Structural studies of HIV-1 Gag p6ct and its interaction with Vpr determined by solution nuclear magnetic resonance

The ability of human immunodeficiency virus type 1 (HIV-1) to egress from human cells by budding with the cell membrane remains a complex phenomenon of unclear steps. HIV-1 viral protein R (Vpr) incorporation in sorting virions relies greatly on the interaction with the group-specific antigen (Gag) C-terminal region, which encompasses protein p6. The complete role of p6 is still undetermined; however, it is thought that p6 interacts with protein core elements from the endosomal sorting complex ESCRT-1, known to sort ubiquitinated cargo into multivesicular bodies (MVB). The three-dimensional structure of the p6 C-terminus (p6ct) comprising amino acids 32-52, determined in this study using NMR methods, includes the region thought to interact with Vpr, i.e., the LXXLF sequence. Here we present new results indicating that the region which interacts with Vpr is the ELY(36) sequence, in the same region where mutational studies revealed that replacing Y36 with a phenylalanine would increase the infectivity of virions by 300-fold. The interaction of Vpr with an egg PC bilayer in the presence of p6ct measured by plasmon waveguide resonance (PWR) is approximately 0.8 microM, approximately 100 times stronger in the absence of p6ct. Our results suggests an interaction based on an ELYP(37) sequence bearing similarities with recently published results, which elegantly demonstrated that the HIV-1 Gag LYPx(n)LxxL motif interacts with Alix 364-702. Moreover, we performed a 60 ns molecular dynamics (MD) simulation of p6ct in DPC micelles. The MD results, supported by differential scanning calorimetry measurements in DMPC, show that p6ct adsorbs onto the DPC micelle surface by adopting a rather stable alpha-helix. Our results provide insights regarding the HIV-1 virion sorting mechanism, specifically concerning the interaction between p6 and Vpr. We also suggest that Gag p6 may adsorb onto the surface of membranes during the sorting process, a property so far only attributed to the N-terminal portion of Gag matrix (MA), which is myristylated. The implications of such a novel event provide an alternative direction toward understanding the assembly and escape mechanisms of virions, which have been undetected so far.