Cells have a defense against latent human cytomegalovirus, but the viral UL138 protein inactivates it to allow lifelong persistence.

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.

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The UL133-138 locus present in clinical strains of human cytomegalovirus (HCMV) encodes proteins required for latency and reactivation in CD34؉ hematopoietic progenitor cells and virion maturation in endothelial cells. The encoded proteins form multiple homo-and hetero-interactions and localize within secretory membranes. One of these genes, UL136 gene, is expressed as at least five different protein isoforms with overlapping and unique functions. Here we show that another gene from this locus, the UL138 gene, also generates more than one protein isoform. A long form of UL138 (pUL138-L) initiates translation from codon 1, possesses an amino-terminal signal sequence, and is a type one integral membrane protein. Here we identify a short protein isoform (pUL138-S) initiating from codon 16 that displays a subcellular localization similar to that of pUL138-L. Reporter, short-term transcription, and long-term virus production assays revealed that both pUL138-L and pUL138-S are able to suppress major immediate early (IE) gene transcription and the generation of infectious virions in cells in which HCMV latency is studied. The long form appears to be more potent at silencing IE transcription shortly after infection, while the short form seems more potent at restricting progeny virion production at later times, indicating that both isoforms of UL138 likely cooperate to promote HCMV latency. IMPORTANCELatency allows herpesviruses to persist for the lives of their hosts in the face of effective immune control measures for productively infected cells. Controlling latent reservoirs is an attractive antiviral approach complicated by knowledge deficits for how latently infected cells are established, maintained, and reactivated. This is especially true for betaherpesviruses. The functional consequences of HCMV UL138 protein expression during latency include repression of viral IE1 transcription and suppression of virus replication. Here we show that short and long isoforms of UL138 exist and can themselves support latency but may do so in temporally distinct manners. Understanding the complexity of gene expression and its impact on latency is important for considering potential antivirals targeting latent reservoirs. Human cytomegalovirus (HCMV) is a betaherpesvirus that causes birth defects and disease in immunocompromised and immunosuppressed patients and has been associated with cancers, cardiovascular disease, and immune dysfunction (1-3). HCMV productively (lytically) infects differentiated cells, such as fibroblasts, endothelial cells, and epithelial cells, and latently infects incompletely differentiated cells of the myeloid lineage, such as monocytes and CD34 ϩ hematopoietic progenitor cells (HPC) (4, 5). Productive infection in multiple cell types within the human host facilitates viral dissemination, while latency ensures lifelong persistence despite an effective immune response against lyticphase antigens. Periodic reactivations of latent infections into the productive phase perpetuate both lifelong infecti...

Chromatin is the nucleoprotein complex that protects and compacts eukaryotic genomes. It is responsible for a large part of the epigenetic control of transcription. The genomes of DNA viruses such as human cytomegalovirus (HCMV) are devoid of histones within virions but are chromatinized and epigenetically regulated following delivery to the host cell nucleus. How chromatin is initially assembled on viral genomes and which variant forms of the core histone proteins are deposited are incompletely understood. We monitored the deposition of both ectopically expressed and endogenous histones H3.1 and H3.2 (collectively, H3.1/2) and H3.3 during lytic and latent HCMV infections. Here, we show that they are deposited on HCMV genomes during lytic and latent infections, suggesting similar mechanisms of viral chromatin assembly during the different infection types and indicating that both canonical and variant core histones may be important modulators of infecting viral genomes. We further show that association of both H3.1/2 and H3.3 occurs independent of viral DNA synthesis or de novo viral gene expression, implicating cellular factors and/or virion components in the formation of chromatin on virion-delivered genomes during both lytic and latent infections. IMPORTANCEIt is well established that infecting herpesvirus genomes are chromatinized upon entry into the host cell nucleus. Why or how this occurs is a mystery. It is important to know why they are chromatinized in order to better understand cellular pathogen recognition (DNA sensing) pathways and viral fate determinations (lytic or latent) and to anticipate how artificially modulating chromatinization may impact viral infections. It is important to know how the genomes are chromatinized in order to potentially modulate the process for therapeutic effect. Our work showing that HCMV genomes are loaded with canonical and variant H3 histones during both lytic and latent infections strengthens the hypothesis that chromatinization pathways are similar between the two infection types, implicates virion or cellular factors in this process, and exposes the possibility that histone variants, in addition to posttranslational modification, may impact viral gene expression. These revelations are important to understanding and intelligently intervening in herpesvirus infections.T he large genomes of eukaryotic cells must be highly compacted in order to fit within the restricted volume of the nucleus. To accomplish this, eukaryotic DNA is packaged into a repeating nucleoprotein structure known as chromatin, the basic subunit of which is the nucleosome (1, 2). The core nucleosome particle consists of approximately 146 bp of DNA wrapped nearly twice around a histone hetero-octamer consisting of two copies each of the four core histone proteins H2A, H2B, H3, and H4 (3). Nucleosomes are further compacted into higher-order chromatin fibers through the incorporation of the linker histone H1, other nonhistone proteins, and structural RNA components (4-6).Wrapping DNA in nucleoso...