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 cysteine-specific crosslinking approach that permits the identification of retroviral Gag protein interactions at particular residues. For analysis, single cysteine creation mutations were made in the context of protease-deficient or protease-competent parental constructs. Cysteine creation mutations were 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 immature virions showed that PrGag proteins were crosslinked at C-terminal CA residues to form dimers while crosslinking of particle-associated N-terminal and N/B region mutant proteins did not yield dimers, but showed evidence of linking to an unknown 140- to 160-kDa partner. Analysis of mature virions demonstrated that both N- and C-terminal CA residues participated in dimer formation, suggesting that processed CA N- and C-termini are free to establish interprotein associations. Interestingly, N/B region mutant residues in mature virus particles did not crosslink to form dimers, but showed a novel crosslinked band, consistent with an interaction between the N/B tropism determining region and a cellular protein of 45-55 kDa.
McDermott
J
J
Vollum Institute and Department of Microbiology, Oregon Health Sciences University, Portland, OR 97201-3098, USA.
Karanjia
S
S
Love
Z
Z
Barklis
E
E
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D003432:Cross-Linking Reagents; D015683:Gene Products, gag; D011506:Proteins; D014763:Viral Matrix Proteins; D016282:Aspartic Acid Endopeptidases; C083201:protease, Moloney murine leukemia virus; D003545:Cysteine