Related Experiment Videos
An infectious cDNA clone of a cytopathic hepatitis A virus: genomic regions associated with rapid replication and
1Department of Medicine, University of North Carolina at Chapel Hill 27599-7030, USA.
Insights
Mutations in the P2 region, specifically in the 2B and 2C proteins, enhance hepatitis A virus (HAV) replication. Full viral replication and cytopathic effects require combined mutations in the 5' non-translated region (5'NTR), P2, and P3 segments.
Area of Science:
- Virology
- Molecular Biology
Background:
- Hepatitis A virus (HAV) variants exhibit varying replication capacities.
- Rapidly replicating, cytopathic (rr/cpe+) HAV variants possess distinct mutations compared to cell culture-adapted strains.
Purpose of the Study:
- To identify specific mutations in the HM175/18f HAV variant responsible for enhanced replication in BS-C-1 cells.
- To elucidate the roles of different viral genomic segments in HAV replication and cytopathic effects.
Main Methods:
- Rescue of chimeric viruses from infectious cDNAs by exchanging genomic segments between HM175/18f (rr/cpe+) and HAV/7 (rr/cpe-) strains.
- Analysis of viral replication foci size and cytopathic effects in BS-C-1 cells infected with chimeric viruses.
Main Results:
- Chimeric viruses with the P2 region from HM175/18f showed increased replication but not to the level of the parental HM175/18f.
- Enhanced replication necessitated mutations in both the 2B and 2C proteins, indicating their functional association.
- Mutations in the 5' non-translated RNA (5'NTR) or P3 proteins alone had no effect but cooperated with P2 mutations.
- Full expression of the rr/cpe+ phenotype required coordinated mutations in the 5'NTR, P2, and P3 segments.
Conclusions:
- Hepatitis A virus replication is modulated by complex interactions between the 5'NTR, P2 (2B and 2C proteins), and P3 regions.
- Specific mutations within the P2 region are critical for enhanced viral replication and cytopathic effects.
- Novel interactions between the 5'NTR and P2 proteins are suggested, impacting HAV replication dynamics.
Abstract:
Rapidly replicating, cytopathic (rr/cpe+) variants of hepatitis A virus (HAV) isolated from persistently infected BS-C-1 cells have numerous mutations from cell culture-adapted rr/cpe- HAV. To determine which mutations in one rr/cpe+ virus, HM175/18f, determine enhanced replication in BS-C-1 cells, a series of chimeric viruses was rescued from infectious cDNAs in which HM175/18f genomic segments were placed within the background of a related rr/cpe- virus, HAV/7. Chimeric viruses containing the P2 region of HM175/18f produced replication foci in BS-C-1 cells that were larger than HAV/7, but not as large as HM175/18f virus. Enhanced viral replication required mutations in both 2B and 2C proteins, suggesting that these proteins remain closely associated during replication. Mutations in 5' nontranslated RNA (5'NTR) or P3 proteins had no independent effect, but acted cooperatively with mutations in P2 proteins to enhance replication and render the virus capable of conventional plaque formation. Cytopathic effects correlated with viral replication capacity and were not the result of any single mutation. Full expression of the rr/cpe+ phenotype required mutations within the 5'NTR, P2, and P3 segments. These results suggest novel interactions between the 5'NTR and P2 proteins during HAV replication and provide useful new infectious cDNA clones.