The Conserved Carboxy Terminus of the Capsid Domain of Human Immunodeficiency Virus Type 1 Gag Protein Is Important for Virion Assembly and Release

Abstract

The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an ␣-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.

Comparative study of the P2X gene family in animals and plants

“…As the result of evolution, some sites remain conserved due to their functional significance, while others are highly variable [41]. Such variability is likely to be advantageous to the organism [42].…”

“…Analyzing and detecting amino acid site under selective pressure is critical for understanding protein function and structure [41]. Past studies have shown that duplicated genes undergo three different kinds of destines after duplication events: neo-functionalization (gain new functions), sub-functionalization (subdivide the functions), and pseudogenization (loss of function) [65][66][67].…”

Mutations in the α-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

“…We adopted the protocol of Melamed et al [39] to analyze the cytoplasmic Gag complex. Briefly, cells were rinsed twice with PBS, pelleted and resuspended in 1 ml TEN buffer containing Complete protease inhibitor cocktail followed by homogenization as described above.…”

“…These data suggest that the C-terminal one-third of the gag sequence contains domains necessary for Gag assembly and release. There is an increasing amount of published data on mapping of the domains responsible for Gag-Gag interactions, and such studies have implicated the C-terminal CA and the downstream NC [36][37][38][39]. In particular, it has been shown that deletion or some substitution mutations in MHR markedly reduce virus particle production [27,38,40].…”

“…In particular, it has been shown that deletion or some substitution mutations in MHR markedly reduce virus particle production [27,38,40]. Several recent studies have shown that the extreme C-terminal sequence of CA and also SP1 play a key role in the process of Gag multimerization and virus assembly in HIV [36,39,41,42]. In a previous study, we found that a 14-codon deletion directly C-terminal to MHR not only markedly reduced virus particle production, but also significantly affected the incorporation of HIV-1 Gag-Pol into wild-type Gag particles [43], suggesting that this region may be functionally involved in both Gag/Gag and Gag-Pol/Gag interaction.…”