Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, largely manifested as central nervous system (CNS) disorders. The principal site of manifestations in the mouse model is the fetal brain's neural progenitor cell (NPC)-rich subventricular zone. Our previous human NPC studies found these cells to be fully permissive for HCMV and a useful in vitro model system. In continuing work, we observed that under culture conditions favoring maintenance of multipotency, infection caused NPCs to quickly and abnormally differentiate. This phenotypic change required active viral transcription. Whole-genome expression analysis found rapid downregulation of genes that maintain multipotency and establish NPCs' neural identity. Quantitative PCR, Western blot, and immunofluorescence assays confirmed that the mRNA and protein levels of four hallmark NPC proteins (nestin, doublecortin, sex-determining homeobox 2, and glial fibrillary acidic protein) were decreased by HCMV infection. The decreases required active viral replication and were due, at least in part, to proteasomal degradation. Our results suggest that HCMV infection causes in utero CNS defects by inducing both premature and abnormal differentiation of NPCs.Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily affecting the central nervous system (CNS). Primary infection during pregnancy poses a 30 to 40% risk of intrauterine transmission, with severe adverse outcomes more likely if the infection occurs within the first half of gestation (46). Each year, approximately 1% of all newborns are congenitally infected with HCMV. Approximately 5 to 10% of these infants manifest signs of serious neurological defects at birth, including deafness, mental retardation, blindness, microencephaly, hydrocephalus, and cerebral calcification (2,4,65). In addition, 10 to 15% of congenitally infected infants who are asymptomatic at birth subsequently develop brain disorders such as sensorineural hearing loss (12, 47, 52). Moreover, accumulating evidence suggests that more subtle changes in human brain development, such as autism and language development, may be related to congenital HCMV infection (68,76,77).Although HCMV can infect a wide range of tissues in vivo (61), the fetal brain is the principal site of the deleterious manifestations of infection. It has been suggested that the severity of neuropathological changes and clinical outcomes may be associated with the stage of CNS development at which congenital infection occurs, with early-gestation infections producing more severe outcomes (3, 46). However, the mechanism of HCMV pathogenesis in the developing CNS remains poorly understood. Studies of HCMV in human subjects have obvious limitations; therefore, model systems of both in vitro and in vivo HCMV infections have been devised to provide insights into infection of the developing brain.Congenital infection studies have been performed principally with the mouse model. Studies of mice revealed that very ea...

Human cytomegalovirus (HCMV) infection stimulates cellular DNA synthesis and causes chromosomal damage. Because such events likely affect cellular proliferation, we investigated the impact of HCMV infection on key components of the cell cycle. Early after infection, HCMV induced elevated levels of cyclin E, cyclin E-associated kinase activity, and two tumor suppressor proteins, p53 and the retinoblastoma gene product (Rb). The steady-state concentration of Rb continued to rise throughout the infection, with most of the protein remaining in the highly phosphorylated form. At early times, HCMV infection also induced cyclin B accumulation, which was associated with a significant increase in mitosis-promoting factor activity as the infection progressed. In contrast, the levels of cyclin A and cyclin A-associated kinase activity increased only at late times in the infection, and the kinetics were delayed relative to those for cyclins E and B. Analysis of the cellular DNA content in the infected cells by flow cytometry showed a progressive shift of the cells from the G 1 to the S and G 2 /M phases of the cell cycle, leading to an accumulation of aneuploid cells at late times. We propose that these HCMV-mediated perturbations result in cell cycle arrest in G 2 /M.

Infection of primary fibroblasts with human cytomegalovirus (HCMV) causes a rapid stabilization of the cellular protein p53. p53 is a major effector of the cellular damage response, and activation of this transcription factor can lead either to cell cycle arrest or to apoptosis. Viruses employ many tactics to avoid p53-mediated effects. One method HCMV uses to counteract p53 is sequestration into its viral replication centers. In order to determine whether or not HCMV benefits from this sequestration, we infected a p53 ؊/؊ fibroblast line. We find that although these cells are permissive for viral infection, several parameters are substantially altered compared to wild-type (wt) fibroblasts.

Congenital human cytomegalovirus (HCMV) infection causes central nervous system structural abnormalities and functional disorders, affecting both astroglia and neurons with a pathogenesis that is only marginally understood. To better understand HCMV's interactions with such clinically important cell types, we utilized neural progenitor cells (NPCs) derived from neonatal autopsy tissue, which can be differentiated down either glial or neuronal pathways. Studies were performed using two viral isolates, Towne (laboratory adapted) and TR (a clinical strain), at a multiplicity of infection of 3. NPCs were fully permissive for both strains, expressing the full range of viral antigens (Ags) and producing relatively large numbers of infectious virions. NPCs infected with TR showed delayed development of cytopathic effects (CPE) and replication centers and shed less virus. This pattern of delay for TR infections held true for all cell types tested. Differentiation of NPCs was carried out for 21 days to obtain either astroglia (>95% GFAP ؉ ) or a 1:5 mixed neuron/astroglia population (␤-tubulin III ؉ /GFAP ؉ ). We found that both of these differentiated populations were fully permissive for HCMV infection and produced substantial numbers of infectious virions. Utilizing a difference in plating efficiencies, we were able to enrich the neuron population to ϳ80% ␤-tubulin III ؉ cells. These ␤-tubulin III ؉ -enriched populations remained fully permissive for infection but were very slow to develop CPE. These infected enriched neurons survived longer than either NPCs or astroglia, and a small proportion were alive until at least 14 days postinfection. These surviving cells were all ␤-tubulin III ؉ and showed viral Ag expression. Surprisingly, some cells still exhibited extended processes, similar to mock-infected neurons. Our findings strongly suggest neurons as reservoirs for HCMV within the developing brain.Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that is the most common cause of virus-induced birth defects. Primary infection during pregnancy poses a 30 to 40% risk of intrauterine transmission, with adverse outcomes more likely if the infection is within the first half of the gestation period (37). Each year, about 1% of all newborns are congenitally infected, and 5 to 10% of these infants manifest signs of serious neurological defects, including deafness, mental retardation, blindness, microencephaly, hydrocephalus, and cerebral calcification (1, 3, 37). An additional 10% of congenitally infected infants are asymptomatic at birth and subsequently develop brain disorders, the most common of which is sensorineural hearing loss (7, 27). Thus, in the United States, some 8,000 children annually suffer the obvious consequences of congenital HCMV infection. Additionally, recent investigations have suggested that more subtle abnormal changes in human brain development, such as autism (with an incidence of 1:200), may in some cases be related to congenital HCMV infection (38,43).Although HCMV has a wide range of ...

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Many viruses (herpes simplex virus type 1, polyomavirus, and human immunodeficiency virus type 1) require the activation of ataxia telangiectasia mutated protein (ATM) and/or Mre11 for a fully permissive infection. However, the longer life cycle of human cytomegalovirus (HCMV) may require more specific interactions with the DNA repair machinery to maximize viral replication. A prototypical damage response to the double-stranded ends of the incoming linear viral DNA was not observed in fibroblasts at early times postinfection (p.i.). Apparently, a constant low level of phosphorylated ATM was enough to phosphorylate its downstream targets, p53 and Nbs1. p53 was the only cellular protein observed to relocate at early times, forming foci in infected cell nuclei between 3.5 and 5.5 h p.i. Approximately half of these foci localized with input viral DNA, and all localized with viral UL112/113 prereplication site foci. No other DNA repair proteins localized with the virus or prereplication foci in the first 24 h p.i. When viral replication began in earnest, between 24 and 48 h p.i., there were large increases in steady-state levels and phosphorylation of many proteins involved in the damage response, presumably triggered by ATM-Rad3-related kinase activation. However, a sieving process occurred in which only certain proteins were specifically sequestered into viral replication centers and others were particularly excluded. In contrast to other viruses, activation of a damage response is neither necessary nor detrimental to infection, as neither ATM nor Mre11 was required for full virus replication and production. Thus, by preventing simultaneous relocalization of all the necessary repair components to the replication centers, HCMV subverts full activation and completion of both double-stranded break and S-phase checkpoints that should arrest all replication within the cell and likely lead to apoptosis.Human cytomegalovirus (HCMV) is the major viral cause of birth defects. Each year approximately 1 to 2% of all newborns are congenitally infected, and of these infants, 5 to 10% manifest signs of serious neurological defects (6,8,16,31). HCMV infection is also a major medical problem in immunocompromised individuals (8).Recent literature points to HCMV as a potential causative agent for the development of certain types of cancers including malignant gliomas, prostate carcinomas, and colorectal cancers (17,35,74). Studies of HCMV infection in nonpermissive rodent cells indicate that the virus can act as a mutagen (1,5,78). In fact, in permissive human cells expression of the immediate-early (IE) gene products by themselves can drive cells into S phase (57). This has led to the suggestion that the IE proteins of HCMV may cause "hit-and-run" mutagenesis. In addition, our earlier findings showed that 15% of fibroblasts infected in S phase were specifically broken in the long arm of chromosome 1 when analyzed during the subsequent mitosis (28). Due to the clinical ramifications of HCMV infection, the study of its interactions w...

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Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem for immunocompromised individuals. Here we show that infection of cells with HCMV during the S-phase of the cell cycle results in two specific chromosome 1 breaks at positions 1q42 and 1q21. We demonstrate that purified virions, and not infected cell supernatant alone, are responsible for the damage. In addition, we show that the specific breaks occur when different sources of fibroblasts and strains of HCMV are used. Incubation of the virus with neutralizing antibody prevents the induction of breaks. However, UV-inactivated virus is as efficient as untreated virus in inducing specific damage to chromosome 1. Thus, there is a requirement for viral adsorption͞penetra-tion, but not new viral gene expression. This HCMV-mediated induction of site-specific damage in actively dividing cells may provide clues for the development of neurological defects in the congenitally infected infant.