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A newly identified MHV-A59 ORF1a polypeptide p65 is temperature sensitive in two RNA negative mutants
S A Hughes1, M R Denison, P Bonilla
1University of Pennsylvania School of Medicine, Phila.
Abstract:
Polypeptide products of MHV-A59 gene 1 have been identified in infected DBT cells and in the products of in vitro translations of genome RNA. In this paper we report the identification in infected cell lysates of a 65-kDa polypeptide (p65) encoded in ORF 1a. Studies on the kinetics of appearance and processing of p65 show that p65 is detectable after p28 but before the appearance of p290, p240 and p50. No homologue of the p65 polypeptide identified in infected cell lysates was immunoprecipitated from in vitro translations of genomic RNA, providing further evidence that in vitro processing of polypeptides encoded in ORF 1a of gene 1 differs from that which occurs late in infection of DBT cells. Although the function of p65 is unknown, two MHV-A59 ts mutants isolated and characterized by Baric et al. (3,4) do not produce detectable levels of p65 at the non-permissive temperature indicating that p65 may play an important role in the virus life cycle.
Insights
Researchers identified a 65-kDa polypeptide (p65) in MHV-A59 infected cells, crucial for viral life cycle. Its absence in mutants suggests p65 plays a vital role in virus replication.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Murine hepatitis virus (MHV-A59) gene 1 encodes essential viral polypeptides.
- Understanding the processing and function of these polypeptides is key to deciphering viral replication mechanisms.
Purpose of the Study:
- To identify and characterize polypeptide products of MHV-A59 gene 1, specifically focusing on a 65-kDa polypeptide (p65) found in infected cells.
- To investigate the kinetics of p65 appearance and processing in relation to other viral proteins.
- To compare in vivo and in vitro processing of gene 1 products.
Main Methods:
- Infected cell lysate analysis using immunoprecipitation.
- In vitro translation of MHV-A59 genomic RNA.
- Kinetic studies of polypeptide appearance and processing.
- Analysis of temperature-sensitive (ts) mutants.
Main Results:
- A 65-kDa polypeptide (p65) encoded in ORF 1a was identified in MHV-A59 infected DBT cell lysates.
- p65 appeared after p28 but before larger polypeptides (p290, p240, p50).
- No p65 homologue was detected in in vitro translations, indicating differences in processing between in vivo and in vitro systems.
Conclusions:
- In vitro processing of MHV-A59 gene 1 products differs from late-stage intracellular processing.
- p65 is likely important for the MHV-A59 virus life cycle, as evidenced by its absence in specific ts mutants at non-permissive temperatures.