Compensatory substitutions restore normal core assembly in simian immunodeficiency virus isolates with Gag epitope cytotoxic T-lymphocyte escape mutations

Abstract

The evolution of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) as they replicate in infected individuals reflects a balance between the pressure on the virus to mutate away from recognition by dominant epitope-specific cytotoxic T lymphocytes (CTL) and the structural constraints on the virus' ability to mutate. To gain a further understanding of the strategies employed by these viruses to maintain replication competency in the face of the intense selection pressure exerted by CTL, we have examined the replication fitness and morphological ramifications of a dominant epitope mutation and associated flanking amino acid substitutions on the capsid protein (CA) of SIV/simian-human immunodeficiency virus (SHIV). We show that a residue 2 mutation in the immunodominant p11C, C-M epitope (T47I) of SIV/SHIV not only decreased CA protein expression and viral replication, but it also blocked CA assembly in vitro and virion core condensation in vivo. However, these defects were restored by the introduction of upstream I26V and/or downstream I71V substitutions in CA. These findings demonstrate how flanking compensatory amino acid substitutions can facilitate viral escape from a dominant epitope-specific CTL response through the effects of these associated mutations on the structural integrity of SIV/SHIV.

FIG. 1.

Amino acid sequence alignment of representative SIV and HIV-2 CA proteins. Sequences were obtained from the Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov) and previously published data. Threonine in position 47 corresponds to position 2 of the immunodominant Mamu-A^*01-restricted Gag p11C, C-M CTL epitope. The SIV p11C, C-M epitope is outlined in a box. Positions 26 and 71, highlighted in gray, are extraepitopic amino acid positions with substitutions that are associated with epitope mutations in position 47. The following representative viral sequences are shown: SHIV-89.6P (accession no. U89134), SAB1C (accession no. U04005), AGMTYO (accession no. AF395567), HIV2ST (accession no. U81836), D205 (accession no. X16109), and ABT96 (accession no. AF208027). The SIVmac239 strains represent sequences that were derived from Mamu-A^*01⁺ chronically infected rhesus monkeys, as published by Friedrich et al. (11).

FIG. 2.

Mutations I26V and I71V in gag restore the level of protein production to that of WT gag, 293T cells were transfected with each noted plasmid and supernatant, and cells were harvested 48 h later. SIV Gag p27 levels in supernatants (A) were determined by ELISA. Western blotting was performed to assess the amount of Gag in soluble cell lysates (B). Full-length unprocessed Gag is 55 kDa.

FIG. 3.

Mutations I26V and I71V restore the replication rate to that of the WT SIV. Rhesus monkey PBMCs were infected with equal amounts of various SIVmac239 clones (WT, I26V, T47I, I71V, I26V/T47I, T47I/I71V, and I26V/T47I/I71V) and supernatants were monitored for SIV p27 antigen. Values illustrated are means of duplicates.

FIG. 4.

Expression and purification of recombinant WT SIV capsid proteins. (A) Proteins were purified from E. coli by anion-exchange chromatography and eluted with a 0 to 1 M NaCl gradient as indicated by the dashed line. (B) Coomassie blue stain of the SDS-PAGE analysis of the following: lane 1, total cellular BL21(DE3)-RIL E. coli proteins prior to induced expression of the capsid protein; lane 2, total cellular bacterial proteins following induction; lanes 3 to 9, SIV CA fractions that were eluted, pooled, and concentrated. The 27-kDa SIV WT CA was optimally eluted at 280 to 320 mM NaCl. The protein preparations were >90% homogeneous. (C) The identity of the proteins was verified by Western blotting using a mouse monoclonal antibody, 55-2F12, directed against SIV CA. The SIV CA is 27 kDa. The positions of migration and molecular mass in kDa are indicated on the left.

FIG. 5.

In vitro assembly of mutant SIV capsid proteins. These panels show representative transmission electron micrographs of negatively stained structures formed by WT CA and CA containing p11C, C-M epitope and extraepitopic mutations. The WT CA polymerized into long and apparently flexible tubules that bend (A and B). Very little assembly was observed with the T47I (C) or I71V CA. The CA containing I26V alone consistently formed long, straight rods (D). I26V/T47I and I26V/T47I/I71V CA formed similar long rods. In contrast, SIV CA containing the T47I/I71V substitutions formed short, rigid rods (E and F). Photographs are shown at the indicated optical magnifications. Scale bars are 100 nm.

FIG. 6.

Transmission electron micrographs of WT and mutant SIVmac239 virions with Gag p11C, C-M and compensatory substitutions. Panels 1 to 4 show detailed magnified views of mature conical cores (panel 1), mature centric cores (panel 2), aberrantly condensed cores (panel 3), and immature/uncondensed cores (panel 4). Panel A shows an overview of a field containing WT viruses. Panel B shows viruses that contain the T47I mutation alone. Note that the majority of viruses contain aberrantly condensed cores, and there are fewer mature conical and centric cores compared to WT viruses. Panel C depicts viruses containing the I71V mutation alone, with the majority of virions containing large cores. Panel D illustrates immature, uncondensed viruses containing the T47A mutation alone, which have a tendency to tether to membranes. Panel E depicts viruses containing the I26V/T47I/I71V mutations. Viruses with I26V/T47I, T47I/I71V, and I26V/T47A/I71V mutations show similar core morphology to what is shown in panel E. Note that the proportion of virions that are immature and contain abnormally large cores is higher in panels B and C than in panels A and E. Thick solid arrows show representative conical cores, bullet arrows indicate representative centric cores, arrowheads point to aberrantly condensed cores, and arrows with thin shafts highlight immature, uncondensed cores. Scale bars are 100 nm.