Global Mapping of Herpesvirus-Host Protein Complexes Reveals a Transcription Strategy for Late Genes

The ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific immediate-early tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other copurified proteins, including prominent ones of ϳ38 kDa and ϳ130 kDa. Mass spectrometry revealed that the 38-kDa protein is viral ORF33 and the 130-kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl-terminal 19 amino acids (aa) of ORF45 and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7 both under transfected conditions and in KSHV-infected cells. Moreover, we noticed ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation that was abolished if either the ORF33-or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45 interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high-molecular-weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference with this interaction may represent a novel strategy to inhibit KSHV lytic replication. K aposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, the most common malignancy in HIV/AIDS patients. It is also associated with two lymphoproliferative disorders: primary effusion lymphoma and multicentric Castleman's disease (1-3). Like other herpesviruses, KSHV exhibits two alternative life cycles, a quiescent latent stage and a productiv...

Section: Discussionsupporting

confidence: 45%

A Survey of the Interactome of Kaposi's Sarcoma-Associated Herpesvirus ORF45 Revealed Its Binding to Viral ORF33 and Cellular USP7, Resulting in Stabilization of ORF33 That Is Required for Production of Progeny Viruses

Gillen

Liang

et al. 2015

“…n betaherpesviruses and gammaherpesviruses, mapped promoters driving late gene transcription are strikingly minimal and their activation is dependent on a set of conserved viral transcription regulatory proteins (1)(2)(3)(4)(5)(6)(7)(8). Studies of the gammaherpesviruses murine gammaherpesvirus 68 (MHV68), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV), as well as the betaherpesvirus human cytomegalovirus (HCMV), identified six viral transactivation factors (vTFs), open reading frame 18 (ORF18), ORF24, ORF30, ORF31, ORF34, and ORF66 (gammaherpesvirus nomenclature) necessary for late gene expression (1-4, 6, 7, 9, 10).…”

mentioning

confidence: 99%

“…Studies of the gammaherpesviruses murine gammaherpesvirus 68 (MHV68), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV), as well as the betaherpesvirus human cytomegalovirus (HCMV), identified six viral transactivation factors (vTFs), open reading frame 18 (ORF18), ORF24, ORF30, ORF31, ORF34, and ORF66 (gammaherpesvirus nomenclature) necessary for late gene expression (1-4, 6, 7, 9, 10). ORF24 is a TATA box-binding protein (TBP) homolog that recruits RNA polymerase II (RNAP II) (2,11), and HCMV UL79 (ORF18 ortholog) contributes to RNAP II elongation (10). However, little is known about the roles of the other individual vTFs or how they are organized to form the late gene transcription preinitiation complex (vPIC).…”

mentioning

confidence: 99%

Interaction between ORF24 and ORF34 in the Kaposi's Sarcoma-Associated Herpesvirus Late Gene Transcription Factor Complex Is Essential for Viral Late Gene Expression

Davis

Hesser

Park

et al. 2016

Self Cite

Transcription of herpesviral late genes is stimulated after the onset of viral DNA replication but otherwise restricted. Late gene expression in gammaherpesviruses requires the coordination of six early viral proteins, termed viral transactivation factors (vTFs). Here, we mapped the organization of this protein complex for Kaposi's sarcoma-associated herpesvirus. Disruption of this complex via point mutation of the interaction interface between the open reading frame 24 (ORF24) and ORF34 vTFs ablated both late gene expression and viral replication.

“…The mechanisms regulating this noncanonical TATA box in betaherpesviruses and gammaherpesviruses have remained elusive, but accumulating evidence indicates that BcRF1 (or its homologs) binds to the noncanonical TATA box (10,12,23), providing specificity to the L promoters. Some other components of the complex are reported to interact with and recruit RNA polymerase II (Pol II) to enhance the transcriptional start of L genes (17,19).…”

mentioning

confidence: 99%

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Watanabe

Narita

Yoshida

et al. 2015

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression. Epstein-Barr virus (EBV) is a human gammaherpesvirus that is present in Ͼ90% the world's population. EBV is associated with infectious mononucleosis and neoplasms such as Burkitt lymphoma, Hodgkin's lymphoma, posttransplant lymphoproliferative disease, nasopharyngeal carcinoma, and gastric carcinoma (1, 2).The EBV BDLF4 gene is an open reading frame (ORF), located between BGLF1 and BDLF3.5. Sequence similarities suggest that it is conserved among the members of the betaherpesvirus and gammaherpesvirus subfamilies (UL92 of human cytomegalovirus [HCMV] and ORF31 of murine gammaherpesvirus 68 and Kaposi's sarcoma-associated herpesvirus [KSHV]) but that it is not present in the alphaherpesviruses. It has been reported that KSHV ORF31, MHV-68 ORF31, HCMV UL92, and MCMV M92 are required for the efficient transcription of viral true late genes (3-7), but the EBV BDLF4 has not been characterized well.BDLF4 expression in B95-8 cells with early kinetics. We first prepared antiserum against EBV BDLF4 by immunizing rabbits with a glutathione S-transferase (GST)-fused BDLF4 protein expressed in bacteria. This anti-BDLF4 antiserum reacted with a protein of about ϳ22 kDa in the lysate from B95-8 cells, treated with tetradecanoyl phorbol acetate (TPA), A23187, and sodium butyrate (T/A/B) for 48 h (Fig. 1A). The BDLF4 protein could be detected readily by 24 h, and expression was increased at 48 h (Fig. 1B). While BALF4 mRNA expression was clearly blocked by phosphonoacetic acid (PAA), the amount of BDLF4 was decreased only slightly by the inhibitor (Fig. 1C), suggesting that the BDLF4 gene is expressed with E kinetics.Generation of BDLF4 knockout virus by frameshifting. To study the biological role of the BDLF4 gene in EBV lytic replication, we constructed a BDLF4-deficient recombinant virus and its revertant, as depicted in Fig. 2A. The integrity of the viral genomes was examined by restriction enzyme analysis, followed by agarose electrophoresis (Fig. 2B), and sequencing confirmed the intended one-nucleotide (nt) deletion.Knockout of BDLF4 decreased viral L gene expression and progeny production. The recombinant EBV-bacterial artificial chromosome (BAC) DNAs were transfected into HEK293 cells, and cells stably and latently containing EBV DNAs were cloned by hygromycin selection. We found that viral DNA synthesis levels of the wild-type, frameshift mutant, and revertant viruses were comparable (Fig. 3A). Disruption of BDLF4 decreased virus yield to approximately 10% to 20% compared with levels seen with the wild-type and revertant viruses (Fig. 3B)...