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...

Congenital human cytomegalovirus (HCMV) infection causes a broad spectrum of central and peripheral nervous system disorders, ranging from microcephaly to hearing loss. These ramifications mandate the study of virus-host interactions in neural cells. Neural progenitor cells are permissive for lytic infection. We infected two induced pluripotent stem cell (iPSC) lines and found these more primitive cells to be susceptible to infection but not permissive. Differentiation of infected iPSCs induced de novo expression of viral antigens. iPSCs can be cultured in three dimensions to generate cerebral organoids, closely mimicking in vivo development. Mock- or HCMV-infected iPSCs were subjected to a cerebral organoid generation protocol. HCMV IE1 protein was detected in virus-infected organoids at 52 days postinfection. Absent a significant effect on organoid size, infection induced regions of necrosis and the presence of large vacuoles and cysts. Perhaps more in parallel with the subtler manifestations of HCMV-induced birth defects, infection dramatically altered neurological development of organoids, decreasing the number of developing and fully formed cortical structure sites, with associated changes in the architectural organization and depth of lamination within these structures, and manifesting aberrant expression of the neural marker β-tubulin III. Our observations parallel published descriptions of infected clinical samples, which often contain only sparse antigen-positive foci yet display areas of focal necrosis and cellular loss, delayed maturation, and abnormal cortical lamination. The parallels between pathologies present in clinical specimens and the highly tractable three-dimensional (3D) organoid system demonstrate the utility of this system in modeling host-virus interactions and HCMV-induced birth defects. IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of central nervous system birth defects, ranging from microcephaly to hearing impairment. Recent literature has provided descriptions of delayed and abnormal maturation of developing cortical tissue in infected clinical specimens. We have found that infected induced pluripotent stem cells can be differentiated into three-dimensional, viral protein-expressing cerebral organoids. Virus-infected organoids displayed dramatic alterations in development compared to those of mock-infected controls. Development in these organoids closely paralleled observations in HCMV-infected clinical samples. Infection induced regions of necrosis, the presence of larger vacuoles and cysts, changes in the architectural organization of cortical structures, aberrant expression of the neural marker β-tubulin III, and an overall reduction in numbers of cortical structure sites. We found clear parallels between the pathologies of clinical specimens and virus-infected organoids, demonstrating the utility of this highly tractable system for future investigations of HCMV-induced birth defects.

Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunocompromised and immunosuppressed individuals. During infection, HCMV is known to employ host transcription factors to facilitate viral gene expression. To further understand the previously observed delay in viral replication and protein expression in p53 knockout cells, we conducted microarray analyses of p53 ؉/؉ and p53 ؊/؊ immortalized fibroblast cell lines. At a multiplicity of infection (MOI) of 1 at 24 h postinfection (p.i.), the expression of 22 viral genes was affected by the absence of p53. Eleven of these 22 genes (group 1) were examined by real-time reverse transcriptase, or quantitative, PCR (q-PCR). Additionally, five genes previously determined to have p53 bound to their nearest p53-responsive elements (group 2) and three control genes without p53 binding sites in their upstream sequences (group 3) were also examined. At an MOI of 1, >3-fold regulation was found for five group 1 genes. The expression of group 2 and 3 genes was not changed. At an MOI of 5, all genes from group 1 and four of five genes from group 2 were found to be regulated. The expression of control genes from group 3 remained unchanged. A q-PCR time course of four genes revealed that p53 influences viral gene expression most at immediate-early and early times p.i., suggesting a mechanism for the reduced and delayed production of virions in p53 ؊/؊ cells.

Many viruses subvert the host cell's ability to mount and complete various DNA damage responses (DDRs) after infection. HCMV infection of permissive fibroblasts activates host DDRs at the time of viral deposition and during replication, but the DDRs remain uncompleted without arrest or apoptosis. We believe this was in part due to partitioning of the damage response and double strand break repair components. After extraction of soluble proteins, the localization of these components fell into three groups: specifically associated with the viral replication centers (RCs), diffused throughout the nucleoplasm and excluded from the RCs. Others have shown that cells are incapable of processing exogenously introduced damage after infection. We hypothesized that the inability of the cells to process damage might be due to the differential association of repair components within the RCs and, in turn, potentially preferential repair of the viral genome and compromised repair of the host genome. To test this hypothesis we used multiple strategies to examine repair of UV-induced DNA damage in mock and virus-infected fibroblasts. Comet assays indicated that repair was initiated, but was not completed in infected cells. Quantitative analysis of immunofluorescent localization of cyclobutane pyrimidine dimers (CPDs) revealed that after 24 h of repair, CPDs were significantly reduced in viral DNA, but not significantly changed in the infected host DNA. To further quantitate CPD repair, we developed a novel dual-color Southern protocol allowing visualization of host and viral DNA simultaneously. Combining this Southern methodology with a CPD-specific T4 endonuclease V alkaline agarose assay to quantitate repair of adducts, we found efficient repair of CPDs from the viral DNA but not host cellular DNA. Our data confirm that NER functions in HCMV-infected cells and almost exclusively repairs the viral genome to the detriment of the host's genome.

Our electron microscopy study found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion.

Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm.

In 2000, we reported HCMV induced specific damage on chromosome 1. The virus' capacity to induce DNA breaks indicated potent interaction between viral protein/s and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12-24 hpi, and continuing throughout infection, steady state (ss) NID1 protein levels were decreased in whole cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor, MG132, to culture media stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found ss NID1 was also reduced in infected ECs and hypothesized virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1 knockout EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal CNS/PNS and is important for neuronal migration, neural network excitability and plasticity and regulates Schwann cell proliferation, migration and myelin production. We found NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus. SIGNIFICANCE: We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein, nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cells' (ECs) secretion of basement membrane proteins is critical for vascular wall integrity and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.

Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM ؊ lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM ؊ lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM ؊ line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM ؊ cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints. T he human cytomegalovirus (HCMV) life cycle involves a complex interplay between the virus and the host, with the virus exploiting the host cellular machinery for many of its own functions and, ultimately, releasing fully infectious virions. During a permissive HCMV infection, after virions have entered the cell, the tegument proteins and virus genome are independently trafficked to the nucleus. In fibroblasts, large bipolar viral replication centers (RCs) are formed within 48 h postinfection (hpi) and certain host cellular proteins become strongly associated with these RCs (1; reviewed in reference 2). These proteins include the regulatory protein p53 (3), as well as numerous components of the host cellular DNA damage response (DDR) and repair pathways (4-8).Many virus infections affect the DDR. The interactions span a range of up-and downregulations and include a complex dynamic between the virus and its host's damage response (as reviewed in references 6 and 9). Some viruses appear to require DDR proteins for efficient replication (10, 11), while for other viruses an efficient DDR can be detrimental to their DNA replication (12-21). Studies from several labs, including our own, have shown that HCMV infection initiates the ataxia telangiectasia mutated (ATM)-dependent double-strand break (DSB) ...