Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6Gag

Abstract

BackgroundHIV-1 formation is driven by the viral structural polyprotein Gag, which assembles at the plasma membrane into a hexagonal lattice. The C-terminal p6Gag domain harbors short peptide motifs, called late domains, which recruit the cellular endosomal sorting complex required for transport and promote HIV-1 abscission from the plasma membrane. Similar to late domain containing proteins of other viruses, HIV-1 p6 is phosphorylated at multiple residues, including a highly conserved serine at position 40. Previously published studies showed that an S40F exchange in p6Gag severely affected virus infectivity, while we had reported that mutation of all phosphorylatable residues in p6Gag had only minor effects.FindingsWe introduced mutations into p6Gag without affecting the overlapping pol reading frame by using an HIV-1 derivative where gag and pol are genetically uncoupled. HIV-1 derivatives with a conservative S40N or a non-conservative S40F exchange were produced. The S40F substitution severely affected virus maturation and infectivity as reported before, while the S40N exchange caused no functional defects and the variant was fully infectious in T-cell lines and primary T-cells.ConclusionsAn HIV-1 variant carrying a conservative S40N exchange in p6Gag is fully functional in tissue culture demonstrating that neither S40 nor its phosphorylation are required for HIV-1 release and maturation. The phenotype of the S40F mutation appears to be caused by the bulky hydrophobic residue introduced into a flexible region.

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

“…Consistent with our previous studies, it has been shown by others that the efficiency of virus release was not affected by mutation of Ser-40 [ 29 , 30 , 31 , 32 ]. In contrast, the E0A mutant, which also increased Gag membrane binding, displayed a severe budding defect.…”

“…Containing 5 positive and 9 negative charges, p6 displays an unusually high charge density, which might also be controlled by phosphorylation. It has already been described that phosphorylation of p6 at position 40 by aPKC [ 36 ] and at residue Thr-23 by Erk-2 [ 35 ] plays a role in virus release and infectivity, although its biological function is still under debate [ 25 , 31 ].…”

“…It has already been shown that mutation of the highly conserved PTAP motif in p6 causes a severe budding defect [ 27 , 28 ]. Additionally, the mutation of Ser-40 in p6 to Phe, which frequently emerges upon antiviral treatment, delays CA-SP1 processing and enhances membrane association of Gag, but causes no defect in virus release [ 25 , 29 , 30 , 31 , 32 ]. The increase in membrane association of p6 mutants correlates with an augmented entry of Gag into the UPS (ubiquitin proteasome system) and results in enhanced MHC-I antigen presentation of Gag-derived epitopes [ 32 , 33 ].…”

The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility

“…Two substitutions that occurred at the same residue, S40F and S40A, were further investigated by engineering the individual substitutions into an HIV genome (pNL4-3). The substitution of F (S40F), where TCC = S to TTC = F, does not alter the sequence of the overlapping pol frame (TTC = F in the WT to TTT = F) but had an impact on several aspects of gag function [ 21 – 25 ]. The alanine (A) substitution (S40A), where TCC = S to GCC = A, in contrast, had a less deleterious effect on Gag processing, polyubiquitination or particle maturation morphology but altered the sequence at the p6*/PR cleavage site in the overlapping pol frame.…”

Post-Translational Modifications of Retroviral HIV-1 Gag Precursors: An Overview of Their Biological Role

“…In a further step, mass spectrometry indicated the phosphorylation of S488 residue [85,86]. Its substitution with a hydrophobic aromatic residue such as phenylalanine (F), which can occur sponta-neously during anti-retroviral treatments, was found to perturb CA-SP1 processing, virus morphogenesis, maturation and virion infectivity [87][88][89]. On the other hand, the substitution of S488 by another non phosphorylable residue, such as asparagine (N), displayed no global impact on infectivity, thus suggesting that the production of non-mature viral particles would not be due to the lack of phosphorylation, but by the substitution itself [89].…”