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Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus
Abstract:
The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.
Insights
Murine cytomegalovirus immediate-early (IE) proteins were identified, with the 89-kd protein being the primary form. Post-translational modifications create smaller IE proteins, and their synthesis negatively regulates IE gene expression.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Murine cytomegalovirus (MCMV) infection involves immediate-early (IE) proteins crucial for viral replication.
- Understanding the nature and regulation of these IE proteins is key to comprehending MCMV pathogenesis.
Purpose of the Study:
- To characterize the immediate-early (IE) infected cell proteins induced by murine cytomegalovirus (Smith strain).
- To elucidate the synthesis, modification, and regulatory roles of MCMV IE proteins.
Main Methods:
- Identification of IE proteins via synthesis during actinomycin D treatment after cycloheximide block.
- Immunoprecipitation using MCMV-specific antiserum to detect polypeptides.
- In vitro translation of IE infected cell RNA.
- Hybrid selection of IE RNA with specific MCMV DNA fragments.
- Cellular localization studies (nuclei and cytoplasm).
Main Results:
- Three abundant IE polypeptides of 89, 84, and 76 kilodaltons (kd) were identified.
- These proteins are phosphorylated but not glycosylated and share antigenic determinants.
- The 84 and 76-kd polypeptides are post-translational products of the 89-kd protein.
- In vitro translation yielded only the 89-kd polypeptide, confirming its primary nature.
- Viral origin of the 89-kd protein's RNA was confirmed via hybrid selection.
- IE polypeptides localize to both nuclei and cytoplasm.
- IE polypeptide synthesis shows negative regulatory effects on IE gene expression.
Conclusions:
- The primary MCMV IE protein is 89-kd, with 84-kd and 76-kd forms resulting from post-translational modifications.
- IE protein synthesis is subject to negative feedback regulation during MCMV infection.