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Persistence of the cytomegalovirus genome in human cells
Abstract:
A small percentage of human fibroblast cells survived high-multiplicity infection by cytomegalovirus and were isolated as persistently infected cultures. Approximately 30% of the cells were in the productive phase of infection, since virus-specific structural antigens and virions were associated with these cells. The remaining cells contained neither viral structural antigens nor particles. Nuclear DNA from these nonproductive cells contained approximately 120 genome equivalents of viral DNA per cell as determined by reassociation kinetics. In situ hybridization confirmed that nuclei from nonproductive cells contained a significant amount of viral DNA that was distributed in most of these cells. Early virus-induced proteins and antigens were also detected. Nonproductive cells continued to grow, and there was a slow, spontaneous transition of some of these cells to productive viral replication. The majority of the viral DNA in nonproductive cells persisted with restricted gene expression. When infectious virus production was eliminated by growing the persistently infected cultures in the presence of anticytomegalovirus serum, approximately 45 genome equivalents of the viral DNA persisted per cell. The reassociation reaction approached completion. After removal of the antiserum and subculturing, infectious virus production resumed. Therefore, it was assumed that all sequences of the viral genome remained associated with these cells. Restriction of cytomegalovirus gene expression in persistently infected cell cultures is discussed.
Insights
Human fibroblast cells surviving high-multiplicity cytomegalovirus infection establish persistently infected cultures. Nonproductive cells harbor significant viral DNA with restricted gene expression, which can spontaneously reactivate.
Area of Science:
- Virology
- Cell Biology
Background:
- Human cytomegalovirus (HCMV) establishes persistent infections in a subset of infected cells.
- Understanding the state of the viral genome and gene expression in these persistently infected cells is crucial.
Purpose of the Study:
- To characterize the viral DNA and gene expression in nonproductively infected human fibroblast cells.
- To investigate the persistence and potential reactivation of HCMV in cell culture.
Main Methods:
- Isolation of persistently infected human fibroblast cell cultures after high-multiplicity HCMV infection.
- Quantification of viral DNA using reassociation kinetics.
- Detection of viral DNA and proteins using in situ hybridization and antigen detection.
- Modulation of viral production using anticytomegalovirus serum.
Main Results:
- A fraction of infected cells entered a nonproductive state, containing approximately 120 genome equivalents of viral DNA per cell.
- Viral DNA in nonproductive cells was distributed throughout the nucleus and associated with early viral protein expression.
- Nonproductive cells could spontaneously transition to productive replication.
- Upon removal of neutralizing antibodies, viral production resumed, indicating the persistence of the complete viral genome.
Conclusions:
- Persistently infected human fibroblast cells harbor significant amounts of HCMV DNA with restricted gene expression.
- The viral genome remains intact and capable of reactivation in these nonproductive cells.
- This model provides insights into HCMV persistence and latency mechanisms.