Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TRdelta4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TRdelta4 displayed a number of defects in early infection processes. Adsorption and entry of TRdelta4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TRdelta4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.     

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.     

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.     

Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication

The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3' untranslated regions (3'-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3'-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.     

Epidemiological studies of HCMV infection in children by restriction analysis of long-PCR products

Sequences of MIE, pol, gB gene longer fragments (2.0-2.6 kb) were amplified in two stage PCR (nested PCR) from 46 PEG-urine extracts. Restriction of particular amplicons with selected endonucleases identified 19 distinct HCMV genotypes: 8 for MIE, 6 for gB and 5 for pol. MIE profiling revealed highest differentiation power. Our data showed the correlation among MIE, pol, and gB genotypes and clinical disease, indicating that genotypes may contribute the course of HCMV infection. For newborns with symptomatic infection, the highest level of variability among three genes analysed was obtained in comparison to samples of newborns with asymptomatic infection. Long PCR technique coupled with restriction analysis may be successfully applied in HCMV genotypes differentiation in samples taken directly from HCMV infected individuals, thus may be useful in prognosis of clinical infection.     

Human cytomegalovirus persistence and latency in endothelial cells and macrophages

Human cytomegalovirus (HCMV) is a clinically significant herpes virus that maintains a lifelong infection in the host. HCMV infection of endothelial cells and macrophages plays an important role in the establishment of latency and persistence, which appears critical for the maintenance of HCMV within the host. HCMV infection is profoundly influenced by endothelial cell origin and the specific pathway of macrophage differentiation. Multiple HCMV genes appear to be involved in enabling virus replication in these two cell types. Although the specific HCMV gene(s) mediating endothelial and macrophage tropism are unclear, a number of genetic determinants required for replication in these two cell types have been identified in the closely related murine cytomegalovirus (MCMV) mouse model, revealing novel mechanisms of virus tropism. This review focuses on recent advances in the understanding of HCMV replication in endothelial cells and macrophages, and the viral determinants that mediate replication in these two important cell types.     

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.     

Early stage of cytomegalovirus infection suppresses host microRNA expression regulation in human fibroblasts

Responses to human cytomegalovirus (HCMV) infection are largely individual and cell type specific. We investigated molecular profiles in 2 primary cell cultures of human fibroblasts, which are highly or marginally sensitive to HCMV infection, respectively. We screened expression of genes and microRNAs (miRs) at the early (3 hours) stage of infection. To assess molecular pathway activation profiles, we applied bioinformatic algorithms OncoFinder and MiRImpact. In both cell types, pathway regulation properties at mRNA and miR levels were markedly different. Surprisingly, in the infected highly sensitive cells, we observed a “freeze” of miR expression profiles compared to uninfected controls. Our results evidence that in the sensitive cells, HCMV blocks intracellular regulation of microRNA expression already at the earliest stage of infection. These data suggest somewhat new functions for HCMV products and demonstrate dependence of miR expression arrest on the host-encoded factors.     

cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.     

Sequence analysis of human cytomegalovirus US28 gene in low-passage clinical isolates from children and AIDS patients

Human cytomegalovirus (HCMV) is often a dangerous opportunistic pathogen that causes significant morbidity and mortality in newborn children and immunocompromised patients. The different symptoms and tissue tropisms of HCMV infection may result from genetic polymorphism. This study investigated the sequence variability of the HCMV US28 ORF, which shows sequence homology to the G protein-coupled receptor. HCMV isolated from suspected pediatric cases and isolates from AIDS patients were compared in order to examine the possible associations between polymorphisms and pathogenesis. Seventy children with suspected congenital HCMV infection, who suffered from jaundice (47), megacolon (10), and microcephaly (13), and 17 AIDS patients, were studied. Mutation was prevalent among the sequences of US28, with a focus on the two ends of US28. The important functional groups of US28 are highly conserved. An unrooted tree showed that all sequences from suspected congenitally infected infants and AIDS patients were divided into three groups. Comparison showed that most of the sequences (12/17) from pediatric patients were included in the first group (G1), whereas most of the sequences (11/17) from AIDS patients were included in the third group (G3). The specific high mutation sites in US28 from children were located at the C terminus of the protein, whereas those from AIDS patients were located at the N terminus. We demonstrated the existence of polymorphisms among the US28 genes of clinical isolates of HCMV from infants with suspected congenital infection. Comparison of US28 sequences from AIDS patients with those from children showed that both sequences have their own specific high mutation points.     

HCMV增殖机制研究进展

人类巨细胞病毒(human cytomegalovirus,HCMV)感染在人群中广泛存在,病毒在感染细胞内增殖会对宿主细胞进行多水平调节和干扰,从而导致各种宏观疾病。本文对HCMV复制周期的基因表达时序、HCMV-DNA复制相关蛋白质及其编码基因、HCMV-DNA复制特点和HCMV必需基因、非必需基因及抑制基因等进行综述。