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Figure 2. Characteristics of HIV capsid assembly are reproduced in the cell-free system. (A) Co-translational myristoylation is required for capsid assembly. Cell-free translation and assembly reactions were programmed with Gag transcript in the absence of added MCoA (−), or with 10 μM MCoA added either at the start of the reaction (0) or at 90 min into the reaction when translation is completed (90). The detergent-treated products of the cell-free reactions were separated into soluble and particulate fractions by centrifugation on step gradients, and radiolabeled protein in each fraction was visualized by SDS-PAGE and autoradiography. The amount of radiolabeled Pr55 in the particulate fraction (which contains assembled capsids) was determined by densitometry of bands and is expressed as a percentage of total Gag protein synthesized. The presence of MCoA had no effect on the total amount of Pr55 synthesized. Values shown are the average of three independent experiments, and error bars indicate standard error. (B) Effect of detergent on capsid assembly. Cell-free translation and assembly reactions containing 10 μM MCoA were programmed with Gag transcript. Nikkol, a nonionic detergent that does not affect protein synthesis, was added at the start of the translation reaction to a final concentration of 0.002 or 0.1%, as indicated. At the end of the incubation, the reactions were analyzed for amount of assembly as in Fig. 2 A. Addition of detergent had no effect on the total amount of Pr55 synthesized. Values shown are the average of three independent experiments, and error bars indicate standard error.
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Figure 4. Both ATP and a subcellular fraction of the cell lysate are required for capsid assembly. (A) Effect of ATP hydrolysis on the posttranslational phase of capsid assembly. As in Fig. 2 B, cell-free translation and assembly reactions were programmed with Pr55 in the presence of 10 μM MCoA. At 50 min into translation, 0.2 μM emetine, a protein synthesis inhibitor, was added to some reactions as indicated. Immediately after emetine treatment, apyrase, an enzyme that hydrolyzes ATP, was added at a concentration of 1 U/μl to one of the emetine-treated reactions. At the end of the incubation (150 min), 1 μl of each reaction was analyzed directly by SDS-PAGE (autoradiographs are shown below bar graph). The remainder of the products was analyzed for amount of assembly as described in Fig. 2. Shown in the bar graph is the amount of Pr55 assembled as a percentage of total Pr55 synthesized in each reaction. Values in the bar graph are the average of three independent experiments, and error bars indicate the standard error. (B) Effect on assembly of depletion and reconstitution of a subcellular fraction. Wheat germ extract was subjected to ultracentrifugation as described in Materials and Methods to generate the HSS, HSP, and HSPd. The HSS was used to program cell-free translation and assembly reactions in the presence or absence of 10 μM MCoA (as indicated). Each of these reactions was treated with the protein synthesis inhibitor emetine for 50 min. After this, the HSP or HSPd was added to aliquots of the reaction as indicated below the bar graph. All reactions were incubated for a total of 150 min. A 1 μl aliquot was removed and analyzed directly by SDS-PAGE (shown below bar graph). The remainder of each reaction was analyzed for amount of assembly as described in Fig. 2 and plotted as a percentage of total Pr55 present in each reaction. The values shown in the bar graph are the average of three independent experiments, and error bars indicate the standard error.
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Figure 5. Pulse–chase analysis of HIV capsid assembly. (A) Analysis of a continuously labeled cell-free reaction by velocity sedimentation. Cell-free translation and assembly of Pr55 were performed as previously described. Upon completion of the cell-free reaction, the products were diluted in 1% NP-40 sample buffer on ice and were analyzed by velocity sedimentation on 13 ml 15– 60% sucrose gradients. Fractions were collected from the top of each gradient as described in Materials and Methods, and the amount of radiolabeled Pr55 protein in each fraction was determined and expressed as a percentage of total Pr55 protein present in the reaction. The calculated positions of 10-, 80-, 150-, 500-, and 750-S complexes are indicated with markers above (see Materials and Methods). 750-S represents the position of authentic immature (de-enveloped) HIV capsids. (B–D) Analysis of a pulse–chase cell-free reaction by velocity sedimentation. Cell-free translation and assembly of Pr55 were performed as previously described, except that [35S]cysteine was used for radiolabeling. At 4 min into translation, an excess of unlabeled cysteine was added to the reaction so that no further radiolabeling would occur. Aliquots of the reaction were collected 25 (C) and 150 min (D) into the reaction. 1 μl of each aliquot was analyzed by SDS-PAGE and autoradiography to reveal the total amount of radiolabeled Pr55 translation product (B, arrow) present at each chase time. The remainder of the aliquots was diluted into 1% NP-40 sample buffer on ice and analyzed by velocity sedimentation on 13 ml 15–60% sucrose gradients (5, C and D, respectively), in the manner described for 5 A above.
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Figure 3. Phenotypes of known mutations in Gag appear to be reproduced in the cell-free system. (A) Diagram of mutations within Gag. Gag is a polyprotein precursor that consists of four domains, referred to as p17, p24, p7, and p6. The Pr46 and Pr41 mutants were constructed by introducing a stop codon truncation at amino acid 435 or at amino acid 363, respectively. In the D2 mutation, amino acids 250 to 260 are deleted. In the GΔA mutation, the glycine at amino acid 2 is substituted with an alanine, thereby blocking myristoylation. The known phenotypes with respect to particle release from cells expressing each of these mutants is indicated to the right. (B) Capsid assembly in cell-free reactions programmed with Gag mutants. Cell-free translation and assembly reactions were programmed with transcript coding for each of the Gag mutants described above, as well as transcript coding for wild-type Gag in the presence or absence of MCoA (labeled WT and −MCoA, respectively). At the end of the reaction period, each sample was detergent treated, fractionated by velocity sedimentation on 13 ml sucrose gradients, and analyzed by SDS-PAGE and autoradiography. The amount of radiolabeled translation product in the position of completed 750-S capsids was quantitated by densitometry and expressed for each reaction as a percentage of total synthesis. The total amount of translation was approximately equal in all reactions.
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Figure 6. Different assembly-defective mutants in Gag accumulate different assembly intermediates. Cell-free translation and assembly reactions were programmed with wild-type Gag (A), the assembly-competent Pr46 truncation mutant (B), the assembly-defective Pr41 truncation mutant (C), the assembly-defective GΔA mutant (D), or the assemblydefective D2 mutant (E). At the end of the incubation, reaction products were detergent treated and analyzed by velocity sedimentation on 13 ml sucrose gradients as in Fig. 5 A. The amount of radiolabeled translation product in each fraction was determined and expressed as a percentage of total synthesis for each reaction.
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Figure 7. Gag-containing complexes in cells transfected with wild-type Gag or assembly-defective Gag mutants. (A) Cos-1 cells were transfected with a transfection vector encoding Pr55 cDNA, as described in Materials and Methods. 4 d later, the medium from the cells was collected. Viral particles in the medium were harvested by ultracentrifugation through a 20% sucrose cushion and then treated with detergent to remove envelopes. The transfected cells were solubilized in detergent to generate the cell lysate (see Materials and Methods). The particles from the medium (right ordinate, open circles) and the detergent lysate of the cells (left ordinate, closed circles) were analyzed in parallel by velocity sedimentation on 13 ml 15–60% sucrose gradients, as in Fig. 5. The amount of Pr55 protein in each fraction of these gradients was determined by immunoblotting and expressed as a percentage of total Pr55 protein present. The calculated positions of 10-, 80-,150-, 500-, and 750-S complexes are indicated with markers above (see Materials and Methods). 750-S represents the position of authentic, immature (de-enveloped) HIV capsids. (B and C) Cos-1 cells were transfected with a transfection vector encoding the Pr41 mutant (B) or the D2 mutant (C). Transfected cells were lysed in detergent, and the lysate was analyzed by velocity sedimentation on 13 ml sucrose gradients, as in A above. The amount of capsid protein in each fraction of these gradients was determined by immunoblotting with anti-Gag antibody, and was expressed as a percentage of total immunoreactive protein present in each reaction.
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Figure 8. A model for the assembly of immature HIV capsids. This model is based on the simplest interpretation of the data presented. Newly synthesized Gag proteins are myristoylated cotranslationally. Nascent Gag polypeptides appear to chase into completed immature capsids by way of a series of Gag-containing complexes (complexes A, B, C, and D). Evidence presented in the text suggests that complexes A, B, and C may constitute assembly intermediates. It is less clear whether complex D constitutes an assembly intermediate or a side reaction. Both ATP and a subcellular fraction of eukaryotic cell extract are required for assembly to take place, while detergent and apyrase are proposed to disrupt assembly at the indicated position. The points along the pathway at which various mutants of Gag are arrested are indicated below the model.
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