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Identification and characterization of a major early cytomegalovirus DNA-binding protein
Abstract:
We characterized a DNA-binding protein with an approximate molecular weight of 129,000 (DB129) which is present in the nuclei of cytomegalovirus- (strain Colburn) infected cells, but not in virus particles. Results of two types of experiments demonstrated that DB129 is a member of the early class of herpesviral proteins. First, time course pulse-labeling experiments showed that its synthesis begins after that of the immediate-early protein IE94, but prior to the appearance of late viral proteins, and was reduced at late times. Second, in the presence of inhibitors of viral DNA replication, DB129 continued to be made and accumulated to elevated levels. A second set of experiments showed that DB129 bound to single-stranded DNA in vitro and was eluted by a NaCl gradient in two peaks, one at about 0.2 M and the second at about 0.6 M. A similar pattern of release was observed when infected-cell nuclei were serially extracted with increasing NaCl concentrations. In addition, treatment of nuclei with DNase I selectively released DB129, along with a small but significant fraction of another DNA-binding protein, DB51. These results suggest that DB129 is associated with DNA in vivo and that it interacts directly with single-stranded DNA. It was also shown that cells infected with human cytomegalovirus (strain Towne) contain a slightly larger counterpart to DB129, which was designated DB140. Similarities between these proteins and the major DNA-binding protein of herpes simplex virus are discussed.
Insights
Researchers identified a novel early herpesviral protein, DNA-binding protein 129 (DB129), in cytomegalovirus-infected cells. DB129 binds single-stranded DNA and is associated with viral DNA in vivo.
Area of Science:
- Virology
- Molecular Biology
- Herpesvirus Research
Background:
- Human cytomegalovirus (HCMV) is a significant pathogen, and understanding its protein synthesis and function is crucial.
- Herpesviral proteins are classified as immediate-early, early, or late, with distinct roles in the viral life cycle.
Purpose of the Study:
- To characterize a newly identified DNA-binding protein (DB129) from HCMV-infected cells.
- To determine the class and DNA-binding properties of DB129.
- To investigate the in vivo association of DB129 with viral DNA.
Main Methods:
- Pulse-labeling experiments to determine protein synthesis timing.
- Inhibition of viral DNA replication to assess protein accumulation.
- In vitro DNA-binding assays using single-stranded DNA and salt gradients.
- Fractionation of infected-cell nuclei with increasing salt concentrations and DNase I treatment.
Main Results:
- DB129 is an early viral protein, synthesized after immediate-early proteins and before late proteins.
- DB129 synthesis is independent of viral DNA replication and accumulates when replication is inhibited.
- DB129 binds to single-stranded DNA in vitro, eluting at 0.2 M and 0.6 M NaCl.
- DB129 is released from infected-cell nuclei by DNase I, indicating association with DNA.
- A larger homolog, DB140, was identified in cells infected with a different HCMV strain.
Conclusions:
- DB129 is an early-acting, single-stranded DNA-binding protein associated with viral DNA in HCMV-infected cells.
- DB129's properties suggest a role in viral DNA processing or replication.
- The findings contribute to understanding the complexity of herpesviral protein functions.