RNase L inhibitor is induced during human immunodeficiency virus type 1 infection and down regulates the 2-5A/RNase L pathway in human T cells

Abstract

The interferon-regulated 2-5A/RNase L pathway plays a major role in the antiviral and antiproliferative activities of these cytokines. Several viruses, however, have evolved strategies to escape the antiviral activity of the 2-5A/RNase L pathway. In this context, we have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein that specifically inhibits RNase L and whose regulated expression in picornavirus-infected cells down regulates the activity of the 2-5A/RNase L pathway. We show here that RLI increases during the course of human immunodeficiency virus type 1 (HIV-1) infection, which may be related to the downregulation of RNase L activity that has been described to occur in HIV-infected cells. In order to establish a possible causal relationship between these observations, we have stably transfected H9 cells with RLI sense or antisense cDNA-expressing vectors. The overexpression of RLI causes a decrease in RNase L activity and a twofold enhancement of HIV production. This increase in HIV replication correlates with an increase in HIV RNA and proteins. In contrast, reduction of RLI levels in RLI antisense cDNA-expressing clones reverses the inhibition of RNase L activity associated with HIV multiplication and leads to a threefold decrease in the viral load. This anti-HIV activity correlated with a decrease in HIV RNA and proteins. These findings demonstrate that the level of RLI, via its modulation of RNase L activity, can severely impair HIV replication and suggest the involvement of RLI in the inhibition of the 2-5A/RNase L system observed during HIV infection.

FIG. 1

RNase L activity during HIV-1 infection. H9 cells were infected with HIV-1 and harvested at the indicated times. (A) Cell extracts (600 μg of proteins) were tested without further fractionation in a 2-5A radiobinding assay. Results are expressed as the percentage of 2-5ApCp bound. 100% is the binding level in uninfected cells at time zero. (B) H9 cells were infected with HIV-1 and harvested at the indicated times. For each time point, 200 μg of protein was analyzed by SDS-PAGE and Western blotting with polyclonal antibody against gp160/gp120. An autoradiograph of the gel is presented.

FIG. 2

Analysis of RNase L and RLI proteins during HIV-1 infection. H9 cells were infected with HIV-1 and harvested at the indicated times. (A) For each time point, 200 μg of proteins was analyzed by SDS-PAGE and Western blotting with polyclonal antibodies against RNase L, RLI, or GAPDH as indicated. (B) A densitometric analysis of the gels is presented (□, RLI; ■, RNase L). Vertical bars represent the standard deviations obtained with three independent experiments. 100% corresponds to the amount of RLI or RNase L protein in untreated cells at time zero.

FIG. 3

Expression of RLI and RNase L activity in H9 cells stably transfected with antisense and sense RLI cDNA. (A) Extracts (200 μg of proteins) from H9 cells stably transfected with the VV empty vector, with the sense RLI cDNA construction (VS1 and VS2), or with the antisense RLI cDNA construction (VAS1 and VAS2) were analyzed by SDS-PAGE and Western blotting with polyclonal antibody against RLI. (B) Cell extracts (600 μg of protein) from the different transfectants were tested without further fractionation in a 2-5A radiobinding assay. Results are expressed as the percentage of 2-5ApCp bound. 100% is the binding level in control VV cells.

FIG. 4

2-5A binding activity in cells stably transfected with RLI sense and antisense constructions during HIV-1 infection. H9 cells transfected with the empty vector (○, VV), with the antisense RLI cDNA construction (◊, VAS), or with the sense RLI cDNA construction (□, VS) were infected with HIV-1 and harvested at the indicated times. Extracts (600 μg of protein) were tested without further fractionation in a 2-5A radiobinding assay. Results are expressed as the percentage of 2-5ApCp bound. 100% is the binding obtained at time zero in uninfected cells. Vertical bars represent the standard deviations obtained with three independent experiments. Clones VV, VAS1, and VS1 are shown in panel A, and clones VV, VAS2, and VS2 are shown in panel B.

FIG. 5

RT activity in clones expressing sense or antisense RLI constructions. H9 cells transfected with the empty vector (○, VV), with the antisense RLI cDNA construction (◊, VAS), or with the sense RLI cDNA construction (□, VS) were infected with HIV-1. Virus production was monitored by supernatant RT assay every day. 100% is the RT activity observed in uninfected cells. Vertical bars refer to the standard deviations obtained with three independent experiments. Clones VV, VAS1, and VS1 are shown in panel A, and clones VV, VAS2, and VS2 are shown in panel B.

FIG. 6

HIV-1 mRNA level and expression of gp160/gp120 during HIV-1 infection in cells transfected with RLI sense and antisense cDNA constructions. H9 cells transfected with the antisense RLI cDNA construction (◊, VAS2), with the sense RLI cDNA construction (○, VS2), or with the empty vector (□, VV) were infected with HIV-1 and harvested at the indicated times. (A) For each time point, 20 μg of total RNA was analyzed by Northern blot. A quantification of total HIV mRNA by the Intelligent Quantifier program is represented. (B) For each time point, 200 μg of protein was analyzed by SDS-PAGE and Western blotting with a polyclonal antibody against gp160/gp120.