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

Abstract

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 β- and γ-herpesviruses, but not in α-herpesviruses. Here, we performed coimmunopreciptation 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 the pM91-pM79 interaction is essential for viral late gene expression. 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 they 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 how CMV regulates late gene expression, and have important implications for the design of antiviral strategies.

The interaction between ORF18 and ORF30 is required for late gene expression in Kaposi’s sarcoma-associated herpesvirus

“…Elucidating the architecture of the six-member vPIC complex is central to understanding the mechanism underlying viral late gene expression in beta- and gammaherpesviruses. Although their functions are largely unknown, each of these viral transcription regulators is essential for late gene promoter activation and evidence increasingly suggests that their ability to form a complex is crucial for transcriptional activity (9, 10, 19). Here, we reveal that selective disruption of an individual protein-protein contact between KSHV ORF18 and ORF30 within the vPIC is sufficient to abrogate K8.1 late gene expression and virion production in infected cells, emphasizing the sensitivity of the complex to organizational perturbation.…”

“…The observation that the majority of the ORF18 point mutations that disrupt the interaction with ORF30 do not affect its binding to ORFs 31, 34, and 66 indicates that these mutations do not significantly alter the overall folding or structure of ORF18. In MCMV, the organization of the vTA complex is similar to KSHV, except that pM92 (homologous to KSHV ORF31) interacts with pM87 (homologous to KSHV ORF24), whereas in KSHV this interaction is bridged through ORF34 (19). Our data complement recent findings in MCMV, in which mutations of the ORF30 homolog (pM91) that perturb the interaction with the ORF18 homolog (pM79) similarly cause a defect in the expression of late genes (19).…”

“…In MCMV, the organization of the vTA complex is similar to KSHV, except that pM92 (homologous to KSHV ORF31) interacts with pM87 (homologous to KSHV ORF24), whereas in KSHV this interaction is bridged through ORF34 (19). Our data complement recent findings in MCMV, in which mutations of the ORF30 homolog (pM91) that perturb the interaction with the ORF18 homolog (pM79) similarly cause a defect in the expression of late genes (19). Thus, mutations that disrupt the ORF18-ORF30 protein-protein interface in either protein cause the same phenotype.…”

“…Studies in both beta-and gammaherpesviruses indicate that the vTAs form a complex, the general organization of which has been mapped in MCMV and KSHV (7, 9, 10, 19) (Figure 1A). Notably, several recent reports demonstrate that specific interactions between the vTAs are critical for late gene transcription.…”

Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

“…Stop mutants of five of the six KSHV vTAs have been generated and tested, revealing that they share a common phenotype in which late gene transcription fails to occur, ultimately preventing release of infectious virions (6, 8-10). Disrupting protein-protein contacts within the complex also prevents late gene transcription, as preventing the incorporation of even the smallest vPIC component, ORF30 (77 amino acids) by disrupting its interaction with its binding partner ORF18 completely prevents late gene transcription in both KSHV and HCMV (11, 12). Although deletion of KSHV ORF66 has not been tested, an EBV mutant lacking the ORF66 homolog (BFRF2) fails to produce infectious virions due to a defect in late gene transcription (4).…”