Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly-defective mutant human immunodeficiency virus type 1 and their association with membranes

Abstract

We have previously identified two distinct forms of putative viral assembly intermediate complexes, a detergent-resistant complex (DRC) and a detergent-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-infected CD4(+) T cells (Y. M. Lee and X. F. Yu, Virology 243:78-93, 1998). In the present study, the intracellular localization of these two viral assembly intermediate complexes was investigated by use of a newly developed method of subcellular fractionation. In wild-type HIV-1-infected H9 cells, the DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC was associated with the membrane fraction. The DRC was also detected in the cytoplasmic fraction in H9 cells expressing HIV-1 Myr- mutant Gag. However, little of the unmyristylated Gag and Gag-Pol proteins was found in the membrane fraction. Furthermore, HIV-1 Gag proteins synthesized in vitro in a rabbit reticulocyte lysate system in the absence of exogenous lipid membrane were able to assemble into a viral Gag complex similar to that of the DRC identified in infected H9 cells. The density of the viral Gag complex was not altered by treatment with the nonionic detergent Triton X-100, suggesting a lack of association of this complex with endogenous lipid. Formation of the DRC was not significantly affected by mutations in assembly domains M and L of the Gag protein but was drastically inhibited by a mutation in the assembly I domain. Purified DRC could be disrupted by high-salt treatment, suggesting electrostatic interactions are important for stabilizing the DRC. The Gag precursor proteins in the DRC were more sensitive to trypsin digestion than those in the DSC. These findings suggest that HIV-1 Gag and Gag-Pol precursors assemble into DRC in the cytoplasm, a process which requires the protein-protein interaction domain (I) in NCp7; subsequently, the DRC is transported to the plasma membrane through a process mediated by the M domain of the matrix protein. It appears that during this process, a conformational change might occur in the DRC either before or after its association with the plasma membrane, and this change is followed by the detection of virus budding structure at the plasma membrane.

Figures

FIG. 1

Subcellular fractionation and detection of DRC in Myr−/H9 cells. Uninfected H9 and Myr−/H9 cells were osmolysed by incubation in hypotonic buffer, and the soluble cytoplasmic fraction (S) of the osmolysed cells was separated from the membrane fraction (P) as described in Materials and Methods. The total proteins in an equal volume of each fraction were precipitated by TCA and separated by SDS-PAGE. Immunoblot analysis with a polyclonal anti-gp41 antiserum (A) and a polyclonal anti-gp120 antiserum (B) was performed to determine the location of the viral Env glycoproteins. Likewise, immunoblotting with a polyclonal anti-HSP70 antiserum (C) and a polyclonal anti-CD4 antiserum (D) was performed to determine the location of HSP70 (indicated by arrowheads) or CD4, respectively. (E) Equal volumes of the S and P fractions were then subjected to the discontinuous sucrose equilibrium-density gradient as previously described (18). After centrifugation, each fraction was collected and analyzed as previously described (18). Viral proteins were visualized by immunoblotting with an HIV-1-positive human serum. The positions of the viral proteins Pr55gag and Pr160gag-pol are indicated on the right, and molecular mass markers (kilodaltons) are indicated on the left.

FIG. 2

Subcellular fractionation of DRC and DSC from HIV-1-infected H9 cells. HIV-1-infected H9 cells expressing wild-type Gag proteins (Myr+) were subjected to subcellular fractionation and discontinuous equilibrium-density centrifugation as described in the legend to Fig. 1. Viral proteins were visualized by immunoblotting with an HIV-1-positive human serum. The positions of the viral proteins as detected in the released virions are indicated on the right, and molecular mass markers (kilodaltons) are indicated on the left. The positions of the DRC and DSC in the sucrose gradients are also indicated. S fraction, soluble cytoplasmic fraction; P fraction, pelleted membrane fraction.

FIG. 3

Formation of DRC by HIV-1 Gag proteins expressed in vitro. The HIV-1 Gag proteins were expressed in a rabbit reticulocyte lysate system as described in Materials and Methods. Reaction samples were then incubated in the absence (A) or presence (B) of Triton X-100 for 10 min at room temperature and subjected to discontinuous equilibrium-density centrifugation as described in the legend to Fig. 1. Viral proteins were visualized by immunoblotting with an HIV-1-positive human serum. The positions of the viral proteins are indicated on the right, and molecular mass markers (kilodaltons) are indicated on the left. Lane V, protein profile of released mature virions.

FIG. 4

Disruption of the DRC into nonpelletable Gag proteins by high-salt (1 M NaCl) treatment. (A) The DRCs in wild-type HIV-1-infected H9 cells were purified by sucrose equilibrium-density gradient centrifugation as described previously (18) and then divided into two equal portions. As a control, one-half was reloaded over a second sucrose equilibrium-density gradient without addition of 1 M NaCl (A, top panel). The other half was treated with 1 M NaCl at room temperature for 10 min prior to centrifugation (A, bottom panel). (B) HIV-1 Gag proteins were expressed in a rabbit reticulocyte lysate system as described in Materials and Methods and divided into two portions. One-half was loaded over a sucrose equilibrium-density gradient without addition of 1 M NaCl (B, top panel). The other half was treated with 1 M NaCl at room temperature for 10 min prior to centrifugation (B, bottom panel). Gradient fractions were pelleted and analyzed by SDS-PAGE and immunoblotting with an HIV-1-positive human serum. Lanes V and M show the protein profile of released mature HIV-1 viral particles (indicated at left) and molecular mass markers in kilodaltons (indicated at right), respectively.

FIG. 5

Sensitivity of the DRC and DSC to trypsin digestion. Homogenates from wild-type HIV-1-infected H9 cells were prepared as described in Materials and Methods and divided into two equal portions. One portion was incubated in the absence of trypsin (top panel), and the other was incubated with 5 μg of trypsin per ml (bottom panel) at 37°C for 30 min. After incubation, excess antitrypsin inhibitor was added to inhibit further proteolytic digestion. Samples were then subjected to sucrose equilibrium-density centrifugation as previously described (18). Viral proteins were analyzed by immunoblotting with an HIV-1-positive human serum. Lanes V and M show the protein profile of released mature HIV-1 viral particles (indicated at left) and molecular mass markers in kilodaltons (indicated at right), respectively.

FIG. 6

Identification of regions in HIV-1 Gag that are important for DRC formation. The efficiency of DRC formation in the cytoplasm of H9 cells expressing the full-length uncleaved Gag and Gag-Pol (Pr−/H9), the full-length uncleaved Gag without Gag-Pol (ΔPol/H9), unmyristylated Gag and Gag-Pol (Myr−/H9), p6gag-truncated Gag (Pr48/H9), and NC-plus-p6gag-truncated Gag (Pr41/H9) was evaluated by sedimentation experiments as described in Materials and Methods. The viral proteins present in the pelletable DRC form (lanes C) and as nonpelletable soluble Gag (lanes S) were analyzed by immunoblotting with an HIV-1-positive human serum.

FIG. 7

Formation of DRC by HIV-1 full-length and NC deletion mutant Gag proteins analyzed by discontinuous equilibrium-density centrifugation. Cytoplasmic lysates from H9 cells expressing the full-length uncleaved Gag (A) or NC-plus-p6gag-truncated Gag (B) were subjected to 16 to 60% discontinuous sucrose equilibrium-density gradients as described in Materials and Methods. Eighteen fractions were collected from each gradient, each fraction was pelleted by a high-speed centrifugation, and fractions 3 to 14 from each gradient were analyzed by SDS-PAGE and immunoblotting with an HIV-1-positive human serum. As a control, total viral Gag proteins present in the cytoplasmic lysates were also analyzed side by side (lanes T).