Regulation of Epstein-Barr Virus Minor Capsid Protein BORF1 by TRIM5α.

TRIM5α is a host anti-retroviral restriction factor that destroys human immunodeficiency virus (HIV) virions and triggers innate immune signaling. TRIM5α also mediates the autophagic degradation of target proteins via TRIMosome formation. We previously showed that TRIM5α promotes Epstein-Barr virus (EBV) Rta ubiquitination and attenuates EBV lytic progression. In this study, we sought to elucidate whether TRIM5α can interact with and induce the degradation of EBV capsid proteins. Glutathione S-transferase (GST) pulldown and immunoprecipitation assays were conducted to identify interacting proteins, and mutants were generated to investigate key binding domains and ubiquitination sites. Results showed that TRIM5α binds directly with BORF1, an EBV capsid protein with a nuclear localization signal (NLS) that enables the transport of EBV capsid proteins into the host nucleus to facilitate capsid assembly. TRIM5α promotes BORF1 ubiquitination, which requires the surface patch region in the TRIM5α PRY/SPRY domain. TRIM5α expression also decreases the stability of BORF1(6KR), a mutant with all lysine residues mutated to arginine. However, chloroquine treatment restores the stability of BORF1(6KR), suggesting that TRIM5α destabilizes BORF1 via direct recognition of its substrate for autophagic degradation. These results reveal novel insights into the antiviral impact of TRIM5α beyond retroviruses.

Expression of Rta in B Lymphocytes during Epstein-Barr Virus Latency.

Rta of Epstein-Barr virus (EBV) is thought to be expressed only during the lytic cycle to promote the transcription of lytic genes. However, we found that Rta is expressed in EBV-infected B cells during viral latency, at levels detectable by immunoblot analysis. Latent Rta expression cannot be attributed to spontaneous lytic activation, as we observed that more than 90% of Akata, P3HR1, and 721 cells latently infected by EBV express Rta. We further found that Rta is sequestered in the nucleolus during EBV latency through its interaction with MCRS2, a nucleolar protein. When Rta is sequestered in the nucleolus, it no longer activates RNA polymerase II-driven transcription, thus explaining why Rta expression during latency does not transactivate EBV lytic genes. Additional experiments showed that Rta can bind to 18S rRNA and become incorporated into ribosomes, and a transient transfection experiment showed that Rta promotes translation from an mRNA reporter. These findings reveal that Rta has novel functions beyond transcriptional activation during EBV latency and may have interesting implications for the concept of EBV latency.

Rta is an Epstein-Barr virus tegument protein that improves the stability of capsid protein BORF1.

Rta, a key transcription factor expressed by Epstein-Barr virus (EBV), primarily acts to induce activation of the EBV lytic cycle. Interestingly, we observed from an immunogold assay that Rta is also present on the EBV capsid in the host cell nucleus, and a centrifugation study further revealed that Rta cofractionates with EBV virions. Importantly, cofractionated Rta showed similar properties as the EBV tegument protein, BGLF4. Glutathione S-transferase (GST)-pulldown and coimmunoprecipitation assays subsequently demonstrated that Rta directly interacts with the EBV capsid protein, BORF1. Rta was observed to colocalize with BORF1 in the nucleus during EBV lytic induction, and this interaction appears to influence BORF1 stability. Moreover, we found that BORF1 is modified by ubiquitin, and Rta reduces this ubiquitination. These results indicate that Rta may act as an inner tegument protein to improve EBV capsid stability and critical to viral infection.

TRIM5α Promotes Ubiquitination of Rta from Epstein-Barr Virus to Attenuate Lytic Progression.

Replication and transcription activator (Rta), a key protein expressed by Epstein-Barr virus (EBV) during the immediate-early stage of the lytic cycle, is responsible for the activation of viral lytic genes. In this study, GST-pulldown and coimmunoprecipitation assays showed that Rta interacts in vitro and in vivo with TRIM5α, a host factor known to be involved in the restriction of retroviral infections. Confocal microscopy results revealed that Rta colocalizes with TRIM5α in the nucleus during lytic progression. The interaction involves 190 amino acids in the N-terminal of Rta and the RING domain in TRIM5α, and it was further found that TRIM5α acts as an E3 ubiquitin ligase to promote Rta ubiquitination. Overexpression of TRIM5α reduced the transactivating capabilities of Rta, while reducing TRIM5α expression enhanced EBV lytic protein expression and DNA replication. Taken together, these results point to a critical role for TRIM5α in attenuating EBV lytic progression through the targeting of Rta for ubiquitination, and suggest that the restrictive capabilities of TRIM5α may go beyond retroviral infections.

RanBPM regulates Zta-mediated transcriptional activity in Epstein-Barr virus.

Epstein-Barr virus (EBV) expresses two immediate-early proteins, Rta and Zta, which are key transcription factors that can form a complex with MCAF1 at Zta-responsive elements (ZREs) to synergistically activate several viral lytic genes. Our previous research indicated that RanBPM interacts with Rta and enhances Rta sumoylation. Here we showed that RanBPM binds to Zta in vitro and in vivo, and acts as an intermediary protein in Rta-Zta complex formation. The Rta-RanBPM-Zta complex was observed to bind with ZREs in the transcriptional activation of key viral genes, such as BHLF1 and BHRF1, while the introduction of RanBPM short hairpin RNA (shRNA) subsequently reduced the synergistic activity of Zta and Rta. RanBPM was found to enhance Zta-dependent transcriptional activity via the inhibition of Zta sumoylation. Interestingly, Z-K12R, a sumoylation-defective mutant of Zta, demonstrated transcriptional activation capabilities that were stronger than those of Zta and apparently unaffected by RanBPM modulation. Finally, RanBPM silencing inhibited the expression of lytic proteins. Taken together, these results shed light on the mechanisms by which RanBPM regulates Zta-mediated transcriptional activation, and point to an important role for RanBPM in EBV lytic progression.