Identification of a second-site mutant capable of rescuing diverse Env-incorporation defective mutants.

<p>(A) Jurkat cells were transfected with the indicated molecular clones. At 2-day intervals the cells were split and samples of media were assayed for RT activity. Virus from the WT and 16EK peaks was normalized by RT then used to infect naïve Jurkat cells and replication of the second passage was followed as described above. Genomic DNA was extracted from cells at the time of peak replication in the 16EK samples after both first and second passage cultures, and the MA coding region was amplified by PCR and subjected to DNA sequencing, revealing the original (16EK) and second-site compensatory (62QR) mutations. (B) Jurkat cells were transfected with the indicated molecular clones and replication was monitored as in (A). (C+E) 293T cells were transfected with the indicated molecular clones. At 24 h, supernatants were filtered then virions were pelleted, lysed, and probed by western blotting for gp41 and CA. (D+F) Supernatants were harvested and assayed for infectivity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Env incorporation was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity and Env incorporation are expressed relative to the WT value. n = 3, +/− SEM.</p

The effect of mutations at the trimer interface on rescue of Env incorporation.

<p>293T cells were transfected with the indicated molecular clones. At 24<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity is expressed relative to the WT value. Supernatants were also filtered and virions pelleted, lysed, and probed by western blotting for gp41 and CA. Env incorporation was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a> and is indicated relative to WT. Representative blots are shown below each graph. n = 5–7, +/− SEM. (A) Ala mutants of S66 and T69. (B) Arg mutants of S66 and T69.</p

Replication of S66 and T69 mutants in Jurkat cells.

<p>Jurkat cells were transfected with the indicated molecular clones. At 2-day intervals the cells were split and samples of media were assayed for RT activity. In each graph of WT pNL4-3, 12LE, 62QR or 12LE/62QR mutations are combined with (A) WT; (B) 66SA; (C) 69TA; (D) 66SR; (E) 69TR. (F) 293T cells were co-transfected with the indicated molecular clones and vectors expressing HIV-1 Env or VSV-G. At 24 h, supernatants were harvested and assayed for infectivity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. n = 3, +/− SEM.</p

Vertical scanning of MA residue 62 to determine effects on Env incorporation and ability to rescue Env-incorporation-defective mutants.

<p>(A) HeLa cells were transfected with the molecular clones indicated. Virus release efficiency was determined by metabolic labeling with ³⁵S[Met/Cys] as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. n = 3, +/− SEM. (B) 293T cells were transfected with the indicated molecular clones. At 24 h, supernatants were filtered then virions pelleted, lysed, and probed by western blotting for gp41 and CA. (C) Supernatants from (B) were harvested and assayed for infectivity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Env incorporation was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity and Env incorporation are expressed relative to the WT value. n = 6, +/− SEM. (D) Jurkat cells were transfected with the indicated molecular clones. At 2-day intervals the cells were split and samples of media were assayed for RT activity.</p

Schematic of MA monomers (blue), organized into a hexamer of trimers, adapted from Alfadhli <i>et al.</i> Virology (2009) [<b>35</b>].

<p>Under normal circumstances the Gag molecules in a particle are homogeneous, all possessing the same sequence (WT or mutant). To examine phenotypic dominance between the WT Gag, Env-incorporation-defective mutants, and 62QR, heterogeneous particles were produced by co-transfecting two proviral DNAs. The hypothetical MA arrangements are indicated as follows: (A) WT MA. (B) The Env-incorporation-defective mutations (red) cluster at the tips of the MA trimer. (C) The location of the Env-incorporation-defective mutations is indicated as for (B); the green circle near the trimer interface indicates the compensatory mutation 62QR. (D+E) Heterogeneous particles based on a 1∶1 mix of either WT with a defective mutant (D) or 62QR with a defective mutant (E). By contrast with the homogeneous particles (A–C), in D and E each MA molecule possesses a maximum of one mutation, it may be either defective or 62QR, but not both.</p

62QR is resistant to dominant-negative inhibition by defective Gag mutants in heterogeneous particles.

<p>(A) 293T cells were co-transfected with molecular clones expressing WT or 62QR Gag with the 12LE molecular clone in the ratios indicated (µg∶µg of DNA). At 24 h, supernatants were harvested and assayed for infectivity as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#s4" target="_blank">Materials and Methods</a>. Infectivity is expressed relative to the WT value. n = 4, +/− SEM. (B–E) 293T cells were co-transfected with molecular clones expressing WT or 62QR Gag with Env-incorporation-defective Gag in the ratios indicated (µg∶µg of DNA). Infectivity relative to WT was determined as described for (A). n = 3, +/− SEM. (F) 293T cells were co-transfected with molecular clones expressing WT or 62QR Gag with d8 gp41 in the ratios indicated (µg∶µg of DNA). Infectivity relative to WT was determined as described for (A) n = 4, +/− SEM.</p

Potential for intersubunit interactions in the MA trimer.

<p>MA trimer as described in Hill <i>et al.</i> PNAS (1996), showing (A) a top-down view and (B) a side-on view <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#ppat.1003739-Hill1" target="_blank">[32]</a>. Env incorporation defects, red; Q62, green; Ser66 and Thr69, cyan. (C) Close-up view of boxed area from (A), showing Q62 side chain (green), and the side chains of S66 and T69 (cyan) of a second MA monomer. Chain a, black; chain b, gray. Distances between the oxygen atoms of Q62 carbonyl group and the S66 and T69 hydroxyl groups are indicated. Modeled configurations for R62 (D) K62 (E), R66 (F), R66, in combination with R62 (G) and R69 (H). Mutagenesis and rendering performed using MacPymol <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003739#ppat.1003739-Pymol1" target="_blank">[70]</a>.</p