[Show abstract][Hide abstract] ABSTRACT: The high-resolution structure of the N-terminal domain (NTD) of the retroviral capsid protein (CA) of Mason-Pfizer monkey virus (M-PMV), a member of the betaretrovirus family, has been determined by NMR. The M-PMV NTD CA structure is similar to the other retroviral capsid structures and is characterized by a six alpha-helix bundle and an N-terminal beta-hairpin, stabilized by an interaction of highly conserved residues, Pro1 and Asp57. Since the role of the beta-hairpin has been shown to be critical for formation of infectious viral core, we also investigated the functional role of M-PMV beta-hairpin in two mutants (i.e., DeltaP1NTDCA and D57ANTDCA) where the salt bridge stabilizing the wild-type structure was disrupted. NMR data obtained for these mutants were compared with those obtained for the wild type. The main structural changes were observed within the beta-hairpin structure; within helices 2, 3, and 5; and in the loop connecting helices 2 and 3. This observation is supported by biochemical data showing different cleavage patterns of the wild-type and the mutated capsid-nucleocapsid fusion protein (CANC) by M-PMV protease. Despite these structural changes, the mutants with disrupted salt bridge are still able to assemble into immature, spherical particles. This confirms that the mutual interaction and topology within the beta-hairpin and helix 3 might correlate with the changes in interaction between immature and mature lattices.
[Show abstract][Hide abstract] ABSTRACT: Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a beta-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the beta-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA.