency.IMPORTANCE LANA, as a key latent protein produced by KSHV, is responsible for episome persistence and regulates viral reactivation. In the present study, our results demonstrated that LANA could bind to the promoter region of the SENP6 gene and inhibit SENP6 expression while the regulated SENP6 could in turn modulate the abundance of LANA through desumoylation. This delicate regulation may provide important insights to explain the abundance of LANA during KSHV latency.

Section: Discussionmentioning

confidence: 59%

The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Inhibits Expression of SUMO/Sentrin-Specific Peptidase 6 To Facilitate Establishment of Latency

Lin

Sun

Zhang

et al. 2017

“…Again, there are further precedents from other viruses: the W protein of Nipah virus is localized to the nucleus and suppresses the activation of promoters responding to the stimulation of the Toll-like receptor 3 (TLR3) by dsRNA (55). Another property of VACV E3 related to its localization is that it covalently conjugates with small ubiquitin-like modifiers, such as SUMO-1 or SUMO-2 via a SUMOinteracting motif (SIM), and colocalizes with SUMO-1 to subnuclear domains (56). This interaction with SUMO proteins reduces transcriptional transactivation of p53-regulated genes by E3.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0

Tseng

Lin

Cheng

et al. 2015

Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2␣ subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCEThe OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. Orf virus (ORFV), a member of the Parapoxvirus genus and the Poxviridae family, is the causative agent of contagious ecthyma in sheep, goats, and other ruminants. The disease is characterized by the development of pustular lesions around the nostrils and mouth with a high incidence rate and a low mortality rate in healthy adult animals. In contrast, infection in immunosuppressed animals or in lambs may be fatal (1). ORFV is also of concern as a source of zoonotic infection because it can cause cutaneous lesions in humans in contact with infected animals. Persistent infection with ORFV can be observed in goats and sheep, and while the severity of lesions is reduced compared with that seen in primary infection, this persistence suggests that the virus is able to evade host immunity (2-4). In line with this observation, ORFV has been shown to encode several proteins that modulate the host response to infection. These include viral homologues of ovine cytokines, such as vascular endothelial growth factor, interleukin-10 (IL-10), and a granulocyte-macrophage colony-stimulating factor (GM-CSF)-inhibiting protein, as well as an apoptosis inhibitor (5-7). ORFV also antagonizes interferon (IFN) signaling, and this is done by the product...

“…Wild-type E3 exhibited a longer half-life than its SIM mutant. It was speculated that the different stabilities observed between wild-type E3 and the SIM mutant were due to the longer ubiquitin chains conjugated on the SIM mutant (74). SUMOylation stabilizes dengue virus NS5 against proteasome degradation, which supports virus replication (72).…”

Section: Discussionmentioning

confidence: 99%

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

Liu

Zheng

Shu

et al. 2016

Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro. Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCEInfection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro. Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication. S mall ubiquitin-like modifier (SUMO) modification, a member of the group of posttranslational modifications (PTMs), is important for the regulation of many cellular proteins and pathways (1). SUMO is a novel...