Molecular Characterization of Proteolytic Processing of the Gag Proteins of Human Spumaretrovirus

Abstract

Molecular characterization of proteolytic processing of the human spumaretrovirus (HSRV) Gag proteins and the precise determination of cleavage sites was performed. For in vitro processing of recombinant HSRV Gag proteins, a recombinant enzymatically active HSRV protease was employed. Recombinant Gag proteins and protease were cloned and expressed as hexa-histidine-tagged proteins in pET-32b and pET-22b vectors, respectively, in the E. coli BL21 expression strain. The recombinant proteins were purified by affinity chromatography on an immobilized metal ion matrix. To determine the precise processing sites, recombinant Gag proteins or synthetic peptides derived from Gag sequences were cleaved in vitro by the recombinant protease. Proteolytic processing reactions were carried out under optimal reaction conditions of HSRV protease in sodium phosphate buffer, pH 6.0, supplied with 2 M NaCl at 37 degrees C. The cleavage sites were determined by amino-terminal amino acid sequencing as well as by matrix-assisted laser desorption/ionization mass spectrometry analysis of the reaction products. Fluorescence spectrophotometry was used to determine cleavage kinetics of peptides mimicking different cleavage sites within the HSRV Gag proteins.

Protease-Dependent Uncoating of a Complex Retrovirus

“…Interestingly, three internal protease-dependent cleavage sites, critical for infectivity, were also characterized in the primate foamy virus (PFV) Gag ( Fig. 1) (31). Although the timing of processing and the role of these consensus cleavage sites have not been studied, the mutation of the first cleavage site at position 310 in the Gag open reading frame prevents subsequent cleavage at the two other sites by the viral PR, reflecting its prominent role (31).…”

“…1) (31). Although the timing of processing and the role of these consensus cleavage sites have not been studied, the mutation of the first cleavage site at position 310 in the Gag open reading frame prevents subsequent cleavage at the two other sites by the viral PR, reflecting its prominent role (31). Moreover, in contrast to animal retroviruses, FV Gag contains three glycine/argininerich basic sequences (the so-called GR boxes) instead of the canonical cysteine-histidine motifs usually found in the NC domain.…”

Identification of a Conserved Residue of Foamy Virus Gag Required for Intracellular Capsid Assembly

“…The majority of viral protease-specific Gag cleavage is limited to a single event near the C terminus of the protein, releasing an approximately 3-kDa protein from the 71-kDa precursor peptide (13,16,35), although it has been proposed that additional cleavage occurs on infection of new cells during viral uncoating (16,34,39). In addition, FV Gag lacks the major homology region found in the capsid proteins of all other retroviruses, as well as the signature Cys-His box motifs found in all retroviral Gag NC proteins (43a).…”

Subviral Particle Release Determinants of Prototype Foamy Virus

“…To a certain extent, the central portion of the LP CyD spanning aa 36 to 47 also contributed to SVP release. Some of these results are reminiscent of the requirements for FV viral particle release, for which the TM CyD is dispensable and the cognate TM MSD and LP CyD are essential (27,39). However, deletion of as few as the five N-terminal amino acids of LP completely abolished PFV viral particle egress, but this was not sufficient to overcome the inhibitory effect on SVP release (27).…”