Three-dimensional structure of a monomeric form of a retroviral protease.

The assembly of Mason-Pfizer monkey virus Gag polyproteins into immature capsids and their cleavage by the encoded protease are temporally and spatially separated processes, making the virus a particularly useful model for investigation of protease activation. Here we present a high resolution NMR structure of a fully folded monomer of a 12 kDa M-PMV protease (wt 12 PR) and of a Cys7Ala/Asp26Asn/Cys106Ala mutant (12 PR(D26N/C7A/C106A)). The overall structures of both wt 12 PR and 12 PR(D26N/C7A/C106A) follow the conservative structural motif of other retroviral proteases. The most prominent difference from the canonical fold of retroviral proteases is the absence of the interfacial beta-sheet, which leads to the loss of the principal force stabilizing the dimer of M-PMV PR. The monomer-dimer equilibrium can be shifted in favor of the dimer by adding a substrate or an inhibitor, partially compensating for the missing role of the beta-sheet. We also show that cysteines C7 and C106 play a crucial role in stabilizing the dimer and consequently increasing the proteolytic activity of M-PMV PR. This is consistent with the role of reversible oxidative modification of the cysteine residues in the regulation of the maturation of assembled M-PMV capsids in the cytoplasm.

Proteinases of betaretroviruses bind single-stranded nucleic acids through a novel interaction module, the G-patch.

Retroviral proteinases (PRs) are essential for retrovirus infectivity but the mechanism of their activity regulation is poorly understood. We investigated possible involvement in this process of the C-terminal domain (CTD) of betaretroviral PRs. We found that the presence of CTD attenuates proteolytic activity of Mason-Pfizer monkey virus PR, while it does not significantly affect the activity of mouse intracisternal A-particle retrovirus PR. However, both PRs bind single-stranded nucleic acids through their CTDs that contain a novel binding motif, the G-patch, whose function is dependent on a single conserved tyrosine residue. Oligonucleotide binding to both PRs does not inhibit their proteolytic activity.

Amino acid residues in the cytoplasmic domain of the Mason-Pfizer monkey virus glycoprotein critical for its incorporation into virions.

Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.