Crosslink analysis of N-terminal, C-terminal, and N/B determining regions of the Moloney murine leukemia virus capsid protein

To analyze contacts made by Moloney murine leukemia virus (M-MULV) capsid ( CA) proteins in immature and mature virus particles, we have employed a cys teine-specific crosslinking approach that permits the identification of ret roviral Gag protein interactions at particular residues. For analysis, sing le cysteine creation mutations were made in the context of protease-deficie nt or protease-competent parental constructs. Cysteine creation mutations w ere chosen near the N- and C-termini of CA and at a site adjacent to the M- MuLV Glu-Ala Fv1 N/B host range determination sequence. Analysis of immatur e virions showed that PrGag proteins were crosslinked at C-terminal CA resi dues to form dimers while crosslinklng of particle-associated N-terminal an d N/B region mutant proteins did not yield dimers, but showed evidence of l inking to an unknown 140- to 160-kDa partner. Analysis of mature virions de monstrated that both N- and C-terminal CA residues participated in dimer fo rmation, suggesting that processed CA N- and C-termini are free to establis h interprotein associations. Interestingly, N/B region mutant residues in m ature virus particles did not crosslink to form dimers, but showed a novel crosslinked band, consistent with an interaction between the N/B tropism de termining region and a cellular protein of 45-55 kDa. (C) 2000 Academic Pre ss.