We have identified an additional role for pUL83 at the start of HCMV infection. UL83Stop, which contains a translational termination codon in the UL83 ORF, replicates with delayed kinetics (Fig. ). Accumulation of IE1 and IE2 proteins is delayed in UL83Stop-infected cells compared to the wild-type parent (Fig. and ). pUL83 alone is capable of inducing expression from the promoter that controls IE1 and IE2 transcription, the MIEP (Fig. ), consistent with a multiplicity-dependent reduction of IE2 RNA within cells infected by the pUL83-deficient virus (Fig. ). pUL83 binds specifically to the cellular IFI16 protein (Fig. and ) and recruits IFI16 to the MIEP (Fig. ). The pUL83-IFI16 interaction plays an important role in the control of transcription from the MIEP, as cells with reduced IFI16 accumulate reduced levels of IE2 RNA and produce progeny with delayed kinetics following infection with wild-type HCMV (Fig. ). The reduced accumulation of IE2 mirrors that seen during infection with UL83Stop (Fig. ) and supports the view that the delayed kinetics of HCMV replication in IFI16-knockdown cells is at least partly the result of a requirement for pUL83 to recruit IFI16 to the MIEP.
It is noteworthy that a UL83-deficient virus has recently been shown to infect macrophages poorly (20
). The defect was localized to a very early event in the infectious cycle, and the authors observed that the mutant failed to incorporate detectable levels of pUL69 and pUL97 into virions. This study raises the possibility that the lack of additional virion proteins at the start of infection could contribute to the multiplicity-dependent growth defect that we have observed for UL83Stop. In this regard, we have previously demonstrated that a pUL69-deficient virus displays a very early defect after infection of fibroblasts (34
Many herpesviruses have been shown to encode virion proteins that facilitate the expression of viral immediate-early genes in the absence of de novo
viral protein synthesis (17
), so it is not surprising that HCMV encodes several such proteins, including pUL82 (also termed pp71). pUL82 is delivered by the virion to the newly infected cell together with other tegument proteins during the initial stages of infection, and it enhances expression from the MIEP (6
). Previously it has been shown that a deletion mutant lacking the UL83 ORF fails to incorporate pUL82 into the virion (79
), and a delay in expression of the IE1 and IE2 proteins was attributed to the absence of pUL82 at the start of infection. Here we show that a virus which lacks pUL83 in the virion but contains pUL82 (Fig. ) is also defective for immediate-early gene expression. Our work demonstrates that pUL83 directly contributes to active transcription from the MIEP. We present several lines of evidence in support of this conclusion: (i) IE2 transcript levels are reduced following infection with UL83Stop virus compared to wild-type virus (Fig. ); (ii) pUL83 alone induces transcription from a reporter construct controlled by the MIEP in a dose-responsive manner (Fig. ); and (iii) pUL83 is physically associated with the MIEP during the immediate-early phase of infection (Fig. ). Together, these results show that pUL83 regulates transcription from the MIEP in a manner distinguishable from its effects on the availability of pUL82. pUL83 might also directly influence the expression of additional viral promoters, but we have not yet tested this possibility.
Enhancement of MIEP activity by pUL83 also can account for the kinetic replication defect of the UL83Stop mutant (Fig. ). After an initial infection at a low input multiplicity, progeny spread to neighboring cells. However, cells infected in the second and subsequent waves of spread receive higher doses of virus than those that occurred in the initial round of infection, allowing UL83Stop to more efficiently express immediate-early proteins. Therefore, as the infection spreads, the replication defect is ameliorated. While pUL82 clearly plays an important role activating the viral transcriptional program, pUL83 independently contributes to transcription of immediate-early genes by acting directly at the MIEP.
The pUL83 interaction partner, IFI16, is a member of the p200 family of proteins (84
) that was originally identified in a screen for proteins whose expression was increased by interferon (84
). Since that time, it has been recognized that IFI16 acts as a modulator of transcription during cell stress (9
). Indeed, its role in the regulation of both the DNA damage response and the innate immune response indicates that IFI16 is an important regulator of stress responses in general. Murine homologs of IFI16 have recently been shown to bind DNA in response to interferon treatment, consistent with the ability of IFI16 to modulate transcription during cell stress. Its role as a transcription modulator is consistent with the presence of IFI16 together with pUL83 at the HCMV MIEP (Fig. ). pUL83 must be present in order for IFI16 to reside at the MIEP (Fig. ), but we do not know if either protein directly contacts viral DNA or whether their association with the MIEP is bridged by other viral or cellular proteins.
IFI16 from extracts of uninfected HeLa cells has previously been found to bind to the HCMV UL54 promoter (53
), and IFI16 inhibits activity of this viral promoter in a reporter assay performed with uninfected cells (40
). We have not yet tested how IFI16 might influence activity of this early promoter within infected cells in which pUL83 is present.
A p200 protein has previously been shown to play an important role during murine cytomegalovirus (MCMV) infection (35
). Replication of MCMV is decreased in cells containing a dominant negative form of the murine p200 family member p204. The replication defect was found to include delayed immediate-early gene expression (35
), suggesting that regulation of viral immediate-early genes by p200 family members is a conserved feature of cytomegalovirus infection. Given the role of p200 family members in regulating the cellular stress response, it seems likely that viruses must subvert the function of these innate immune sensors to successfully circumvent host cell defenses, and it is possible that additional p200 family members are targeted by HCMV proteins.
IFI16 may affect transcription by modulating the NF-κB pathway (14
). Loss of IFI16 expression resulted in decreased levels of NF-κB DNA binding activity in response to tumor necrosis alpha (TNF-α) treatment (76
). In addition, overexpression of IFI16 resulted in decreased expression of the NF-κB regulatory protein IκBα, independent of IκB kinase (IKK) activation (14
). NF-κB activity is induced by HCMV infection (15
), and infection with a pUL83-deficient virus results in enhanced NF-κB activity, as evidenced by increased nuclear localization of NF-κB subunits and increased NF-κB DNA binding activity (12
). Given the ability of both pUL83 and IFI16 to modulate NF-κB activity, it is tempting to speculate that the regulation of NF-κB activity during HCMV infection results from the interaction of pUL83 with IFI16. The HCMV MIEP contains four NF-κB sites (77
), which have been shown to stimulate the MIEP under some conditions (reference 77
and references therein). Thus, the pUL83-IFI16 complex at the MIEP might act to stimulate transcription through NF-κB.
In addition to the ability of pUL83 to activate the HCMV MIEP described here, pUL83 inhibits the ability of infected cells to respond to interferon and limits the expression of cellular inflammatory genes during the immediate-early phase of HCMV infection (1
) (Fig. ). How does pUL83 simultaneously antagonize expression of inflammatory genes and activate the MIEP? The promoters of inflammatory genes generally include NF-κB binding elements (36
), as does the MIEP (77
). Possibly, then, pUL83 redirects IFI16 from NF-κB at inflammatory genes to the MIEP, or it could modify the activity of IFI16 so that it stimulates NF-κB activity in the context of the MIEP but blocks activity at inflammatory genes. Uncharacterized pUL83 interactions might also contribute to the two seemingly contradictory effects. Whatever the mechanism, pUL83 might hijack a cellular innate protective response, NF-κB activation, simultaneously inducing the expression of HCMV immediate-early proteins and blocking the expression of cellular inflammatory genes.
In summary, we provide multiple lines of evidence supporting the conclusion that the HCMV pUL83 protein enhances transcription from the MIEP during HCMV infection and that this regulation requires the cellular IFI16 protein.