Human Cytomegalovirus pUL83 Stimulates Activity of the Viral Immediate-Early Promoter through Its Interaction with the Cellular IFI16 Protein

Abstract

The human cytomegalovirus (HCMV) virion protein pUL83 (also termed pp65) inhibits the expression of interferon-inducible cellular genes. In this work we demonstrate that pUL83 is also important for efficient induction of transcription from the viral major immediate-early promoter. Infection with a mutant virus containing a premature translation termination codon in the UL83 open reading frame (ORF) (UL83Stop) resulted in decreased transcription from the major immediate-early promoter in a time-and multiplicitydependent manner. Expression of pUL83 alone is capable of transactivating the promoter in a reporter assay, and pUL83 associates with the promoter in infected cells. To investigate the mechanism by which the protein regulates the major immediate-early promoter, we utilized a mutant virus expressing an epitope-tagged pUL83 from its own promoter to identify protein binding partners for pUL83 during infection. We identified and confirmed the interaction of pUL83 with cellular IFI16 family members throughout the course of HCMV infection. pUL83 recruits IFI16 to the major immediate-early promoter, and IFI16 binding at the promoter is dependent upon the presence of pUL83. Consistent with the results obtained with the UL83Stop virus, infection of IFI16 knockdown cells with wild-type virus resulted in decreased levels of immediate-early transcripts compared to those of control cells. These data identify a previously unknown role for pUL83 in the initiation of the human cytomegalovirus gene expression cascade.Viral infection is marked by a race between the competing interests of the virus and the host cell. Efficient initiation of viral gene expression is critical to circumvent host defenses aimed at blocking viral gene expression. Human cytomegalovirus (HCMV), a betaherpesvirus encoding nearly 200 predicted proteins (57, 59), has evolved multiple means to evade the initial host cell response to infection. The first viral proteins expressed, the immediate-early proteins, play an important role in this process. Immediate-early proteins are detected in fibroblasts within 4 h of infection and thus are available to function at very early stages in the viral life cycle to block antiviral signaling events. For example, the IE1 protein binds to and inhibits STAT1 and STAT2 (64), two host cell proteins critical for the activation of interferon-inducible gene expression; and IE2 has also been implicated in regulation of transcription of antiviral genes (80). pTRS1 blocks the activation of protein kinase R (PKR), an important regulator of protein translation in response to innate immune signals (16,33,54,85), and pUS3 inhibits antigen presentation by infected cells by sequestering and degrading the major histocompatibility complex (MHC) class I heavy-chain complex (39,48,55).In addition to their role in subverting the host response to viral infection, immediate-early proteins are critical for the induction of viral gene expression. IE1 binds to and inhibits histone deacetylases (HDACs) to ensure a chromatin structure on viral...

Restriction of Human Cytomegalovirus Replication by ISG15, a Host Effector Regulated by cGAS-STING Double-Stranded-DNA Sensing

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Differential Role for Host Translation Factors in Host and Viral Protein Synthesis during Human Cytomegalovirus Infection

“…The abundance of specific mRNAs in total cellular RNA was quantified by reverse transcriptase realtime PCR (qRT-PCR) as previously described (24). Briefly, frozen cell pellets were resuspended in TRIzol (Invitrogen).…”