Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies. Whether excessive lytic EBV infection increases the risk of EBV-induced cancers is not clear. A particular variant (Zp-V3) of the viral promoter driving expression of the EBV immediate-early BZLF1 (Z) protein that mediates lytic viral reactivation has been reported to be over-represented (relative to the prototype Zp-P form of the promoter) in certain EBV-positive malignancies, but no functional difference between the two promoter variants has been reported. Here we show that the malignancy-associated Zp-V3 variant (but not the Zp-P variant) contains a binding site for the cellular NFATc1 (nuclear factor of activated T cells c1) transcription factor that allows it to be activated by NFATc1-inducing stimuli such as B-cell receptor stimulation. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV genome greatly enhances lytic viral reactivation in response to the NFATc1-inducing stimuli. We also find that the Zp-V3 variant is over-represented in EBV-positive Burkitt lymphomas and gastric carcinomas, and in lymphoblastoid cell lines transformed by EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These findings suggest that the Zp-V3 version of the EBV BZLF1 promoter increases the likelihood of EBV-induced malignancies by increasing lytic EBV infection.

The human cytomegalovirus pp71 protein is packaged within the tegument of infectious virions and performs multiple functions in host cells to prime them for productive, lytic replication. Here we review the known and hypothesized functions of pp71 in regulating proteolysis, infection outcome (lytic or latent), histone deposition, transcription, translation, immune evasion, cell cycle progression, and pathogenesis. We also highlight recent advances in CMV-based vaccine candidates informed by an improved understanding pp71 function.

Significance: Until now, it was not known if, how, or why pathogenic human viruses might modulate the de novo production of the replication-dependent (RD) histone proteins that decorate their DNA genomes within infected cells. Our finding that human cytomegalovirus (HCMV) inhibits RD histone production affirms that a virus targets this fundamental cellular process. Furthermore, our revelation that HCMV induces, relocalizes, and then commandeers the stem loop–binding protein (SLBP) for a purpose other than RD histone synthesis to support productive replication illuminates the potential for other functions of this highly conserved protein. The critical nature of SLBP for HCMV infection and of RD histone synthesis for cellular DNA replication highlights this process as a target for future antiviral and chemotherapeutic interventions.

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

Repression of human cytomegalovirus (HCMV) immediate-early (IE) gene expression is a key regulatory step in the establishment and maintenance of latent reservoirs. Viral IE transcription and protein accumulation can be elevated during latency by treatment with histone deacetylase inhibitors such as valproic acid (VPA), rendering infected cells visible to adaptive immune responses. However, the latency-associated viral protein UL138 inhibits the ability of VPA to enhance IE gene expression during infection of incompletely differentiated myeloid cells that support latency. UL138 also limits the accumulation of IFNβ transcripts by inhibiting the cGAS-STING-TBK1 DNA-sensing pathway. Here, we show that, in the absence of UL138, the cGAS-STING-TBK1 pathway promotes both IFNβ accumulation and VPA-responsive IE gene expression in incompletely differentiated myeloid cells. Inactivation of this pathway by either genetic or pharmacological inhibition phenocopied UL138 expression and reduced VPA-responsive IE transcript and protein accumulation. This work reveals a link between cytoplasmic pathogen sensing and epigenetic control of viral lytic phase transcription and suggests that manipulation of pattern recognition receptor signaling pathways could aid in the refinement of MIEP regulatory strategies to target latent viral reservoirs.