Functional domains within the human immunodeficiency virus type 2 envelope protein required to enhance virus production

Abstract

Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.

FIG. 1.

Vpu and HIV-2 Env enhance the production of diverse retroviral particles. (A) Western blot analysis of cell lysates and virus supernatants from HeLa cells expressing HIV-1, MLV, or EIAV particles. CA proteins were detected with specific antibodies for each virus. (B) Quantitation of effects on budding caused by coexpression of Vpu, HIV-1 Env, or HIV-2 Env. The intensity of the CA-reacting bands on the Western blots was measured, and the ratio of the signal in virions to that in lysates was obtained. The enhancement of budding was calculated by normalizing all values to the Gag-Pol-only controls. Data are averages for three independent experiments.

FIG. 2.

Virus production from HeLa and Cos-7 cells. (A) Vpu and HIV-2 Env stimulate HIV-1 budding from HeLa cells but not Cos-7 cells. The exposures of the Western blots shown were chosen to highlight the fact that for similar levels of virus proteins in cell lysates, baseline virus yield from Cos-7 cells is higher than from HeLa cells. (B) Comparison of baseline HIV-1 particle production from heterokaryons formed between HeLa, Cos-7, or Cos-7 plus HeLa cells. The ratios of the CA signal in virions to that in lysates were calculated and normalized to the HeLa cell value. The HeLa cell phenotype of restricted virus production is dominant in the mixed heterokaryons. Data are averages for two independent experiments. (C) Enhancement of virus production from heterokaryons in the presence of Vpu or HIV-2 Env. Virus production from Cos-7 plus HeLa heterokaryons is stimulated by both proteins to an extent similar to that in the HeLa cell-only control. Data are averages for two independent experiments.

FIG. 3.

A tyrosine-based motif in the cytoplasmic tail of HIV-2_ROD10 Env is necessary for activity. (A) Schematic of the HIV-2_ROD10 tail, showing the position of the conserved GYXXθ motif (bold) and the truncations and point mutations used in this study. (B) Western blot analysis demonstrating the stimulation of HIV-1 and MLV particle production by HIV-2_ROD10 Env and its derivatives. Antibodies were used to detect the specific CA proteins, and the HIV-2 Env proteins (gp160/gp120) were visualized with an anti-ST Env antiserum that cross-reacts with the HIV-2_ROD10 protein. Env proteins could be clearly seen in cell lysates but were difficult to detect in viral particles. Loss of the GYXXθ motif by truncation or point mutation blocks activity.

FIG. 4.

Activity of HIV-2_ST Env. (A) Schematic of the HIV-2_ST tail, showing the position of the conserved GYXXθ motif (bold) and the truncations used in this study. (B) HIV-2_ST Env stimulates virus production to the same extent as the HIV-2_ROD10 Env. Loss of the GYXXθ motif by truncation blocks activity.

FIG. 5.

Chimeric proteins formed between HIV-1_BH10 and HIV-2_ROD10 Envs. (A) Schematic of proteins used in the study. E, ectodomain; M, membrane-spanning region; T, cytoplasmic tail. (B) Western blot analysis of effects of the chimeric proteins on HIV-1 particle release reveals that the HIV-1 tail can substitute for the HIV-2 tail, but there is a specific requirement for the HIV-2 Env ectodomain.

FIG. 6.

Chimeric proteins formed between HIV-2_ROD10 Env and CD8. (A) Schematic of proteins used in the study. (B) Enhancement of HIV-1 particle release. Data are averages for three experiments. (C) Coexpression of E8M8T2 and E2M2T8 does not result in enhanced virus production.

FIG. 7.

Coexpression of HIV-2_ROD10 Env and defective mutants. Transfection with different amounts of functional HIV-2_ROD10 Env causes a dose-response effect on virus release. Cotransfection with different amounts of the nonenhancing mutant Env proteins reduced the positive effect of the wild-type Env, indicating a trans-negative effect. Data are from one representative experiment.

FIG. 8.

Incorporation of truncation mutants of HIV-2_ROD10 Env into retroviral vector particles. MLV-based particles were produced in 293T cells, and the extent of HIV-2 Env incorporation was assessed by Western blotting. Truncation of the cytoplasmic tail significantly increased vector incorporation. However, there is no correlation with the ability to stimulate virus production from HeLa cells (compare Fig. 3B).