Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein

Abstract

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.

Different capsid-binding patterns of the β-herpesvirus-specific tegument protein pp150 (pM32/pUL32) in murine and human cytomegaloviruses

“…The observations that ΔM32 grew in NIH3T3 cells indicate that M32 is not required for MCMV replication in vitro . Thus, in contrast to its HCMV homolog pUL32, which is essential for HCMV replication (29, 31), M32 is not essential—though important—for MCMV replication in vitro .…”

“…To generate ΔM32, we adopted our previously-published protocols for generating gene-deletion mutants of HCMV to mutagenize a BAC clone of the wild-type MCMV (Smith strain) genome (MCMV BAC ) by deleting the M32 open reading frame (28-30). Furthermore, rescued viral mutant, R-M32, was generated from ΔM32, by restoring the M32 sequence, following the procedures as described previously (29, 31). Deletion of the M32 gene in ΔM32 and the restoration of the M32 sequence in R-M32 were confirmed by PCR and Southern blot analysis.…”

“…Mutant ΔM32, which contained a deletion of the entire coding sequence of M32, was derived from MCMV BAC using a two-step mutagenesis protocol (29, 31). In the first step, we inserted at the M32 sequence with a cassette (tet/str) containing the tetracycline resistance gene tetA and rpsL gene conferring streptomycin susceptibility, following the mutagenesis procedures as described previously (29, 31). The bacteria harboring the mutant BAC constructs were electroporated with the PCR-amplified tet/str cassette.…”

Repurposing approved drugs on the pathway to novel therapies

“…However, many viruses are not sensitive to the IFN response or produce factors that interfere with IFN and interferon‐stimulated genes (ISGs) . As IFN levels in uninfected individuals are typically vanishingly low, the high persistent presence of IFNs and/or their induced genes (ISGs) may also complicate or even cause other diseases, such as lupus or atopic dermatitis.…”

Viral gene drive in herpesviruses

“…UL23, the viral gene targeted by our drive, was initially thought to be dispensable for hCMV replication in fibroblasts (13,18). Serendipitously, it was later showed that UL23 is a tegument protein that blocks antiviral interferon-γ (IFN-γ) responses by interacting with human N-myc interactor (Nmi) protein (19). As a result, the growth of UL23-knockout viruses is severely inhibited in infected cells treated with IFN-γ.…”