Mutation of the highly conserved Ser-40 of the HIV-1 p6 gag protein to Phe causes the formation of a hydrophobic patch, enhances membrane association, and polyubiquitination of Gag.

Friedrich Hahn,Sara Solbak,Ulrich Schubert,Jörg Votteler,Torgils Fossen,Pia Rauch,Melanie Friedrich,Christian Setz,Nils Frøystein,Petra Henklein

doi:10.3390/v6103738

Abstract

The HIV-1 p6 Gag protein contains two late assembly (l-) domains that recruit proteins of the endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane fission between the nascent virion and the cell membrane. It was recently demonstrated that mutation of the highly conserved Ser-40 to Phe (S40F) disturbs CA-SP1 processing, virus morphogenesis, and infectivity. It also causes the formation of filopodia-like structures, while virus release remains unaffected. Here, we show that the mutation S40F, but not the conservative mutation to Asp (S40D) or Asn (S40N), augments membrane association, K48-linked polyubiquitination, entry into the 26S proteasome, and, consequently, enhances MHC-I antigen presentation of Gag derived epitopes. Nuclear magnetic resonance (NMR) structure analyses revealed that the newly introduced Phe-40, together with Tyr-36, causes the formation of a hydrophobic patch at the C-terminal α-helix of p6, providing a molecular rationale for the enhanced membrane association of Gag observed in vitro and in HIV-1 expressing cells. The extended exposure of the S40F mutant to unidentified membrane-resident ubiquitin E3-ligases might trigger the polyubiquitination of Gag. The cumulative data support a previous model of a so far undefined property of p6, which, in addition to MA, acts as membrane targeting domain of Gag.

Highlights

The Gag polyprotein Pr55 of HIV-1 contains all structural components that are essential and sufficient for the formation of virus like particles (VLPs) [1,2]
We and others reported that specific mutation of the highly conserved serine 40 to phenylalanine (S40F) within p6 leads to impaired CA-SP1 processing resulting in an abnormal core morphology and in a reduced replication capacity, whereas virus release and binding of ALIX to p6 were not reduced [30,31]
Since phosphorylation is known to trigger ubiquitination [35], we investigated whether Ser-40 affects this modification of Gag, which was previously shown to be dependent only on the integrity of the PTAP late assembly (L-)domain [26,38,55]

Summary

Introduction

The Gag polyprotein Pr55 of HIV-1 contains all structural components that are essential and sufficient for the formation of virus like particles (VLPs) [1,2]. MA mediates targeting and binding of Gag to the plasma membrane and lines the inner shell of the mature virus particle [3,4]. S40F mutation into the psyngag background enhances the Gag ubiquitination at a similar rate when compared to the situation when Gag was expressed in the context of the HIV-1 genome (Figure 2B). The level of Gag ubiquitination for the S40F mutant was almost indistinguishable from that observed for the ∆PTAP mutant. HIV-1 proteins, and this ubiquitination occurs to an extent in cell and VLP fraction that is comparable to that of the ∆PTAP mutant. It has only been demonstrated that the phosphorylation of Thr-23 by Erk-2 is important for budding and maturation of virus particles [29], whereas the aPKC mediated phosphorylation of Ser-40 facilitates Vpr incorporation [28]

Methods

Results

Conclusion