- Short report
- Open Access
Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication
© Vana et al; licensee BioMed Central Ltd. 2005
- Received: 01 February 2005
- Accepted: 27 September 2005
- Published: 27 September 2005
A series of amino acid substitutions (M239F, M239G, P240F, V241G) were placed in the p10-CA protease cleavage site (VVAM*PVVI) to change the rate of cleavage of the junction. The effects of these substitutions on p10-CA cleavage by RSV PR were confirmed by measuring the kinetics of cleavage of model peptide substrates containing the wild type and mutant p10-CA sites. The effects of these substitutions on processing of the Gag polyprotein were determined by labeling Gag transfected COS-1 cells with 35S-Met and -Cys, and immunoprecipitation of Gag and its cleavage products from the media and lysate fractions. All substitutions except M239F caused decreases in detectable Gag processing and subsequent release from cells. Several of the mutants also caused defects in production of the three CA proteins. The p10-CA mutations were subcloned into an RSV proviral vector (RCAN) and introduced into a chick embryo fibroblast cell line (DF-1). All of the mutations except M239F blocked RSV replication. In addition, the effects of the M239F and M239G substitutions on the morphology of released virus particles were examined by electron microscopy. While the M239F particles appeared similar to wild type particles, M239G particles contained cores that were large and misshapen. These results suggest that mutations affecting cleavage at the p10-CA protease cleavage site block RSV replication and can have a negative impact on virus particle morphology.
- Media Fraction
- Peptide Cleavage
- V241G Mutant
- Lysate Fraction
- Chick Embryo Fibroblast Cell
The structural proteins of retroviruses are encoded by the gag gene and are translated as a single polyprotein. During or subsequent to virus budding, the Gag polyprotein is cleaved by the viral protease (PR), thereby releasing the mature structural proteins. Gag processing leads to morphological changes in the virus particle, including condensation of the capsid core, and is associated with the appearance of infectious particles . It has previously been demonstrated that proper processing at several protease sites throughout RSV Gag is required for production of infectious virus [2, 3]. However, the protease site separating the C-terminus of p10 and the N-terminus of CA has not been examined.
Multiple studies have highlighted the importance of cleavage at the N-terminus of retrovirus CA proteins in particle assembly and maturation. Structural studies have identified a β hairpin structure at the N-terminus of RSV CA that is thought to form after proteolysis at the p10-CA site and liberation of the N-terminus of CA . Moreover, a conserved Pro residue at the extreme N-terminus of RSV CA forms a salt bridge with an internal Asp residue, thereby stabilizing the β-hairpin structure . These Pro and Asp residues are highly conserved among many retrovirus CA proteins, suggesting that the β-hairpin is a common structural feature of retrovirus CA proteins [5–8]. Mutating the conserved Asp residue in HIV-1 CA (Asp51) or murine leukemia virus CA (MLV, Asp63) causes a loss in virus infectivity . In addition, blocking protease cleavage at the N-terminus of MLV CA results in the production of virus that is non-infectious . It has also been demonstrated that the N-terminus of CA and the residues immediately upstream of CA have a role in determining the shape of assembling retrovirus particles [8, 10–13]. More specifically for RSV, it has been demonstrated that the presence of p10 on the N-terminus of CA-NC converts the in vitro assembly phenotype from cylindrical particles to spherical particles that resemble wild type immature RSV particles [10, 11].
The effects of the p10-CA substitutions on Gag processing were tested by introduction of the mutations into the context of full-length Gag and expressing the wild type or mutant Gag proteins in COS-1 cells [2, 3]. Gag and its cleavage products were immunoprecipitated from the media and lysate fractions from transfected cells following metabolic labeling and were separated using SDS-PAGE (Fig. 1B, top). By comparison to wild type (Fig. 1B, top lanes 2), all of the p10-CA substitutions except M239F caused processing defects. The banding pattern in the lysate and media fractions from cells transfected with M239F (Fig. 1B, top, lanes 3) was very similar to wild type, suggesting that the M239F substitution did not affect Gag processing. In contrast, a novel and stable band representing a p10-CA fusion protein was present in the lysate and media fractions from cells transfected with the M239G (Fig. 1B, top, lanes 4) and P240F (Fig. 1B, top, lanes 5) mutants that was not present in fractions from cells transfected with wild type Gag (lanes 2 top). The presence of a p10-CA fusion indicated that these mutations resulted in a reduction in the ability of PR to cleave the p10-CA site within Gag.
In cells transfected with wild type Gag, three CA species were detected (CA1, CA2, and CA3) in the media and lysate fractions (Fig. 1B, top, lanes 2) [2, 3]. These species are the result of processing of CA at its C-terminus at different sites. In contrast, in cells transfected with the M239G mutant, CA2 and CA3 were detected in the media fraction, but CA1 was not (Fig. 1B, top, lanes 4). Furthermore, mature CA proteins were not detected in the lysate. Similarly, none of the mature CA proteins were detected in the media or lysate fractions from cells transfected with the P240F (Fig. 1B, top, lanes 5) mutant, and CA1 made up the majority of the CA protein in the media and lysate fractions from cells transfected with the V241G (Fig. 1B, top, lanes 6) mutant. There also appeared to be a reduction in the amount of Gag released into the media from cells transfected with the V241G mutant compared to cells transfected with wild type Gag (Fig. 1B, top, lanes 6 and 2). This effect was most apparent when examining the signal of PR in the lysate and media fractions. The amount of PR in the lysate fraction from cells transfected with the V241G mutant was similar to wild type, but the amount of PR in the media fraction from cells transfected with the V241G mutant was greatly reduced compared to wild type. In order to determine whether the reduction in particle release observed with the V241G mutant was due to impaired Gag processing, a D37S mutation in the PR domain was constructed in the context of the p10-CA Gag mutants. COS-1 cells were transfected with the p10-CA/PR-D37S mutants and full-length Gag was immunoprecipitated from the media and lysate fractions. A similar level of Gag release was observed with all of the p10-CA/PR-D37S mutants when compared to PR-D37S (Fig. 1B, bottom), suggesting that the particle release defect observed with the V241G substitution was due to impaired Gag processing. Taken together, these results indicate that mutations to the p10-CA site of Gag affect processing of the C-terminus of CA.
This work was supported in part by United States Public Health Service grant CA52047 (to J.L.), CA58166 (to I. W.), and GM60170 (to E.B.), the Hungarian Science and Research Fund, OTKA F35191 (to P.B.), and the Cancer Biology Fellowship Program, Chicago Baseball Cancer Charities, from the Robert H. Lurie Comprehensive Cancer Center (to M.L.V.). Peptides were a generous gift of Dr. Terry Copeland, NCI, Frederick, Maryland.
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