Murine Cytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral Late Gene Expression.

Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies.

Human Cytomegalovirus Protein pUL38 Prevents Premature Cell Death by Binding to Ubiquitin-Specific Protease 24 and Regulating Iron Metabolism.

Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death.IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases.