Contributions of charged residues in structurally dynamic capsid surface loops to Rous sarcoma virus assembly

Extensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible loop region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation and early replication events. We have now defined the contributions of charged residues in the flexible loop (FL) region of the Rous sarcoma virus CA to particle assembly. Effects of mutations on assembly were assessed in vivo and in vitro and analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on inter-hexamer interactions involving helix α7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that 1) permits the packing of NTD domains in the outer layer of the Gag shell, 2) directs the maturational rearrangements of the NTDs that yield a functional core structure, and 3) supports capsid function during the early stages of virus infection.