Real-Time Visualization of HIV-1 GAG Trafficking in Infected Macrophages

HIV-1 particle production is driven by the Gag precursor protein Pr55Gag. Despite significant progress in defining both the viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of assembly in the infected cell remains to be elucidated. The Gag trafficking itinerary in primary monocyte-derived macrophages is especially poorly understood. To define the site of assembly and characterize the Gag trafficking pathway in this physiologically relevant cell type, we have made use of the biarsenical-tetracysteine system. A small tetracysteine tag was introduced near the C-terminus of the matrix domain of Gag. The insertion of the tag at this position did not interfere with Gag trafficking, virus assembly or release, particle infectivity, or the kinetics of virus replication. By using this in vivo detection system to visualize Gag trafficking in living macrophages, Gag was observed to accumulate both at the plasma membrane and in an apparently internal compartment that bears markers characteristic of late endosomes or multivesicular bodies. Significantly, the internal Gag rapidly translocated to the junction between the infected macrophages and uninfected T cells following macrophage/T-cell synapse formation. These data indicate that a population of Gag in infected macrophages remains sequestered internally and is presented to uninfected target cells at a virological synapse

Global rescue of defects in HIV-1 envelope glycoprotein incorporation: implications for matrix structure.

The matrix (MA) domain of HIV-1 Gag plays key roles in membrane targeting of Gag, and envelope (Env) glycoprotein incorporation into virions. Although a trimeric MA structure has been available since 1996, evidence for functional MA trimers has been elusive. The mechanism of HIV-1 Env recruitment into virions likewise remains unclear. Here, we identify a point mutation in MA that rescues the Env incorporation defects imposed by an extensive panel of MA and Env mutations. Mapping the mutations onto the putative MA trimer reveals that the incorporation-defective mutations cluster at the tips of the trimer, around the perimeter of a putative gap in the MA lattice into which the cytoplasmic tail of gp41 could insert. By contrast, the rescue mutation is located at the trimer interface, suggesting that it may confer rescue of Env incorporation via modification of MA trimer interactions, a hypothesis consistent with additional mutational analysis. These data strongly support the existence of MA trimers in the immature Gag lattice and demonstrate that rescue of Env incorporation defects is mediated by modified interactions at the MA trimer interface. The data support the hypothesis that mutations in MA that block Env incorporation do so by imposing a steric clash with the gp41 cytoplasmic tail, rather than by disrupting a specific MA-gp41 interaction. The importance of the trimer interface in rescuing Env incorporation suggests that the trimeric arrangement of MA may be a critical factor in permitting incorporation of Env into the Gag lattice

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

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

Evidence that Productive Human Immunodeficiency Virus Type 1 Assembly Can Occur in an Intracellular Compartment▿ †

Human immunodeficiency virus type 1 (HIV-1) assembly occurs predominantly at the plasma membrane of infected cells. The targeting of assembly to intracellular compartments such as multivesicular bodies (MVBs) generally leads to a significant reduction in virus release efficiency, suggesting that MVBs are a nonproductive site for HIV-1 assembly. In the current study, we make use of an HIV-1 Gag-matrix mutant, 29/31KE, that is MVB targeted. We previously showed that this mutant is severely defective for virus particle production in HeLa cells but more modestly affected in primary macrophages. To more broadly examine the consequences of MVB targeting for virus production, we investigated 29/31KE particle production in a range of cell types. Surprisingly, this mutant supported highly efficient assembly and release in T cells despite its striking MVB Gag localization. Manipulation of cellular endocytic pathways revealed that unlike Vpu-defective HIV-1, which demonstrated intracellular Gag localization as a result of Gag endocytosis from the plasma membrane, 29/31KE mutant Gag was targeted directly to an MVB compartment. The 29/31KE mutant was unable to support multiple-round replication; however, this defect could be reversed by truncating the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 and by the acquisition of a 16EK change in matrix. The 16EK/29/31KE matrix mutant replicated efficiently in the MT-4 T-cell line despite maintaining an MVB-targeting phenotype. These results indicate that MVB-targeted Gag can be efficiently released from T cells and primary macrophages, suggesting that under some circumstances, late endosomal compartments can serve as productive sites for HIV-1 assembly in these physiologically relevant cell types

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