Impact of Mutations in Highly Conserved Amino Acids of the HIV-1 Gag-p24 and Env-gp120 Proteins on Viral Replication in Different Genetic Backgrounds

<div><p>It has been hypothesized that a single mutation at a highly conserved amino acid site (HCS) can be severely deleterious to HIV in most if not all isolate-specific genetic backgrounds. Consequently, potentially universal HIV-1 vaccines exclusively targeting highly conserved regions of the viral proteome have been proposed. To test this hypothesis, we examined the impact of 10 Gag-p24 and 9 Env-gp120 HCS single mutations on viral fitness. In the original founder sequence of the subject in whom these mutations were identified, all Gag-p24 HCS mutations significantly reduced viral replication fitness, including 7 that were lethal. Similar results were obtained at 9/10 sites when the same mutations were introduced into the founder sequences of two epidemiologically unlinked subjects. In contrast, none of the 9 Env-gp120 HCS mutations were lethal in the original founder sequence, and four had no fitness cost. Hence, HCS mutations in Gag-p24 are likely to be severely deleterious in different HIV-1 subtype B backgrounds; however, some HCS mutations in both Gag-p24 and Env-gp120 fragments can be well tolerated. Therefore, when designing HIV-1 immunogens that are intended to force the virus to nonviable escape pathways, the fitness constraints on the HIV segments included should be considered beyond their conservation level.</p></div

Impact of non-lethal HCS mutations on viral replication fitness.

<p>A) Net growth rates of individual <i>gag</i> viruses in competitions. B) Net growth rate differences between the mutants and <i>vifB</i> founder <i>gag</i> viruses in competitions. gagVifA and gagVifB have founder virus <i>gag</i>-p24 (HIV-1_HXB2 nucleotides 1089–2022) inserted into the NL4-3 backbone and differ only by 6 synonymous site mutations in the <i>vif</i> gene. Mutant and founder viruses in the <i>vifA</i> backbone were competed against founder viruses in the <i>vifB</i> backbone. Reported are the means and 95% confidence intervals from competition experiments carried out in triplicate. * indicates significant differences (<i>p</i><0.05, calculated using the method developed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Liu2" target="_blank">[19]</a>) between (net growth rate difference between the mutant and <i>vifB</i> founder viruses) and (net growth rate difference between <i>vifA</i> and <i>vifB</i> founders). A value of that was significantly smaller was interpreted as a fitness cost from the mutation; a value of that was significantly greater was interpreted as a fitness gain; otherwise it was interpreted as no fitness impact. C) Correlation between viral replication fitness and TCID₅₀ in Env-gp120 HCS mutants.</p

HCS mutations located in known CTL^a.

a<p>In the examined regions, 44 Gag and 17 Env CTL epitopes have been optimally defined <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Llano1" target="_blank">[33]</a>, among which 4 Gag and 3 Env epitopes are known to be restricted by HLA alleles of PIC87014 (A*0201, A*2501, B*1801, B*5101, Cw*0102, and Cw*1203) and all 7 were recognized by the subject <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Liu3" target="_blank">[20]</a>. PIC87014 also recognized 2 additional Env-gp120 epitopes (NW9, Env 88–96 and TL9, Env 341–349, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone-0094240-g002" target="_blank">Figure 2</a>).</p>b<p><u>F</u>RDYVD<u>R</u>FYK (FK10, Gag 293–302) is B*1801 restricted. YCAPAGFAI<u>L</u> (YL10, Env 271–226) is Cw*0102 restricted. The underlined letters indicate the location of the HCS mutations.</p

Env-gp120 founder sequence and HCS mutations of PIC87014.

<p>The Env founder sequence (HIV-1_HXB2 Env 1–525) of PIC87014 is shown. All sequences were obtained from previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Troyer1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Liu3" target="_blank">[20]</a>. # indicates the start of the sequence that was incorporated into the NL4-3 backbone. The letter marked with a ∇represents the HCS (Env 312_HXB2) in the founder sequence (alanine) that is different from the group M or subtype B consensus (glycine). The sequences of the following optimally defined epitopes were different from the founder sequence: A*0202 restricted <u>RG</u>PGRAF<u>VTI</u> (311–320) and A*3002 restricted HI<u>G</u>PGRAFY (Env 310–318). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone-0094240-g001" target="_blank">Figure 1</a> for other conventions.</p

Known roles of the examined HCS in HIV-1 structure and function.

a<p>MHR is a sequence found in many retroviruses and in the yeast transposon Ty3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Patarca1" target="_blank">[40]</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Hansen1" target="_blank">[42]</a>.</p

Impact of Gag-p24 and Env-gp120 HCS mutations on viral growth and compensation by VS mutations.

<p>cDNA copy numbers (black bars), p24 levels (gray bars) and TCID₅₀ (open bars) derived from transfection supernatants of the <i>gag</i> founders and mutants of PIC87014 (A), PIC71101 (B) and PIC83747 (C), as well as the <i>env</i> founder and mutants of PIC87014 (D). Mutations that resulted in undetectable TCID₅₀ (<38 IU/ml, the dotted horizontal lines represent this threshold) were considered lethal. E) Compensatory impact of VS mutations that were linked to GagI261V. Gag215V, Gag223I, Gag268L and Gag383F are mutants of PIC87014gag821-5 with individual VS mutations reverted to the PIC87014 founder state. The +/− sign underneath each virus represents the presence/absence of the indicated mutation. ND, not determined. F) Frequencies of the VS mutations in the plasma-derived viral sequences of PIC87014 over time.</p

Gag-p24 founder sequences and HCS and VS mutations.

<p>Gag-p24 sequences (HIV-1_HXB2 Gag 94–411) from PIC87014 (F, founder sequence from day 8 DPS and 821-5, sequence of clone 5 from 821 DPS), and founder sequences from PIC71101 (7 DPS) and PIC83747 (6 DPS) are shown. All sequences were obtained from previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Troyer1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094240#pone.0094240-Liu3" target="_blank">[20]</a>. Numbers in the right column correspond to amino acid positions in HIV-1_HXB2 Gag. * indicates the start and end of p24. All HCS are underlined, and the examined HCS and VS mutations are in red and blue boxes, respectively. The yellow highlights represent the CTL epitopes recognized by PIC87014, with their HLA restricting elements indicated (HLA alleles of PIC87014 are A*0201, A*2501, B*1801, B*5101, Cw*0102, and Cw*1203). The bracketed regions define the boundaries of optimally defined epitopes containing the HCS examined here, with the HLA restricting elements indicated. The sequences of the following optimally defined epitopes were different from the founder sequence of PIC87014: B*3501 restricted PPIPVG<u>D</u>IY (Gag 254–262), Cw*18 restricted VRMYSP<u>V</u>SI (Gag 274–282), A*2402 restricted RDYVDRF<u>F</u>KTL (Gag 294–304) and B*1503/Cw*0303/Cw*0304 restricted YVDRF<u>F</u>KTL (Gag 296–304), the underlined letters indicating the differences.</p