Interactions of HIV-1 Gag with Assembly Cofactors

Abstract

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.

HIV-1 Gag: Flipped out for PI(4,5)P 2

“…Analysis of phosphoinositidebinding preferences may reveal whether the effect of HIV-1 MA mutations on Gag localization can be explained by a switch in phosphoinisitide binding from PI(4,5)P 2 to other members of this family of lipid molecules. Given that residues in the highly basic domain of MA appear to be crucial for PI(4,5)P 2 binding (9,26) and that analogous basic patches are evident in the MA domains of a number of other retroviruses (27), it will be of great interest to determine the importance of PI(4,5)P 2 in promoting the assembly of retroviruses other than HIV-1. From a more practical perspective, the structural information provided by Saad et al (9) may help in the design of inhibitors that disrupt HIV-1 replication by interfering with the interaction between Gag and PI(4,5)P 2 .…”

Traveling-wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking

“…The observed y-ion series from the product ion spectrum of the ATD Region 1 for P1 (Figure 1c) is consistent with a dead-end modification on the second Lys residue (unmodified y 4 and modified y 5 *, y 6 *, y 7 *, and y 8 * ions). Conversely, the ATD Region 2 product ion spectrum (Figure 1d) displays unmodified y 4 , y 5 , y 6 , y 7 , and y 8 ions, demonstrating the presence of the dead-end modification on the first Lys residue. No unmodified y 5 , y 6 , y 7 , or y 8 ions were observed in the spectrum of ATD Region 1 (Figure 1c), neither were modified y 5 *, y 6 *, y 7 *, y 8 * ions in the spectrum of ATD Region 2 (Figure 1d), indicating no contamination of the spectrum of one isomer with fragment ions of the other and vice versa.…”

“…Combining over ATD Regions 1 and 2 separately results in different spectra, compatible with two isomeric structures containing the dead-end modification on different Lys residues, which may be present but are not as separated as observed for P1. For P2, the ATD Region 1 product ion spectrum (Figure 2c) displays unmodified y 5 and modified y 6 *, y 7 *, and y 8 *, whereas the ATD Region 2 product ion spectrum (Figure 2d) displays unmodified y 5 , y 6 , y 7 , and y 8 ions. As no complete mobility separation was achieved for P2, one extracted product ion spectrum contains minor, low intensity fragment ions from the other isomer and vice versa, but these fragments have intensities below 10%.…”

“…The spectrum obtained by combining over ATD Region 2 (Figure 3b) shows unmodified y 4 , y 5 , y 6 , y 7 , and y 8 ions (Figure 3d), which demonstrates that the dead-end modification is either at the N-terminus or first Lys residue. Inspection of the low m/z region of the spectrum of Figure 3d reveals unmodified b 2 and b 3 ions, as well as modified b 1 * and b 2 * ions, indicating that ATD Region 2 represents dead-end modifications that occurred either at the N-terminus or first Lys residue.…”

“…As each one of these species gathers different structural features regarding protein structure [19], the correct detection and annotation of which residue is linked to the cross-linker is essential to obtain reliable data for protein structure elucidation [20 -24]. Dead-end species can be used as structural probes as they may yield information about residue exposure or solvent accessibility [6,7,25,26].…”