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Human cytomegalovirus (HCMV), a member of the human herpesvirus family, is a common opportunistic virus causing severe ailments and deaths in people with immature or compromised immune systems. UL23 is a virion protein found in the tegument and is expressed in the cytoplasm in HCMV infected cells. However, UL23 is dispensable for viral replication in cultured cells and little is currently known about its function. In order to further study of UL23, polyclonal antibody of UL23 was prepared. UL23 gene fragment was cloned from HCMV Towne by PCR and ligated into pET28a (+). The recombinant plasmid pET28a (+)-UL23 was transformed into E.coli BL21(DE3) to induce expression of the target protein. Then we efficiently purified the recombinant protein affinity chromatography under unique denaturation conditions. Recombinant UL23 protein was used as immunogen to inoculate New Zealand white rabbits and the sera was collected after the fourth immunization. UL23 Polyclonal antibody was purified from antisera using CNBr-activated Sepharose 4 beads. Our UL23 Polyclonal antibody showed specific reaction with UL23 in ELISA, Western-blot and immunofluorescence. More importantly, UL23 Polyclonal antibody could specifically recognize UL23 protein in HCMV infected cells, which laid a foundation for further study of HCMV UL23.

Interferon-γ (IFN-γ) represents one of the most important innate immunity responses in a host to combat infections of many human viruses including human herpesviruses. Human N-myc interactor (Nmi) protein, which has been shown to interact with signal transducer and activator of transcription (STAT) proteins including STAT1, is important for the activation of IFN-γ induced STAT1-dependent transcription of many genes responsible for IFN-γ immune responses. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi. This interaction was identified through a yeast two-hybrid screen and co-immunoprecipitation in human cells. We observed that Nmi, when bound to UL23, was not associated with STAT1, suggesting that UL23 binding of Nmi disrupts the interaction of Nmi with STAT1. In cells overexpressing UL23, we observed (a) significantly reduced levels of Nmi and STAT1 in the nuclei, the sites where these proteins act to induce transcription of IFN-γ stimulated genes, and (b) decreased levels of the induction of the transcription of IFN-γ stimulated genes. UL23-deficient HCMV mutants induced higher transcription of IFN-γ stimulated genes and exhibited lower titers than parental and control revertant viruses expressing functional UL23 in IFN-γ treated cells. Thus, UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ induced responses and an increase of viral resistance to IFN-γ. Our results further highlight the roles of UL23-Nmi interactions in facilitating viral immune escape from IFN-γ responses and enhancing viral resistance to IFN antiviral effects., Author summary Interferon-γ (IFN-γ) responses are vital for a host to combat infections of many human viruses including human herpesviruses. Upon treatment of IFN-γ, transcription of many genes responsible for IFN-γ immune responses is activated primarily by the signal transducer and activator of transcription (STAT) proteins such as STAT1 protein. Human N-myc interactor (Nmi) protein has been shown to interact with STAT proteins including STAT1 and activate IFN-γ induced STAT-dependent transcription. However, no proteins encoded by herpesviruses have been reported to interact with Nmi and inhibit Nmi-mediated activation of IFN-γ immune responses to achieve immune evasion from IFN-γ responses. In this study, we show strong evidence that the UL23 protein of human cytomegalovirus (HCMV), a human herpesvirus, specifically interacts with Nmi protein. UL23 appears to interact directly with Nmi and inhibit nuclear translocation of Nmi and its associated protein STAT1, leading to a decrease of IFN-γ responses and an increase of viral resistance to IFN-γ. Blocking UL23 expression led to higher transcription of IFN-γ stimulated genes and significant inhibition of viral growth in infected cells. These results suggest that interfering with Nmi function may represent an effective mechanism for a herpesvirus to block Nmi-mediated IFN-γ responses and increase viral resistance to IFN-γ. This also provides a potentially new therapeutic strategy to treat HCMV infection by modulating Nmi activity with blocking the expression of a viral protein.