Interactions of HIV-1 Gag with assembly cofactors.
HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.
Shkriabai
Nick
N
Center for Retrovirus Research, College of Pharmacy, The Ohio State University Health Sciences Center, Columbus, Ohio 43210, USA.
Datta
Siddhartha A K
SA
Zhao
Zhuojun
Z
Hess
Sonja
S
Rein
Alan
A
Kvaratskhelia
Mamuka
M
D000595:Amino Acid Sequence; D019921:Biotinylation; D004277:DNA, Single-Stranded / Q000737:chemistry; D015683:Gene Products, gag / Q000737:chemistry*; D015497:HIV-1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D023302:Protein Footprinting / Q000379:methods; D012343:RNA, Transfer / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004277:DNA, Single-Stranded; D015683:Gene Products, gag; D019269:Phosphatidylinositol 4,5-Diphosphate; D012343:RNA, Transfer