Human Cytomegalovirus Infection Causes Premature and Abnormal Differentiation of Human Neural Progenitor Cells

Abstract

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...

Effects of cytomegalovirus infection in human neural precursor cells depend on their differentiation state

“…On the other hand, when we infected SK-N-MC with the MOI of 0.01, cellular prolongations were observed suggesting cellular differentiation to a neuron-like morphology, an unexpected finding since we did not add any other stimulus that may account for these changes, and this normally does not occur in an spontaneous way (Higgins et al 2009). This induction of differentiation is in agreement with the recent observation of abnormal, spontaneous, and premature differentiation of NPC induced by HCMV (Odeberg et al 2006;Luo et al 2010). The virus concentrations that we used were much lower than those in previous studies, what may explain the delayed effect observed.…”

“…Altogether, these observations suggest that cellular differentiation is stimulated in early stages of infection and that cells tend to fuse forming syncytia as viral concentration increases or time after infection progresses. A possible explanation for this latter effect can be a distinct regulation of transcription factors such as SOX2 which is related to embryonic development and stem cell self-renewal and expression of some receptors like EGFR as well as cytoskeleton components (Jafferji et al 2009;Luo et al 2010). Nevertheless, the relevance of HCMV-induced differentiation in the context of CNS development and finding why it is reversed when viral concentration increases are a matter for further investigation.…”

“…Because our main objective was to describe the earliest effects of HCMV infection, we have used significantly lower MOI than those employed in other reports (Cheeran et al 2005;Odeberg et al 2006Odeberg et al , 2007Luo et al 2008Luo et al , 2010. Instead of showing the deepest impact of the infection, we decided to monitor the progression in different time points, in order to follow slight effects.…”

“…In addition, cytopathic effect (CPE) in these cells at the multiplicity of infection (MOI) of three plaque formation units (PFU) per cell consisted in formation of syncytia, which can be the result of viral cell to cell dissemination (Luo et al 2008). Infection of these cells may increase the apoptosis frequency and inhibit neuronal and glia differentiation; however, this effect remains controversial since it has also been reported that CMV infection may cause a spontaneous, premature, and abnormal NPC differentiation (Odeberg et al 2006(Odeberg et al , 2007Luo et al 2010).…”

Immunobiology of congenital cytomegalovirus infection of the central nervous system—the murine cytomegalovirus model

“…38,46,47,53,63 CMV infection causes NSPCs to alter their differentiation program, and it inhibits the proliferation and induces the apoptosis of NSPCs. 47,62,64 Whole-genome expression analysis found rapid downregulation of the mRNA levels of several genes important for maintaining NSPC multipotency and for establishing their neural identity (nestin, doublecortin, sex-determining home box 2 and glial fibrillary acidic protein). 64 It is thus possible that HCMV infection causes in utero CNS defects by inducing both premature and abnormal differentiation of NSPCs.…”