Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication

Abstract

The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4(+) T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge.

FIG. 1.

Nef is specifically required for efficient replication of X4-tropic HIV-1 in activated primary CD4⁺ T cells. Wild-type and nef-defective X4-tropic (A) and R5-tropic (B) virus clones were cultured in activated highly purified CD4⁺ T lymphocytes as described in Materials and Methods. Results shown are the averages of duplicate growth curves from a representative of two experiments. Filled squares, nef⁺ HIV-1; open diamonds, nef-deficient HIV-1.

FIG. 2.

The requirement for Nef in HIV-1 replication maps to the V3 loop of gp120. NL4-3-based V3 loop chimeras conferring different tropisms were cultured in activated CD4⁺ primary T lymphocytes as described in Materials and Methods. Results shown are the averages of duplicate growth curves from a representative of eight experiments. Filled squares, nef⁺ HIV-1; open diamonds, nef-deficient HIV-1.

FIG. 3.

Nef is required for optimal infectivity of X4-tropic virions released from HIV-1-infected primary T lymphocytes. Viruses were harvested 5 days following initial inoculation, and infectivity was analyzed on P4/CCR5⁺ reporter cells as described in Materials and Methods. Values shown are the number of blue cells per nanogram of p24 of input virus. Results shown are the mean values and standard deviations and are a representative of six experiments. Filled bars, nef⁺ HIV-1; shaded bars, nef-deficient HIV-1.

FIG. 4.

Nef enhances the gp120 content of X4-tropic virions purified from primary T lymphocytes. X4-tropic (A) and R5-tropic (B) viruses were purified 8 days postinfection, and ELISAs specific for p24 and gp120 were performed as described in Materials and Methods. Shown is a representative of four experiments. Values shown are nanograms per milliliter for both p24 and gp120. Shaded bars, p24; open diamonds, gp120.

FIG. 5.

Nef does not affect gp41 content in X4- and R5-tropic virions purified from primary T lymphocytes. ELISAs were performed on gradient fractions containing purified T-cell virus as described in Materials and Methods. (A) Levels of gp41 from a representative purification of NL4-3 virions. Shaded bars, p24; open diamonds, gp120. (B) Total gp41 in peak fractions was divided by total p24 to calculate virion-associated gp41. Shown are the results of a representative of four experiments. Filled bars, nef⁺ HIV-1; shaded bars, nef-deficient HIV-1.

FIG. 6.

Nef prevents incorporation of CD4 into X4- and R5-tropic virions produced in CD4⁺ primary T lymphocytes. CD4 capture ELISAs were performed on gradient-purified virions as described in Materials and Methods. The total of the CD4 in peak fractions was divided by the total p24 to calculate virion-associated CD4. Shown are the results of a representative of four experiments. Filled bars, nef⁺ HIV-1; shaded bars, nef-deficient HIV-1.

FIG. 7.

sCD4 preferentially inhibits infection by X4-tropic HIV-1. X4- and R5-tropic viral isolates (A) and V3 loop mutants (B) were produced in 293T cells. Virions were treated with the indicated concentrations of sCD4 and assayed for infection on P4/CCR5⁺ reporter cells as described in Materials and Methods. Values are expressed as a percentage of the infectivity of the corresponding untreated virus. Shown is a representative of up to three experiments. Filled symbols, X4-tropic HIV-1; open symbols, R5-tropic HIV-1.