Nef enhances HIV-1 infectivity via association with the virus assembly complex

Abstract

The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle.

Fig. 1

Protein constructs used in this study and their expression in 293T cells. (A) Schematic of the five proteins used in this study. (B) Expression of the proteins in 293T cells. Cell lysates were analyzed by immunoblotting. Proteins in the left panel were detected with rabbit antibody for Nef, and proteins in the right panel were detected with rat antibody for HA tag. Abbreviations are as follows: C, control vector; WT, wild type HIV-1; CypA, cyclophilin A; HA: hemagglutinin epitope tag; CA, viral capsid protein.

Fig. 2

CypA-Nef is incorporated into Nef-defective HIV-1 particles and enhances their infectivity. Viruses were produced in 293T cells by cotransfection with a Nef-defective HIV-1 provirus together with plasmids encoding the indicated proteins. Wild type (WT) HIV-1 was tested as a control. Virus supernatants were harvested and assayed for p24 and titrated on P4 indicator target cells. Infectivity was determined as the number of infected cells per ng of p24 in the respective inocula. (A) Shown are the relative infectivity values obtained from 3 independent experiments, with error bars representing one standard deviation. Infectivity values were normalized to that of the control Nef-defective virus. (B) The infectivity results from a representative single experiment. Error bars represent the standard deviation of triplicate assays of each virus. (C) Wild type and Nef-defective virions were produced by cotransfection with constructs encoding Nef or CypA-Nef. The resulting supernatants were assayed for infectivity on P4 indicator cells. Infectivity is shown relative to the corresponding Nef-defective and wild type control viruses to illustrate the differential effects of expression of Nef and CypA-Nef on the two viruses. (D) Immunoblot analysis of pelleted virus particles with antibodies specific for Nef and CA. The results shown in this figure are representative of 2 independent experiments.

Fig. 3

CypA-Nef copurifies with HIV-1 cores. Concentrated HIV-1 particles were sedimented through a layer containing 0.5% Triton X-100 into a linear sucrose density gradient. (A) Fractions were collected from the top and p24 levels were quantified by ELISA. Top panel: Nef-defective HIV-1; bottom panel: Nef-defective HIV-1 produced by contransfection with the CypA-Nef expression construct. (B) Immunoblot analysis of purified HIV-1 cores. Cores present in the peak fraction (fraction 8) from each gradient were pelleted and analyzed by immunoblotting using antibodies specific for Nef and CA. Lane 1: Nef-defective HIV-1 cores; lane 2: cores isolated from Nef-defective HIV-1 particles produced in cells expressing wild type Nef; lane 3: cores isolated from Nef-defective HIV-1 particles produced in cells expressing CypA-Nef; lane 4: wild type HIV-1 cores. The upper and lower panels show the proteins detected with antibodies to Nef and CA, respectively.

Fig. 4

Point mutations in the Nef portion of CypA-Nef abolish infectivity enhancement. Viral supernatants were harvested and assayed for infectivity on P4 cells. The results shown are representative of 3 independent experiments. (A) Infectivity of HIV-1 mutants encoding substitutions in nef. (B) Infectivity of virions generated by cotransfection of the CypA-Nef mutants and nef-defective provirus. (C) Plot of infectivity of Nef mutant viruses (abscissa) vs. viruses complemented with the corresponding mutant CypA-Nef proteins (ordinate). (D) Immunoblot analysis of CypA-Nef expression in 293T cells (upper panel) and incorporation into Nef-defective HIV-1 particles (lower panel).

Fig. 5

CsA inhibits HIV-1 incorporation of CypA-Nef and its ability to enhance viral infectivity. Viruses were produced in transfected cells cultured in the presence and absence of 5 μM CsA. (A) Infectivity of viruses on P4 indicator cells. Inset: fold inhibition of infectivity by CsA. Shown are the mean values of triplicate infections; results are representative of two independent experiments. (B) Immunoblot analysis of cell lysates and pelleted viruses using antibodies specific for Nef and CA.

Fig. 6

Mutations in the CypA-binding site in Gag inhibit HIV-1 incorporation of CypA-Nef and infectivity enhancement. Viruses were produced by cotransfection of Nef-defective proviruses containing the indicated mutations in Gag with plasmids encoding Nef or CypA-Nef. (A) Infectivity of the viruses. Inset: ratio of infectivity of viruses produced with and without coexpression of CypA-Nef. (B) Immunoblot analysis of cell lysates and pelleted viruses using antibodies specific for Nef and CA.

Fig. 7

CypA-Nef enhances the infectivity of Nef-defective virions bearing native HIV-1 Env but not VSV-G. Viral particles were produced by transfecting 293T cells with R7.nef- provirus and Nef or CypA-Nef expression plasmids in trans (VC, vector control). For HIV-1(VSV-G) pseudotyped particles, the R7.nef-env- proviral construct was cotransfected with a VSV-G expression plasmid and the other indicated expression constructs. (A) Analysis of infectivity. Shown are the mean values of triplicate determinations, with error bars representing one standard deviation. The infectivity of each virus relative to its respective vector control is shown in panel (B).