Mutations in the alpha-helix directly C-terminal to the major homology region of human immunodeficiency virus type 1 capsid protein disrupt Gag multimerization and markedly impair virus particle production.
The X-ray crystallographic structure of HIV-1 capsid protein suggests that the dimer interface of the dimerization domain is mainly formed from a putative alpha-helix structure of 14 amino acids (Gag residues 311-324) and lies directly C-terminal to the capsid major homology region. We found that a deletion mutation in the alpha-helix drastically reduces virus particle production. Alanine-scanning mutagenetic analysis indicated that substitution mutations at residues Q311, V313, K314, W316, and M317 all impair virus particle production markedly. Membrane flotation assays suggested that some mutations in the dimer interface have slight effects on the efficient binding of Gag to membranes. Indirect immunofluorescence studies revealed that mutants defective in virus production exhibit a subcellular distribution pattern similar to that of wild-type. However, velocity sedimentation analysis showed that mutations significantly impairing virus particle production were also detrimental to Gag multimerization, suggesting that the impaired virus production may be due to a defect in Gag multimerization. These results support the proposal that residues in the capsid dimer interface play a crucial role in promoting Gag multimerization, possibly by facilitating stable Gag-Gag interactions.
Chu
Hung-Hao
HH
Department of Medical Research and Education, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei, 11217, Taiwan.
Chang
Yu-Fen
YF
Wang
Chin-Tien
CT
D036022:Capsid Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D015683:Gene Products, gag / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D014771:Virion; D014779:Virus Replication / Q000235:genetics
D036022:Capsid Proteins; D015683:Gene Products, gag