Mechanistic independence of Nef and cyclophilin A enhancement of human immunodeficiency virus type 1 infectivity

Abstract

Optimal HIV-1 infectivity requires the presence of both the viral factor Nef and the cellular protein cyclophilin A (CyPA) during virion assembly. These two proteins are integral components of HIV-1 particles. Both CyPA and Nef facilitate a step in the viral life cycle occurring between penetration and reverse transcription, suggesting a common mechanism of action. Experiments were performed to test the potential interplay of Nef- and CyPA-mediated enhancement of HIV-1 infectivity. In single-cycle infection assays, nef-defective virions were partially resistant to cyclosporin A (CsA), a drug that inhibits the binding of CyPA to the HIV-1 Gag precursor and CyPA incorporation into virions. Genetic dissection of the relative contributions of Nef and the cyclophilin A-Gag interaction to HIV-1 infectivity demonstrated the independence of these two effects. Nef was not required for incorporation of CyPA into HIV-1 virions and vice-versa. Surprisingly, CyPA-deficient virions remained sensitive to inhibition by CsA, in a manner that depended strongly on the presence of a functional nef gene. These results demonstrate that Nef and CyPA act independently to render HIV-1 particles fully infectious. They further suggest that in addition to blocking the CyPA-Gag interaction, CsA can also inhibit HIV-1 replication through a novel mechanism involving suppression of Nef-directed enhancement of virus infectivity.

Fig. 1

Effects of cyclosporin A on wild-type and nef-defective HIV-1 infectivity. Viruses were produced by culturing transfected 293T cells in the presence of the indicated concentrations of cyclosporin A. Culture supernatants were assayed for reverse transcriptase activity and their titers determined by infecting P4-2 indicator cells. Panel A: The ordinate values represent the number of infectious units per unit reverse transcriptase activity. R9: wild-type HIV-1; R9ΔN: nef-defective HIV-1. Shown are the mean values of triplicate infections, with error bars representing one standard deviation from the mean. The data are representative of two independent experiments. In Panel B, the data shown in Panel A are represented as the percentage by which viral infectivity is reduced by the indicated concentrations of CsA.

Fig. 2

Levels of CyPA in wild-type and nef-defective HIV-1 particles. Virions produced by transfection of 293T cells were purified by centrifugation through 20% sucrose. The pellets were lysed and subjected to Western blot analysis using aliquots of cytoplasmic extracts normalized for p24 content by ELISA. Proteins were detected with (A) rabbit anti-cyclophilin A; (B) mouse anti-HIV-1 CA, and (C) rabbit anti-Nef; blots were developed using chemiluminiscent detection. Lane 1: wild-type HIV-1, lane 2: wild-type HIV-1 produced in the presence of 10 µM cyclosporin A; lane 3: nef-defective HIV-1, lane 4: env-defective HIV-1; lane 5: env-defective, nef-defective HIV-1.

Fig. 3

Nef and the CyPA-Gag interaction independently enhance HIV-1 infectivity. Viruses were produced by transfection of 293T cells and were assayed for infectivity using P4-2 cells as targets. Shown are the mean values of triplicate infections, with error bars representing one standard deviation. The paired solid and open bars are the results of independent virus stocks. R7:wild-type HIV-1; R7ΔN:nef-defective HIV-1. R7.221 and ΔN.221 contain a mutation of Gly221 of capsid to Ala; R7.222 and ΔN.222 contain a mutation of Pro222 to Ala.

Fig. 4

Effects of gag mutations of HIV-1 packaging of CyPA. Virions produced by transfection of 293T cells with proviral DNA constructs and were purified by ultracentrifugation through 20% sucrose. Viral pellets were dissolved in lysis buffer, assayed for p24 content by ELISA, and subjected to immunoblot analyses. The blots were probed sequentially with rabbit anti-CyPA (panel A) and mouse anti-CA (panel B), and protein bands were visualized using chemiluminescent detection.

Fig. 5

Inhibition of HIV-1 infectivity by CsA correlates with the expression of Nef. Viruses were produced by culturing transfected 293T cells for two days in the presence or absence of 10 µM CsA. Virus stocks were assayed for reverse transcriptase activity and their titers determined by infecting P4 cells. Panel A: shown are the mean values of triplicate infections, with error bars representing one standard deviation. The results are representative of two experiments. Shown in panel B are the ratios of the infectivity values obtained for viruses produced in the absence of CsA to those of viruses produced in the presence of CsA.

Fig. 6

Cyclosporin A inhibits replication of mutant HIV-1 lacking CyPA. CEM cells were inoculated with aliquots of viruses containing 30,000 cpm reverse transcriptase activity. After overnight culturing, the cells were pelleted to remove input virus, and cultured in 2 ml media in the presence (dashed lines) and absence (solid lines) of 2.5 µM CsA. Virus production was monitored periodically by assaying for reverse transcriptase activity in the supernatants. Panel A: wild-type HIV-1; C: HIV-1 containing a mutation of Pro222 to Ala in capsid; E: HIV-1 containing a mutation of Gly221 to Ala in capsid. Replication of the nef-defective variants of R7, R7.222, and R7.221 are shown in panels B, D, and F, respectively.