The late stage of human immunodeficiency virus type 1 assembly is an energy-dependent process

Abstract

Several recent studies have indicated the involvement of host cell factors in human immunodeficiency virus type 1 (HIV-1) assembly. To ascertain whether ATP-dependent factors play a role in this process, we quantified virus-like particle (VLP) production by ATP-depleted cells. Pharmacological ATP depletion abrogated VLP production without affecting cell viability or inducing degradation of HIV-1 Gag protein. This effect occurred even when the ATP-depleting agents were added 1 h into the assembly process, and it was reversed by removal of these agents. ATP depletion did not affect Gag membrane binding or multimerization. Density gradient analysis indicated that HIV-1 assembly intermediates were stalled late in the assembly process. This conclusion was further supported by electron microscopy analysis, which revealed a preponderance of plasma membrane-associated stalk-like structures in the ATP-depleted cells. Since no HIV-1 proteins bind or hydrolyze ATP, these findings indicate that an ATP-requiring cellular factor is an obligatory participant late in the HIV-1 assembly process.

FIG. 1

Cellular ATP depletion inhibits HIV-1 VLP production. (A) Kinetics of cellular ATP depletion. COS-1 cells were incubated in glucose-free medium containing 2-deoxyglucose and NaN₃. Cellular ATP levels were quantified, as described in Materials and Methods, before treatment began and at various times after treatment. ATP levels are plotted as a percentage of the original level. (B) VLP production in untreated and ATP-depleted cells. Transfected COS-1 cells were pulse-labeled for 20 min with Tran³⁵S-label and chased for various time periods (5 min, 1 h, 2 h, or 4 h) either in glucose-containing medium (untreated; squares) or in glucose-free medium containing 2-deoxyglucose and NaN₃ (ATP depleted; diamonds). VLPs were isolated from the medium, and radioactivity from the labeled Gag protein in cells and VLPs was quantified by RIPA with anti-CA. VLP production was expressed as the ratio of the percentage of Gag radioactivity found in VLPs to the total Gag radioactivity (cells plus VLPs).

FIG. 2

VLP production is restored by reversal of cellular ATP depletion. (A) Rebound in cellular ATP levels. COS-1 cells were depleted of ATP for 2 h as described in the legend to Fig. 1A and then washed and incubated in glucose-containing medium. Cellular ATP was quantified at various time points after addition of the glucose-containing medium and is plotted as a percentage of the original ATP level (time zero). (B) VLP production after removal of ATP-depleting agents. Transfected COS-1 cells were pulse-labeled for 20 min with Tran³⁵S-label, chased for 2 h in the presence of ATP-depleting agents, washed, and chased for an additional 2 or 4 h in glucose-containing medium (depleted/reversed; circles). For untreated (squares) or ATP-depleted (diamonds) cells, the entire chase was performed in glucose-containing medium or with ATP-depleting agents, respectively. VLP production was expressed as described in the legend to Fig.1. For the depleted/reversed samples, VLPs were isolated from both the chase medium from the first 2 h of the chase and the glucose-containing chase medium from the subsequent chase time. Counts of labeled Gag from the two medium samples were combined and then divided by the total Gag counts (cells plus VLPs) to obtain the value for VLP production.

FIG. 3

Cellular Gag counts in untreated and ATP-depleted cells. Transfected COS-1 cells were pulse-labeled and chased in control medium (untreated; squares) or in medium containing ATP-depleting agents (depleted; diamonds) as described in the legend to Fig. 1B. Cell lysates were immunoprecipitated with anti-CA and analyzed by SDS-PAGE and exposure to phosphorimager screens. Counts from labeled cellular Gag were expressed as a percentage of the counts in the pulse-labeled cells.

FIG. 4

ATP depletion of cells does not affect Gag membrane binding assay. Transfected COS-1 cells were pulse-labeled for 5 min and chased for 2, 20, or 60 min in control medium (untreated) or in medium containing ATP-depleting agents (ATP depleted). Denucleated P100 fractions were prepared and subjected to sucrose flotation as described in Materials and Methods. Labeled Gag in the gradient fractions was quantified after immunoprecipitation, SDS-PAGE, and phosphorimaging. (A) A representative experiment showing labeled Gag in each gradient fraction. (B) Graphic depiction of data from three independent experiments. The counts from the labeled Gag in the interface fractions (fractions 4 and 5) were divided by the total counts in the gradient fractions to determine the percentage of Gag that was membrane bound.

FIG. 5

ATP depletion does not affect Gag multimerization. Transfected COS-1 cells were pulse-labeled and either harvested immediately (pulse) or chased for 2 h in control medium (untreated) or in medium containing ATP-depleting agents (ATP depleted). P100 fractions were prepared and fractionated over Optiprep gradients containing a nonionic detergent, as described in Materials and Methods. Labeled Gag in the gradient fractions was quantified by analyses of immunoprecipitates by SDS-PAGE followed by phosphorimaging.

FIG. 6

ATP depletion results in accumulation of dense Gag complexes at the bottom of Optiprep gradients performed in the absence of detergent. (A and B) Fractionation of Gag protein from untreated and from ATP-depleted cells on detergent-free Optiprep gradients. Transfected COS-1 cells were subjected to pulse-chase and ATP depletion as described in the legend to Fig. 5. P100 fractions were prepared and fractionated over detergent-free Optiprep gradients, as described in Materials and Methods. Gag in the gradient fractions was detected by Western blotting with anti-p24 CA antiserum. (A) A representative experiment. (B) Percentages of Gag in the bottom two fractions (density, ≥1.11 g/ml) in several experiments. Similar results were obtained by labeling cells at steady state with Tran³⁵S-label, chasing in the presence or absence of ATP-depleting agents, and visualizing labeled Gag protein (data not shown). (C) Results of a VLP budding experiment like that shown in Fig. 1B, except that the ATP-depleting agents were not added until 1 h into the chase period (arrow).

FIG. 7

EM images of Gag-producing untreated and ATP-depleted cells. Transfected untreated (A) or ATP-depleted (B and C) cells were washed in STE and then fixed, stained, and examined by transmission EM as described in Materials and Methods. HIV-1 budding structures are indicated by arrowheads. Cross-sections of ∼20 untreated and ∼20 ATP-depleted cells were examined to obtain the estimated frequencies of the budding structures in the untreated and depleted cells that are stated in the text. Bars, 500 nm.