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...

Human cytomegalovirus DNA is packaged in virions without histones but associates with histones upon reaching the nucleus of an infected cell. Since transcription is modulated by the interplay of histone modifications, we used chromatin immunoprecipitation to detect acetylation and methylation of histone H3 at viral promoters at different times during the viral replication cycle. Histone H3 at immediate-early promoters is acetylated at the start of infection, while it is initially methylated at early and late promoters. Acetylation at immediate-early promoters is dynamic, with a high level of activating modifications at 3 and 6 h postinfection (hpi), followed by a marked reduction at 12 hpi. All viral promoters, as well as nonpromoter regions, are modified with activating acetylations at 24 to 72 hpi. The transient reduction in histone H3 acetylation at the major immediate-early promoter depends on the cis-repressive sequence to which the UL122-coded IE2 protein binds. A mutant virus lacking this element exhibited decreased IE2 binding at the major immediate-early promoter and failed to show reduced acetylation of histone H3 residing at this promoter at 12 hpi. Our results demonstrate that cytomegalovirus chromatin undergoes dynamic, promoter-specific histone modifications early in the infectious cycle, after which the entire chromosome becomes highly acetylated.A high percentage of the population has been infected with human cytomegalovirus (HCMV), the prototypical member of the Betaherpesviridae family, and carries the virus in a latent state. For most immunocompetent people, infection does not cause serious disease. In contrast, primary infection or reactivation in immunocompromised individuals can be associated with serious pathology or mortality (42).HCMV genes, like those of other herpesviruses, are expressed sequentially upon infection of permissive cells (60). The first genes expressed are the immediate-early genes. The major immediate-early promoter (MIEP) controls the production of mRNAs encoding the viral IE1 and IE2 proteins which interact with a variety of cellular proteins to regulate subsequent viral gene expression (7,15,17,34). IE2 also binds DNA directly to modulate transcription (1,8,29,30). The next group to be transcribed are the early genes, some of which are required for viral DNA replication (58). Once viral DNA replication occurs, it leads to the transcription of the late genes; this group includes most of the structural proteins that make up the capsid and the tegument (16).The transcriptional regulation of cellular genes is controlled at the level of chromatin by the interplay among numerous modifications of histones (61). For instance, acetylation of certain lysine residues in histones H4 and H3 is associated with active transcription (9, 41). While histone H4 is acetylated at lysines 5, 8, 12, and 16 (67), it appears that lysine 16 acetylation may be the founding modification event (57). In the case of histone H3, acetylation at lysine 9 and 14 lead to active transcription (33,47). Be...

New world hemorrhagic fever arenaviruses infection of humans results in 15–30% mortality. We performed a high throughput siRNA screen with Junín virus glycoprotein-pseudotyped viruses to find potential host therapeutic targets. Voltage-gated calcium channels (VGCC) subunits, for which there are FDA-approved drugs, were identified in the screen. Knockdown of VGCC subunits or treatment with channel blockers diminished Junín virus-cell fusion and entry into cells and thereby decreased infection. Gabapentin, an FDA-approved drug used to treat neuropathic pain that targets the α2δ2 subunit, inhibited infection of mice by the Candid 1 vaccine strain of the virus. These findings demonstrate that VGCCs play a role in virus infection and have the potential to lead to therapeutic intervention of new world arenavirus infection.

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs.

Junín virus is the causative agent for Argentine hemorrhagic fever, and its natural host is the New World rodent Calomys musculinus. The virus is transmitted to humans by aerosolization, and it is believed that many of the clinical symptoms are caused by cytokines produced by sentinel cells of the immune system. Here we used the Junín virus vaccine strain Candid 1 to determine whether mouse cells could be used to study virus entry and antiviral innate immune responses. We show that Candid 1 can infect and propagate in different mousederived cell lines through a low-pH-dependent, transferrin receptor 1-independent mechanism, suggesting that there is a second entry receptor. In addition, Candid 1 induced expression of the antiviral cytokines tumor necrosis factor alpha and beta interferon in macrophages, and this induction was independent of viral replication. Using Candid 1, as well as virus-like particles bearing the viral glycoprotein, to infect different primary cells and established macrophage cell lines with deletions in the Toll-like receptor (TLR) pathway, we show that TLR2 is a cellular sensor of both the Parodi and Candid 1 viral glycoproteins. Because Junín virus is highly lethal in humans, the use of an experimentally tractable model system, such as the mouse, could provide a better understanding of the antiviral innate cellular responses to Junín virus and the role of these responses in pathogenesis.