Disruption of the actin cytoskeleton can complement the ability of Nef to enhance human immunodeficiency virus type 1 infectivity

Abstract

The human immunodeficiency virus (HIV) protein Nef has been shown to increase the infectivity of HIV at an early point during infection. Since Nef is known to interact with proteins involved in actin cytoskeleton rearrangements, we tested the possibility that Nef may enhance HIV infectivity via a mechanism that involves the actin cytoskeleton. We find that disruption of the actin cytoskeleton complements the Nef infectivity defect. The ability of disruption of the actin cytoskeleton to complement the Nef defect was specific to envelopes that fuse at the cell surface, including a variety of HIV envelopes and the murine leukemia virus amphotropic envelope. In contrast, the infectivity of HIV virions pseudotyped to enter cells via endocytosis, which is known to complement the HIV Nef infectivity defect and can naturally penetrate the cortical actin barrier, was not altered by actin cytoskeleton disruption. The results presented here suggest that Nef functions to allow the HIV genome to penetrate the cortical actin network, a known barrier for intracellular parasitic organisms.

FIG. 1.

Actin depolymerization complements the infectivity defect of ΔNef virions. Magi+/+ cells were infected in the presence of 3 μM CytD, 5 μM LatB, or dimethyl sulfoxide without drug (−) as a negative control. Infection was quantified using a liquid β-Gal assay (A) or by fixing infected cells and counting the number of β-Gal-positive infected foci (B). (C) Cells were infected in the presence of 125 nM Jas or in the absence of Jas (−) as a negative control, and β-Gal expression was quantified using a liquid β-Gal assay. Bold numbers above the bars represent the fold increase in infection relative to the no-drug infection (−) (negative control). OD 405 nm, optical density at 405 nm.

FIG. 2.

CytD, LatB, and Jas disrupt cortical actin structures. Magi+/+ cells expressing a YFP-actin fusion protein were imaged before (0 min) and after a 60-min treatment with 3 μM CytD, 5 μM LatB, or 125 nM Jas.

FIG. 3.

Actin depolymerization complements the reverse transcription defect of ΔNef virions. (A) Magi+/+ cells were infected in the presence of 3 μM CytD, 5 μM LatB, 5 μg of Aph per ml, or 10 μM nevirapine (Nev), or in the absence of drug (−) as a negative control. (B) Magi+/+ cells were infected in the presence of 3 μM CytD, 5 μM LatB, or 125 nM Jas or in the absence of drug (−) as a negative control. Genomic DNA was isolated from the target cells, and the number of viral genomes present in each samples was calculated. This value was normalized to the number of genomic actin copies present in an identical sample to calculate the number of late reverse transcription products per cell. Bold numbers above the bars represent the fold increase in infection relative to infection in the absence of drug (−).

FIG. 4.

Actin depolymerization complements the infectivity defect of ΔNef virions independently of coreceptor tropism and in other viral strains. (A) Cells were infected with equivalent amounts of WT and ΔNef viruses in which the envelope has been deleted that were pseudotyped with the JRFL envelope protein in the presence of 3 μM CytD or in the absence of drug (−) as a negative control. Infection was measured 36 h p.i. using a fixed cell β-Gal assay. (B) Cells were also infected with equivalent amounts of NL43 and NL43ΔNef in the presence of 3 μM CytD, 5 μM LatB, or dimethyl sulfoxide (negative control) (−). Infection was assayed using a liquid β-Gal assay. OD 405nm, optical density at 405 nm.

FIG. 5.

Actin depolymerization does not complement the infectivity defect of virions produced in the presence of CsA. Cells were infected with equivalent amounts of WT virus, ΔNef virus, or WT virus produced in 1 μM CsA. Cells were infected in the presence of 3 μM CytD or in the absence of drug (−) as a negative control. Infection was quantified using a liquid β-Gal assay. OD 405nm, optical density at 405 nm.

FIG. 6.

Actin depolymerization complements the infectivity defect of ΔNef HIV-1 virions in GHOST cells. GHOST cells were infected in duplicate in the presence of 3 μM CytD or 5 μM LatB or in the absence of drug (−) as a negative control. Cells were fixed in 1% paraformaldehyde and analyzed for GFP expression following infection with WT LAI and LAIΔNef virions (A) or Env-deficient versions of WT and ΔNef virions pseudotyped with VSV-g envelope protein (B) or amphotropic MLV envelope protein (C). The bars represent the mean percentages of infected cells of duplicate infections determined by analyzing more than 5,000 cells/infection.

FIG. 7.

LAI and LAIΔNef HIV-1 virions productively enter cells at similar levels and in the presence or absence of actin-depolymerizing agents. HOS CD4 cells were infected for 3 h with equivalent amounts of LAI or LAIΔNef virus containing the Vpr-β lactamase fusion protein in the presence of 3 μM CytD or 5 μM LatB or in the absence of drug as a negative control. Fusion was measured as cleavage of CCF2 substrate as previously described (10). The percent fusion is indicated by the bold number below the positive fusion population. Data are representative of at least three independent experiments.

FIG. 8.

Model for Nef's infectivity enhancement of HIV-1 virions. Following fusion at the plasma membrane, viral cores encounter and must traverse a dense layer of cortical actin capable of hindering virion mobility. (A) Depending on the local actin environment encountered after fusion, a great number of ΔNef virions may be unable to traverse this barrier. (B) WT virions rely on the ability of Nef to alter the host cell actin cytoskeleton following fusion and are therefore more likely to cause infection. Following depolymerization of the actin cytoskeleton of the target cell, both WT and ΔNef virions have similar infectivity. (C) This model also explains why HIV-1 virions pseudotyped with pH-dependent envelope protein no longer require Nef for maximal infectivity. Trafficking of the endosome induced by the pH-dependent envelope would complement Nef-deficient virions, since the endosome would be trafficked past cortical actin structures by cellular endocytic machinery.