Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

Abstract

BackgroundThe HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6.ResultsConsistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site.ConclusionsOverall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.

Comprehensive Mutational Analysis Reveals p6 Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

“…Votteler et al (26) reported a processing and infectivity phenotype for an S40F variant of p6. This nonconservative mutation was chosen to avoid potentially confounding changes in the overlapping pol region.…”

“…1A and B (see Figure S1 and Tables S1 and S2 in the supplemental material), we selected residues S14, T23, S25, and S51 for mutagenesis. Residue S40 was omitted because a detailed analysis of an HIV-1 p6 mutant, S40F, had recently been performed (26). Residue T8 was not included because it is part of the essential PTAP motif (11), and residue S43 was not included because an S43A mutation had no apparent effect on HIV-1 release (16).…”

“…Residue T23 of p6 can be phosphorylated by Erk kinase, and mutation of this residue was reported to result in a late-domain-deficient phenotype, with viral buds arrested at the plasma membrane and reduced bulk particle release (25). More recently, Votteler and coworkers (26) reported that an S40F mutation in p6 resulted in aberrant HIV-1 core formation and reduced viral infectivity, while bulk release efficiency was unaffected. A mechanistic connection between phosphorylation at either of these sites and HIV-1 release has not been identified, however.…”

Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression

“…The overrepresentation of the 483L/484Y motif in the intersubtype recombinants suggests that it is advantageous and may partially contribute to the enhanced replication capacity of these viruses. Residues 483 and 484 are essential residues in a late domain for binding the Alix host protein (65), which acts in concert with the primary budding factor Tsg101 to mediate viral budding (66). Mutants with disrupted Alix binding have significantly reduced particle production and infectivity, demonstrating an important role for Alix in HIV-1 replication (66).…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…Gag may interrupt the proteolytic cleavage between capsid Gag and SP1 Gag (552). (iii) Neutralizing responsiveness to Nef and glycosylated Gag is determined by GP120 V1/V2 regions (344), whereas it remains unclear whether GP120 physically interacts with Nef or glycosylated Gag.…”