Abstract
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.
Highlights
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 [1]
Summary
Amino acid substitutions were made in the first two N-terminal residues of CA and the last C-terminal amino acid of p10 in order to alter cleavage at the p10-CA site and examine the role of p10-CA cleavage in Gag processing and RSV replication (Fig. 1A). The ability of RSV PR to cleave peptides containing the p10CA amino acid substitutions compared to a peptide containing the wild type p10-CA site was tested using an in vitro protease assay [16].
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