Viral FGARAT Homolog ORF75 of Rhesus Monkey Rhadinovirus Effects Proteasomal Degradation of the ND10 Components SP100 and PML

Abstract

Nuclear domain 10 (ND10) components restrict herpesviral infection, and herpesviruses antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. The rhesus monkey rhadinovirus (RRV) shares many key biological features with the closely related Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) and readily infects cells of both human and rhesus monkey origin. We used the clustered regularly interspaced short palindromic repeatCas9 (CRISPR-Cas9) technique to generate knockout (ko) cells for each of the four ND10 components, PML, SP100, DAXX, and ATRX. These ko cells were analyzed with regard to permissiveness for RRV infection. In addition, we analyzed the fate of the individual ND10 components in infected cells by immunofluorescence and Western blotting. Knockout of the ND10 component DAXX markedly increased RRV infection, while knockout of PML or SP100 had a less pronounced effect. In line with these observations, RRV infection resulted in rapid degradation of SP100, followed by degradation of PML and the loss of ND10 structures, whereas the protein levels of ATRX and DAXX remained constant. Notably, inhibition of the proteasome but not inhibition of de novo gene expression prevented the loss of SP100 and PML in cells that did not support lytic replication, compatible with proteasomal degradation of these ND10 components through the action of a viral tegument protein. Expression of the RRV FGARAT homolog ORF75 was sufficient to effect the loss of SP100 and PML in transfected or transduced cells, implicating ORF75 as the viral effector protein. IMPORTANCEOur findings highlight the antiviral role of ND10 and its individual components and further establish the viral FGARAT homologs of the gammaherpesviruses to be important viral effectors that counteract ND10-instituted intrinsic immunity. Surprisingly, even closely related viruses like KSHV and RRV evolved to use different strategies to evade ND10-mediated restriction. RRV first targets SP100 for degradation and then targets PML with a delayed kinetic, a strategy which clearly differs from that of other gammaherpesviruses. Despite efficient degradation of these two major ND10 components, RRV is still restricted by DAXX, another abundant ND10 component, as evidenced by a marked increase in RRV infection and replication upon knockout of DAXX. Taken together, our findings substantiate PML, SP100, and DAXX as key antiviral proteins, in that the first two are targeted for degradation by RRV and the last one still potently restricts replication of RRV. T he rhesus monkey rhadinovirus (RRV) is a gamma-2-herpesvirus (rhadinovirus) naturally occurring in rhesus macaques (Macaca mulatta) and is closely related to the only human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV) (1). While RRV is usually apathogenic, at least isolate 17577 of RRV has been demonstrated to induce lymphomas and mesenchymal tumors after experimental RRV infection in the context of simian immunodeficiency vir...

EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus

“…Generation of knockout cell pools. Knockout cell pools were generated using the lentiCRISPRv2 system (37) as described previously (38), with the exception that transfection was carried out using polyethylenimine (PEI) MAX (Polysciences) (39). In short, BJAB cells were transduced with lentiviruses harboring the indicated sgRNAs.…”

“…Two monoclonal cell lines per target (nontargeting [sgNT-1] and EphA7 [sgEPHA7-3 and sgEPHA7-4]) were transduced with a lentivirus carrying an EphA7-Strep expression cassette (transcript variant 1 [NM_004440]) (pLenti-CMV-BLAST-EphA7-Strep) or an empty vector control. Lentiviral particles were generated as described previously (38), with the exception that transfection was carried out using PEI. Two days postransduction the selection antibiotic blasticidin (Invivogen) was added to a final concentration of 10 g/ml.…”

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells

“…GAPDH served as an internal RNA loading control. 59 and 34 (lanes 3 and 7) did not express a detectable level of ORF50 protein. These data indicate that lack of ORF57 expression, at least in single-cell clones 6, 2, and 39, was not due to the lack of successful KSHV lytic induction but rather to the specific ORF57 KO from all copies of the virus genome.…”

Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor To Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling

“…Additionally, R12 protein expression during RRV vIRF-KO infection could restore the full disruption and loss of PML-NBs, suggesting a critical role for R12 in this process. A previous publication on PML-NB disruption by RRV implicated the RRV tegument protein encoded by ORF75 and found that while RRV infection of RhF cells resulted in a loss of SP100 protein by 8 hpi, PML protein was not lost until 24 hpi, and this could be rescued with cycloheximide treatment (24). Thus, the inhibition of R12 expression by cycloheximide treatment could explain why PML protein levels were rescued.…”