Subcellular fractionation of wt Gag and M domain mutants.
The first set of experiments was designed to study the localization of Pr55Gag using biochemical fractionation techniques. Transfected COS-1 cells expressing Pr55Gag and M domain Gag mutants were homogenized as described in Materials and Methods, and P-10, P-100, and S-100 fractions were prepared by differential ultracentrifugation. Aliquots from each subcellular fraction were subjected to SDS-gel electrophoresis, and Pr55Gag was detected by Western blotting using an anti-p24CA polyclonal antibody. The results are shown in Table . Approximately 95% of wt Gag and 8N Gag fractionated in the P-10 and P-100 fractions. Most (75%) of the 8N Gag partitioned into the P-10 fraction, which may reflect formation of large aggregates or the interaction of 8N Gag with denser membranes due to altered subcellular localization (see below). In contrast, at least 40% of G2A Gag was found in the S-100 (cytosolic) fraction. The remaining G2A Gag in the P-10 and P-100 fractions may represent protein that is either transiently membrane bound or present in large cytoplasmic complexes.
TABLE 1 Subcellular localization of wt and mutant Pr55Gag in COScells
Subcellular fractionation by velocity sedimentation does not resolve macromolecular cytoplasmic complexes or aggregates from membrane-bound protein. Therefore, we performed sucrose flotation analyses of transfected cells expressing wt Gag and Gag mutant proteins (33
). As depicted in Fig. A, fractionation of S1 homogenates from cells expressing wt Pr55Gag
showed that virtually all of the protein floated up to the 10 to 65% sucrose interface. The blots were then stripped and reprobed with an anti-gp120Env polyclonal antibody. Greater than 97% of the HIV-1 Env protein (Fig. A) also floated up to the interface. We then subjected the individual subcellular P-10, P-100, and S-100 fractions to flotation analysis. Nearly all wt Gag from both P-10 and P-100 fractions floated up through the sucrose cushion to the 10 to 65% sucrose interface (Fig. A). In contrast, the small amount of wt Gag that was found in the S-100 fraction remained predominantly in the bottom part of the gradient after ultracentrifugation, consistent with this fraction representing membrane-free complexes.
FIG. 1 Fractionation of wt Pr55Gag by sucrose flotation analysis. COS-1 cells transfected with pHXB2ΔBalID25S, which bears the gene encoding wt Pr55Gag, were harvested 48 h posttransfection. Cell homogenates were fractionated and subjected to sucrose (more ...)
Flotation analyses were also performed on wt Gag expressed in HeLa S3 cells, where Gag expression levels are about 10-fold lower than in COS cells. Cells were transfected with pHXB2ΔBalID25S. Forty-eight hours posttransfection, S-1 cell homogenates were prepared and membranes were isolated by sucrose flotation assays as described in Materials and Methods. Greater than 74% of the Gag floated to the 10 to 65% sucrose interface (Fig. A). Taken together, these results clearly indicate that, at steady state, most of wt Pr55Gag is membrane bound.
Analysis of M domain Gag mutants on sucrose gradients.
We next analyzed the behavior of the M domain Gag mutants by sucrose flotation assays. Greater than 95% of the G2A Gag from the P-100 (Fig. B) or P-10 (not shown) fractions remained at the bottom of the gradient. This result is in agreement with that of others (14
) and is consistent with the nonmyristoylated protein being present in large cytosolic complexes. The gradient profile obtained with the 8N Gag mutant showed that the majority of the protein in the P-100 fraction (Fig. B) and the P-10 fraction (not shown) floated to the 10 to 65% sucrose interface. Since the 8N mutant contains an intact myristoylation site, it was likely that the presence of myristate at the N terminus contributed to membrane association. To verify that myristate was responsible for the membrane binding of 8N Gag, the protein was expressed in the presence of the myristoylation inhibitor 2-OH-Myr. Cells were homogenized as described above, and subcellular fractions were subjected to sucrose flotation. In the presence of 2-OH-Myr, the distribution of 8N Pr55Gag
in either the P-100 fraction (Fig. B) or the S-1 fraction (not shown) shifted to the bottom of the gradient, with only ~10 to 15% of the total 8N Gag remaining at the 10 to 65% interface. Likewise, treatment of COS cells expressing wt Gag in the presence of 2-OH-Myr resulted in a shift of nearly all of the protein to the bottom of the tubes (not shown). We conclude that both wt Gag and 8N Gag proteins are membrane associated, whereas nonmyristoylated Gag is largely present in cytosolic complexes. The membrane binding of wt and 8N Gag proteins is dependent on the presence of myristate, implying that the myristate moiety is exposed and available for insertion into the lipid bilayer.
Confocal microscopy reveals the presence of wt Pr55Gag primarily at the plasma membrane.
In order to determine the subcellular localization of Gag more precisely, we analyzed cells expressing wt Gag and Gag mutants by laser scanning confocal microscopy. A Rev-independent construct expressing Pr55Gag fused to green fluorescent protein (pGag-EGFP) was used to observe Gag directly in live cells. This construct expresses Pr55Gag without other viral proteins; viral particles are released at levels similar to those obtained with the proviral pHXB2 expressing wt Pr55Gag (not shown). COS-7 cells transfected with pHXB2-derived constructs expressing wt Pr55Gag, G2A Gag, and 8N Gag were fixed 48 h posttransfection and stained for immunofluorescence.
Figure shows a z stack of wt Pr55Gag expressed from live cells transfected with pGag-EGFP (top). The wt Pr55Gag displayed a punctate pattern localized at the cell surface; the strongest FITC fluorescence was observed in the earlier z-stack slices most distal from the coverslip, where the nuclear fluorescence was minimal. Deeper slices inside the cell, where nuclear staining was maximal, showed diminished Gag fluorescence. Surface staining of wt Gag was confirmed by comparison with Nomarski optics (not shown). Identical results were obtained from cells transfected with pHXB2 expressing wt Gag that had been fixed prior to staining (not shown). We also examined HeLa S3 cells, which have a more spherical morphology. HeLa S3 cells transfected with either pHXB2 expressing wt Pr55Gag or pGag-EGFP exhibited distinct plasma membrane fluorescence (Fig. , bottom). These results clearly indicate that Pr55Gag is predominantly localized at the plasma membrane.
FIG. 2 Immunofluorescence of Pr55Gag in live and fixed COS-7 and HeLa S3 cells. (Top) Confocal microscopy analysis of live COS-7 cells transfected with wt Gag-EGFP and stained with Hoechst as described in Materials and Methods. A composite z stack corresponding (more ...) M domain Pr55Gag mutants exhibit altered cellular localization.
We next examined the cellular localization of the Gag mutants. COS-7 cells were transfected with pHXB2 expressing G2A Gag or 8N Gag and analyzed by confocal microscopy. Single confocal sections are shown in Fig. . The nonmyristoylated G2A Gag mutant (B) was dispersed throughout the cell except in the nucleus. The staining pattern of G2A Gag appeared flocculent and differed from the diffuse pattern seen with the soluble green fluorescent protein (Fig. A). Superimposition of the FITC images with Nomarski optics confirmed that the G2A staining did not extend to the plasma membrane (not shown). Identical results were obtained with live COS cells transfected with pGag-EGFP and treated with 2-OH-Myr (Fig. C) or with 2-OH-Myr-treated COS cells transfected with pHXB2 expressing wt Gag (not shown) or 8N Gag (Fig. F). Thus, it is likely that the images in Fig. B represent G2A Gag in intracellular cytoplasmic aggregates.
FIG. 3 Immunofluorescence of Pr55Gag and M domain Gag mutants. Single confocal sections of transfected COS-7 cells are shown. (A) Cells transfected with pEGFP; (B) cells transfected with pHXB2ΔBalID25S expressing G2A Gag; (C) cells transfected with pGag-EGFP (more ...)
In cellular fractionations about 40% of G2A Gag is present in the soluble S-100 fraction. If the nonmyristoylated Gag present in the S-100 fraction represents small cytosolic complexes, it should be released by gentle cell permeabilization. pGag-EGFP-transfected COS cells were left untreated or were treated with 2-OH-Myr, followed by incubation with the membrane permeabilizer streptolysin O (25
). The untreated wt pGag-EGFP-transfected cells that were permeabilized with streptolysin O maintained the punctate staining at the cell surface (not shown). In contrast, permeabilization of live cells expressing 2-OH-Myr-treated (nonmyristolyated) wt Gag-EGFP with streptolysin O revealed a different pattern from that of the unpermeabilized cells, in that the remaining nonmyristoylated Gag was distributed in very large aggregates throughout the cytoplasm (compare Fig. C and D). These data support the notion that nonmyristoylated Gag forms cytosolic complexes, some of which are small enough to be released through the 30-nm pores created by streptolysin O.
Analysis of the cellular distribution of 8N Pr55Gag showed intracellular perinuclear staining polarized on one side of the nucleus (Fig. C), which is consistent with the finding that 8N Gag was enriched in the cellular fraction produced by centrifugation at 10,000 × g (Table ). The morphology of the perinuclear fluorescence in 8N Pr55Gag-transfected COS cells varied considerably. Most cells displayed a crescent shape fluorescence pattern, while some cells displayed a very round, cylindrical structure reminiscent of the Golgi apparatus and other cells presented fragmented fluorescence distributed around the nucleus and included vesicle-like structures dispersed through the cytosol. These observations were consistently seen and cannot be attributed to differences in protein expression levels among the cells. Some minor surface staining was seen as well, perhaps a reflection of the ability of the 8N Gag mutant to produce viral particles at less than 10% of the levels obtained with wt Pr55Gag.
In order to determine the intracellular localization of the 8N Pr55Gag protein more precisely, monoclonal antibodies against cellular markers for ER (Grp78), cis and medial Golgi (p115 and GM130), and coatomer (COP-I) were used. A plasmid encoding TGN38 fused to the cytoplasmic domain of the interleukin-2 receptor (CD25) was used in cotransfection experiments to visualize the TGN. The fusion protein was visualized by staining with an anti-CD25 monoclonal antibody. Comparison of 8N Gag localization with Grp78, a resident ER protein, is shown in Fig. . Image rendering of the 8N Gag-Grp78 confocal data files was done through deconvolution methods and showed no overlap between 8N Gag and the ER. The staining pattern of p115, a medial Golgi marker protein, is also shown in Fig. (middle). No costaining of 8N Gag with p115 or any of the other Golgi markers used was observed. It should be noted that cells expressing 8N Pr55Gag from the HXB2 proviral constructs displayed an abnormal Golgi morphology relative to nonexpressing cells. This may be attributed to the expansion of the Golgi to accommodate the processing of other HIV proteins such as Env in the ER and Golgi compartments. The location of 8N Gag relative to the Golgi varied among the three distinct morphologies seen (Fig. , middle). In the cells displaying round, compact staining, 8N Gag fluorescence was surrounded by the Golgi; in the cells with the crescent 8N Gag pattern, the Golgi became diffused and expanded and its localization was found to be internal to, but not to overlap, the 8N Gag fluorescence. Finally, in cells where 8N Gag exhibited a scattered, vesicle-like morphology, there was no colocalization between the Golgi and 8N Gag. Incubation of 8N Gag-transfected cells with the Golgi-disrupting agent brefeldin A showed no obvious change in the 8N Gag fluorescence, confirming that the 8N Gag mutant localization does not overlap the Golgi apparatus (not shown). Fluorescence staining of 8N Pr55Gag-transfected cells partially overlapped the TGN; however, the z stacking of these cells revealed that the majority of the TGN was directly below the 8N Gag in the cells analyzed (Fig. , bottom). We conclude that membrane-bound 8N Gag localizes at or near the Golgi and TGN-proximal region. Although there is partial overlap of 8N Gag with TGN38 fluorescence, the origin of the membranes to which 8N Gag is bound is unclear.
FIG. 4 Intracellular localization of 8N Gag and costaining with ER and Golgi markers. Single confocal sections are shown for cells transfected with pHXB2 expressing and 8N Gag stained with cellular markers for ER (rhodamine) and Golgi and TGN (Cy 5) (left column), (more ...) Colocalization of wt Pr55Gag with Env at the cell surface.
Since Env glycosylation and processing occur in the ER and Golgi compartments, we used double labeling to examine the localization of 8N Gag and Env by confocal microscopy. Partial overlap of 8N Gag with Env was observed in cells permeabilized and stained for 8N Gag and Env (not shown). We then examined the distribution of Env at the cell surface, which represents a small fraction of the total envelope protein expressed in COS cells. Formalin-fixed cells were first incubated with an anti-Env monoclonal antibody and a rhodamine-conjugated secondary antibody to label Env proteins exposed at the cell surface. Cells were then permeabilized and stained with anti-Gag and an FITC-conjugated antibody. Interestingly, results of surface staining of Env differed significantly between cells transfected with pHXB2 expressing wt Gag and 8N Gag. In cells expressing wt Gag, Env surface staining was punctate and overlapped the punctate staining of wt Gag (Fig. , top). In contrast, redirection of 8N Gag assembly to intracellular sites resulted in diffuse staining of Env at the surface of the cell (Fig. , bottom), with no overlap between 8N Gag and Env staining. Similarly, diffuse surface staining of Env was also observed in cells expressing G2A Gag (not shown).
FIG. 5 wt Gag directs surface localization of Env to the sites of viral assembly. Cell surface staining of Env in COS cells transfected with pHXB2 expressing wt Gag (top) and 8N Gag (bottom) is shown. Transfected cells were fixed and stained for plasma membrane-associated (more ...)
In order to confirm that 8N Gag expression did not alter the integrity of the ER and/or Golgi, cells were fixed and stained with antibodies against endogenous plasma membrane proteins β-glycan (transforming growth factor β RIII) and HLA class I (β2-microglobulin), which traffic through the secretory pathway. Confocal analysis showed no change in expression patterns of either marker in pHXB2-transfected or nontransfected cells, ruling out a functional defect in the Golgi caused by 8N Gag (not shown). These findings strongly suggest that the presence of Gag at the plasma membrane promotes the recruitment of Env to the site of assembly.