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

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

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

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

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

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

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

In COS Cells Vpu Can Both Stabilize Tetherin Expression and Counteract Its Antiviral Activity

<div><p>The interferon-inducible cellular protein tetherin (CD317/BST-2) inhibits the release of a broad range of enveloped viruses. The HIV-1 accessory protein Vpu enhances virus particle release by counteracting this host restriction factor. While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood. Here, we show that human tetherin is expressed at low levels in African green monkey kidney (COS) cells. However, Vpu markedly increases tetherin expression in this cell line, apparently by sequestering it in an internal compartment that bears lysosomal markers. This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin. Although Vpu stabilizes human tetherin in COS cells, it still counteracts the ability of tetherin to suppress virus release. The enhancement of virus release by Vpu in COS cells is associated with a modest reduction in cell-surface tetherin expression, even though the overall expression of tetherin is higher in the presence of Vpu. This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.</p></div

Vpu induces the degradation of human tetherin in 293T cells but stabilizes its expression in COS cells.

<p>293T (A) or COS (B) cells were transfected with vectors expressing HA-tagged human, mouse, rhesus, and agm tetherins without or with Vpu expression vector at a tetherin:Vpu DNA ratio of 1∶5. Total transfected DNA was held constant with empty vector. Truncated human tetherins that lack the cytoplasmic tail (delCT) or putative GPI anchor (delGPI) were also used to identify the regions of tetherin required for Vpu-mediated degradation. One day posttransfection, cells were lysed and subjected to western blot analysis with the indicated antibodies. Vpu decreased tetherin expression by 3.1 fold in 293T cells whereas it increased tetherin expression by ∼5-fold in COS cells; levels of the delCT mutant were increased ∼3-fold in COS cells and 1.6 fold in 293T cells by Vpu. (C) COS cells were transfected with HA-tagged human tetherin expression vector and cell lysates were subjected to western blot analysis with anti-HA or anti-tubulin antibodies. Vpu was also co-expressed to compare the pattern of tetherin expression. Molecular mass markers are shown on the right (in kDa). In this experiment, proteasomal inhibitors (MG132 and ALLN) increased the expression of the ∼23 kDa tetherin species by 3.2 fold, whereas lysosomal inhibitors (bafilomycin and concanamycin) rescued the expression of the ∼26 kDa tetherin species by 2.6 fold and the ∼23 kDa tetherin species by 1.7 fold compared to the no inhibitor control. Co-expression of Vpu increased the expression of the ∼26 kDa tetherin species by 5.2 fold and the ∼23 kDa tetherin species by 1.7 fold. (D) 293T, COS, and Vero cells were transfected with HA-tagged human tetherin expression vector in the absence and presence of Vpu expression vector, and one-day posttransfection cells were lysed and subjected to Western blot analysis as above. Vpu reduced the expression of tetherin by 2.4 fold in 293T cells, whereas the levels were increased by ∼20-fold in COS cells and ∼4-fold in Vero cells. Similar results were obtained in an independent experiment.</p

Vpu induces the sequestration of tetherin in COS cells.

<p>293T and COS cells were transfected with vector expressing HA-tagged tetherin alone or in combination with Vpu expression vector (1∶5 DNA ratio). Twenty-four h post-transfection, cells were fixed with 4% formaldehyde, permeabilized with methanol and stained with anti-HA (green), anti-Vpu (red), or DAPI (blue) and were analyzed with a Delta-Vision RT deconvolution microscope. Numbers represent the Pearson correlation coefficient (R) ± SD from 10–15 cells. Scale bars, 15 µm.</p