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Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149
Abstract:
The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis in vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982) J. Biol. Chem. 257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982) J. Biol. Chem. 257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.
Insights
Adenovirus DNA polymerase (Pol) is crucial for viral DNA synthesis. Temperature-sensitive mutants reveal Pol, not the terminal protein precursor (pTP), is essential for replication, suggesting Pol is virally encoded.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Adenovirus (Ad) serotype 5 mutants with temperature-sensitive DNA synthesis were studied.
- A 140,000-dalton DNA polymerase (Pol) copurified with the 80,000-dalton terminal protein precursor (pTP).
Purpose of the Study:
- To investigate the role of DNA polymerase (Pol) and terminal protein precursor (pTP) in adenovirus DNA replication.
- To determine if Pol is a virally encoded protein.
Main Methods:
- Utilized temperature-sensitive Ad serotype 5 mutants (N complementation group).
- Assayed viral DNA synthesis in cell extracts at nonpermissive temperatures.
- Purified Pol from wild-type Ad-infected cytosol.
- Employed glycerol gradient sedimentation to analyze protein complexes.
- Tested urea lability of DNA synthesis reactions.
Main Results:
- Extracts from H5ts149 mutant-infected cells failed to synthesize viral DNA at nonpermissive temperatures.
- Addition of purified Pol restored DNA synthesis, while pTP alone did not.
- Glycerol gradients showed reduced pTP-Pol complex in H5ts149 extracts.
- H5ts149 extracts showed increased lability of initiation and elongation to urea.
Conclusions:
- The 140,000-dalton DNA polymerase (Pol) is essential for Ad DNA replication.
- The data strongly suggest that the Ad DNA polymerase (Pol) is a virally encoded protein, similar to pTP.
- The H5ts149 mutation likely affects the viral DNA polymerase.