Research has shown that RNA virus populations are highly variable, most likely due to low fidelity replication of RNA genomes. It is generally assumed that populations of DNA viruses will be less complex and show reduced variability when compared to RNA viruses. Here, we describe the use of high throughput sequencing for a genome wide study of viral populations from urine samples of neonates with congenital human cytomegalovirus (HCMV) infections. We show that HCMV intrahost genomic variability, both at the nucleotide and amino acid level, is comparable to many RNA viruses, including HIV. Within intrahost populations, we find evidence of selective sweeps that may have resulted from immune-mediated mechanisms. Similarly, genome wide, population genetic analyses suggest that positive selection has contributed to the divergence of the HCMV species from its most recent ancestor. These data provide evidence that HCMV, a virus with a large dsDNA genome, exists as a complex mixture of genome types in humans and offer insights into the evolution of the virus. Human Cytomegalovirus (HCMV) is a dsDNA virus that is the leading source of birth defects associated with an infectious agent. There is currently no effective HCMV vaccine and few treatment strategies for congenital infections exist. Thus, a better understanding of HCMV infections is warranted. Limited data has shown that HCMV exists as a mixture of a few genotypes in human hosts. Here, we describe our use of high throughput sequencing to study the extent of genome wide variability within HCMV infections sampled from congenital infections. Surprisingly, we find that HCMV populations are as variable as quasispecies RNA viruses; it is commonly believed that DNA viruses are more genetically stable than RNA viruses, and thus produce homogenous populations. Additionally, we find evidence of evolutionary pressures acting on the HCMV genome, both within and among populations. These results provide the first evidence that diversity of DNA virus populations can be comparable to that of RNA virus populations.

The stringent regulation of cell cycle progression helps to maintain genetic stability in cells. MicroRNAs (miRNAs) are critical regulators of gene expression in diverse cellular pathways, including developmental patterning, hematopoietic differentiation and antiviral defense. Here, we show that two c-Myc-regulated miRNAs, miR-17 and miR-20a, govern the transition through G1 in normal diploid human cells. Inhibition of these miRNAs leads to a G1 checkpoint due to an accumulation of DNA double-strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. Surprisingly, gross changes in E2F1 levels were not required to initiate the DNA damage response and checkpoint, as these responses could occur with a less than twofold change in E2F1 protein levels. Instead, our findings indicate that the precise timing of E2F1 expression dictates S-phase entry and that accurate timing of E2F1 accumulation requires converging signals from the Rb/E2F pathway and the c-Myc-regulated miR-17 and miR-20a miRNAs to circumvent a G1 checkpoint arising from the untimely accumulation of E2F1. These data provide a mechanistic view of miRNA-based regulation of E2F1 in the context of the emerging model that miRNAs coordinate the timing of cell cycle progression.

Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ∼88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (>90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13–18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting ≥34 of the 167 open reading frames (∼20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The application of population genetic methods to these data provides clinically useful information, such as the timing of infection and compartment colonization. The large, dsDNA virus Human cytomegalovirus (HCMV) is the most genetically complex viral pathogen of humans. HCMV populations are highly variable, which may allow the virus to evolve in human hosts on short timescales. We tested this hypothesis by longitudinally sampling HCMV populations from the urine and/or plasma of congenitally infected infants. We found that HCMV is generally stable within a compartment, but rapidly evolves when crossing host compartments. In fact, HCMV sampled from two compartments of the same host is as different as HCMV collected from unrelated hosts. We used mathematical modeling and population genetic analysis to show that both a bottleneck (i.e., a reduction in population size) associated with compartment colonization as well as positive selection are necessary to explain the observed differences between compartments. We also conclude from these data that fetal infection in these patients occurred between 13–18 weeks gestational age, consistent with the timing of symptomatic congenital HCMV infections. This study is the most detailed investigation of DNA virus evolution in human hosts to date, provides a framework for the study of other viral infections using similar techniques, and will aid in the development of new antiviral therapies and vaccines.

Significance: Identifying cell-surface receptors required for viral infection is important for developing antiviral therapies and effective vaccines. Human cytomegalovirus (HCMV) infects epithelial cells during interhost transmission. The pentameric protein complex (PC) was identified as being required for infection of this clinically relevant cell type. The majority of neutralizing antibody responses are directed against the PC. Here we describe an HCMV receptor, OR14I1, which binds to the PC and defines epithelial tropism. Importantly, we identified a peptide representing the N terminus of OR14I1 that blocks HCMV infection of epithelial cells. Moreover, an adenylate cyclase signaling pathway functioning downstream of OR14I1 contributes to HCMV infection and endocytosis. These findings reveal information useful for the development of new anti-HCMV therapies and vaccines.

Significance: Human cytomegalovirus (HCMV) is the leading cause of birth defects associated with infections and a leading cause of transplantation failure. This study reveals the patterns and limits of HCMV genomic diversity by performing a large-scale analysis of HCMV sequences sampled from human hosts, identifying the hot and cold spots of variability. We find that the diversity is unevenly distributed across three host compartments and show that HCMV populations of vascular compartments are genetically constrained while enriched for polymorphisms of glycoproteins and regulatory proteins. This work significantly advances our understanding of the genomic diversity of HCMV in humans and has clear implications for the development of therapeutics against HCMV.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in several disorders, including an association between HCMV reactivation and the overproliferation of arterial smooth muscle cells observed in restenosis. Although HCMV can mediate a growth-arrest phenotype in infected cells, the virus can also promote an environment conducive to proliferation. Here, we present evidence that the HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, may be responsible for inducing this proliferative environment by altering cell cycle control. We find that expression of either of these IE proteins can alter the cell cycle distribution of randomly cycling cells towards S and G ₂ /M phases. Additionally, we find that expression of IE2-86, but not IE1-72, induces quiescent cells into S phase and delays cell cycle exit. In the absence of p53, IE1-72 expression can induce S phase and delay cell cycle exit. We also demonstrate that p53 protein levels increase in fibroblasts following the expression of IE1-72. The observed accumulation of p53 protein in IE1-72-expressing cells may account for the inability of IE1-72 to induce S phase and delay cell cycle exit. Our data suggest that expression of HCMV IE1-72 and IE2-86 is sufficient to alter the cell cycle to generate an environment conducive to proliferation.