HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 µM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 µM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

The stability of HIV-2 Vpx and Vpr proteins is regulated by the presence or absence of zinc-binding sites and poly-proline motifs with distinct roles.

The Vpx and Vpr proteins of human immunodeficiency virus type 2 (HIV-2) are important for virus replication. Although these proteins are homologous, Vpx is expressed at much higher levels than Vpr. Previous studies demonstrated that this difference results from the presence of an HHCC zinc-binding site in Vpx that is absent in Vpr. Vpx has another unique region, a poly-proline motif (PPM) of seven consecutive prolines at the C-terminus. Using PPM point mutants of Vpx, this study demonstrated that these seven consecutive prolines are critical for suppressing proteasome degradation of Vpx in the absence of Gag. Both the PPM and the zinc-binding site stabilize Vpx but do so via different mechanisms. PPM and zinc-binding site mutants overexpressed in Escherichia coli aggregated readily, indicating that these motifs normally prevent exposure of the hydrophobic region outside the structure. Furthermore, introduction of the zinc-binding site and the PPM into Vpr increased the level of Vpr expression so that it was as high as that of Vpx. Intriguingly, HIV-2 has evolved to express Vpx at high levels and Vpr at low levels based on the presence and absence of these two motifs with distinct roles.

Zinc-binding site of human immunodeficiency virus 2 Vpx prevents instability and dysfunction of the protein.

Human immunodeficiency virus 2 Vpx coordinates zinc through residues H39, H82, C87 and C89. We reported previously that H39, H82 and C87 mutants maintain Vpx activity to facilitate the degradation of SAMHD1. Herein, the expression of Vpx mutants in cells was examined in detail. We demonstrated that the zinc-binding site stabilizes the protein to keep its function in virus growth when low levels of Vpx are expressed. At higher levels of expression, Vpx aggregation could occur, and zinc binding would suppress such aggregation. Among the amino acids involved in zinc coordination, H39 plays the most critical role. In summary, zinc binding appears to mitigate flexibility of the three-helix fold of Vpx, thereby preventing dysfunction.