Felicia D Goodrum Sterling

Viral persistence is the rule following infection with all herpesviruses. The β-herpesvirus, human cytomegalovirus (HCMV), persists through chronic and latent states of infection. Both of these states of infection contribute to HCMV persistence and to the high HCMV seroprevalence worldwide. The chronic infection is poorly defined molecularly, but clinically manifests as low-level virus shedding over extended periods of time and often in the absence of symptoms. Latency requires long-term maintenance of viral genomes in a reversibly quiescent state in the immunocompetent host. In this review, we focus on recent advances in the biology of HCMV persistence, particularly with respect to the latent mode of persistence. Latently infected individuals harbour HCMV genomes in haematopoietic cells and maintain large subsets of HCMV-specific T-cells. In the last few years, impressive advances have been made in understanding virus-host interactions important to HCMV infection, many of which will profoundly impact HCMV persistence. We discuss these advances and their known or potential impact on viral latency. As herpesviruses are met with similar challenges in achieving latency and often employ conserved strategies to persist, we discuss current and future directions of HCMV persistence in the context of the greater body of knowledge regarding α- and γ-herpesviruses persistence.

We have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3' coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3' end of each transcript and is preceded by an extensive 5' sequence (approximately 0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5' ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5' of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.

T cell responses to viruses are initiated and maintained in tissue sites; however, knowledge of human antiviral T cells is largely derived from blood. Cytomegalovirus (CMV) persists in most humans, requires T cell immunity to control, yet tissue immune responses remain undefined. Here, we investigated human CMV-specific T cells, virus persistence and CMV-associated T cell homeostasis in blood, lymphoid, mucosal and secretory tissues of 44 CMV seropositive and 28 seronegative donors. CMV-specific T cells were maintained in distinct distribution patterns, highest in blood, bone marrow (BM), or lymph nodes (LN), with the frequency and function in blood distinct from tissues. CMV genomes were detected predominantly in lung and also in spleen, BM, blood and LN. High frequencies of activated CMV-specific T cells were found in blood and BM samples with low virus detection, whereas in lung, CMV-specific T cells were present along with detectable virus. In LNs, CMV-specific T cells exhibited quiescent phenotypes independent of virus. Overall, T cell differentiation was enhanced in sites of viral persistence with age. Together, our results suggest tissue T cell reservoirs for CMV control shaped by both viral and tissue-intrinsic factors, with global effects on homeostasis of tissue T cells over the lifespan.

Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function.

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.