ATRX restricts Human Cytomegalovirus (HCMV) viral DNA replication through heterochromatinization and minimizes unpackaged viral genomes.

ATRX limits the accumulation of human cytomegalovirus (HCMV) Immediate Early (IE) proteins at the start of productive, lytic infections, and thus is a part of the cell-intrinsic defenses against infecting viruses. ATRX is a chromatin remodeler and a component of a histone chaperone complex. Therefore, we hypothesized ATRX would inhibit the transcription of HCMV IE genes by increasing viral genome heterochromatinization and decreasing its accessibility. To test this hypothesis, we quantitated viral transcription and genome structure in cells replete with or depleted of ATRX. We found ATRX did indeed limit viral IE transcription, increase viral genome chromatinization, and decrease viral genome accessibility. The inhibitory effects of ATRX extended to Early (E) and Late (L) viral protein accumulation, viral DNA replication, and progeny virion output. However, we found the negative effects of ATRX on HCMV viral DNA replication were independent of its effects on viral IE and E protein accumulation but correlated with viral genome heterochromatinization. Interestingly, the increased number of viral genomes synthesized in ATRX-depleted cells were not efficiently packaged, indicating the ATRX-mediated restriction to HCMV viral DNA replication may benefit productive infection by increasing viral fitness. Our work mechanistically describes the antiviral function of ATRX and introduces a novel, pro-viral role for this protein, perhaps explaining why, unlike during infections with other herpesviruses, it is not directly targeted by a viral countermeasure in HCMV infected cells.

NFκB and Cyclic AMP Response Element Sites Mediate the Valproic Acid and UL138 Responsiveness of the Human Cytomegalovirus Major Immediate Early Enhancer and Promoter.

The major immediate early enhancer and promoter (MIEP) of human cytomegalovirus (HCMV) drives the transcription of the immediate early one (IE1) and IE2 genes, whose encoded proteins stimulate productive, lytic replication. The MIEP is activated by the virally encoded and tegument-delivered pp71 protein at the start of de novo lytic infections of fully differentiated cells. Conversely, the MIEP is silenced at the start of de novo latent infections within incompletely differentiated myeloid cells in part because tegument-delivered pp71 is sequestered in the cytoplasm in these cells, but also by viral factors that repress transcription from this locus, including the UL138 protein. During both modes of infection, MIEP activity can be increased by the histone deacetylase inhibitor valproic acid (VPA); however, UL138 inhibits the VPA-responsiveness of the MIEP. Here, we show that two families of cellular transcription factors, NF-κB and cAMP response element-binding protein (CREB), together control the VPA-mediated activation and UL138-mediated repression of the HCMV MIEP. IMPORTANCE Artificial regulation of the HCMV MIEP, either activation or repression, is an attractive potential means to target the latent reservoirs of virus for which there is currently no available intervention. The MIEP could be repressed to prevent latency reactivation or induced to drive the virus into the lytic stage that is visible to the immune system and inhibited by multiple small-molecule antiviral drugs. Understanding how the MIEP is regulated is a critical part of designing and implementing either strategy. Our revelation here that NF-κB and CREB control the responsiveness of the MIEP to the viral UL138 protein and the FDA-approved drug VPA could help in the formulation and execution of promoter regulatory strategies against latent HCMV.