Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments in the newborn. It is likely that the timing of infection as well as the range of susceptible cells at the time of infection will affect the severity of the disease. A major goal of our research is to understand at a high-resolution the effects of HCMV infection on the neural lineage specification and maturation of stem and progenitor cells. Elucidation of the genes and cellular processes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
This past year, we have made significant progress in accomplishing the goals of this project. We used human embryonic stem cells-derived primitive pre-rosette neural stem cells (pNSCs) maintained in chemically defined conditions to study host-HCMV interactions in early neural development. Infection of pNSCs with HCMV was largely inefficient and non-progressive. At low multiplicity of infection (MOI), we observed severe defects with regards to the proportion of cells expressing the major immediate-early proteins (IE) despite an optimal viral entry, thus indicating the existence of a blockade to specific pre-IE events. IE expression, even at high MOI, was found to be restricted to a subset of cells negative for the expression of the forebrain marker FORSE-1. Treatment of pNSCs with the caudalizing agent retinoic acid rescued IE expression, suggesting that the hindbrain microenvironment might be more permissive for the infection. Transactivation of the viral early genes was found to be severely debilitated and expression of the late genes was barely detectable even at high MOI. Differentiation of pNSCs into primitive neural progenitor cells (pNPCs) restored IE expression but not the transactivation of early and late genes. Increasing the number of viral particles bypassed this barrier to early gene expression and thus permitted expression of the late genes in pNPCs. Consequently, viral spread was only observed at high MOI but was largely restricted to one cycle of replication as secondarily infected cells failed to efficiently express early genes. Of note, virions produced in pNPCs and pNSCs were exclusively cell-associated. Finally, we found that viral genomes could persist in pNSCs culture up to a month after infection despite the absence of detectable IE expression by immunofluorescence. Clonogenic expansion of infected pNSCs revealed that the presence of viral DNA and IE proteins were insufficient to block host cell division therefore allowing the survival of viral genomes via cellular division rather than viral replication. These results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. To study in greater depth the molecular basis of the interaction of HCMV with cell of the neural lineage, we also have initiated high-throughput genomics approaches to analyze HCMV microRNAs, alterations in cellular microRNA and gene expression profiles, and global defects in host alternative splicing in infected and uninfected pNSC-derived NPCs. Interestingly, although there are many changes in host cell gene expression in the infected cells, there was only a small overlap with the set of changes we had found in infected human fibroblasts. This highlights the importance of performing these studies in the relevant targets of the virus in the developing fetus.
We expect that the results of these studies will provide an unprecedented resolution of the effects on neurogenesis when HCMV infects a newborn, serve as a foundation for future therapeutic efforts in preventing the birth defects due to HCMV, and provide insight into the serious potential problem of disseminated HCMV in immunosuppressed individuals receiving transplanted allogeneic stem cells.