Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication.
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.
Vana
Marcy L
ML
Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. mvana@stanford.edu
Chen
Aiping
A
Boross
Peter
P
Weber
Irene
I
Colman
Dalbinder
D
Barklis
Eric
E
Leis
Jonathan
J
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000502:physiology*; D001358:Avian Sarcoma Viruses / Q000502:physiology*; D019556:COS Cells; D036022:Capsid Proteins / Q000737:chemistry* / Q000502:physiology; D002522:Chlorocebus aethiops; D015683:Gene Products, gag / Q000378:metabolism*; D009154:Mutation; D013329:Structure-Activity Relationship; D014771:Virion / Q000502:physiology; D019065:Virus Assembly; D014779:Virus Replication
D036022:Capsid Proteins; D015683:Gene Products, gag; D016282:Aspartic Acid Endopeptidases; C072891:Rous sarcoma virus protease