Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response

Abstract

Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.

FIG. 1.

PKR response of astrocytes modulates HIV-1 infection. (A) PKR immunoprecipitation and subsequent immunoblot analysis of U251MG astrocytoma cells to determine level of PKR phosphorylation in the presence of HIV-1 NL4-3 virus or NL4-3-positive IFN-α. Matched amounts of U251MG astrocytoma cell lysate were immunoprecipitated with PKR 71/10 monoclonal antibody, and probed with either the PKR F9 mouse monoclonal antibody (PKR) or a phosphorylation-specific PKR polyclonal antibody (P-PKR). (B) Relative PKR phosphorylation was calculated by quantifying the ratio of phosphorylated PKR to total PKR in PKR immunoprecipitation-immunoblot assay from panel A by densitometry. (C) Reverse transcriptase assay of transfection supernatants from U251MG astrocytoma cells cotransfected with pNL4-3 proviral plasmid and either the catalytically inactive (PKR K296R) or the N-terminal PKR (PKR-N) mutants. (D) Titration of PKR-specific siRNA into U251MG astrocytoma cells increased virus detected in culture supernatants using an assay for HIV-1 reverse transcriptase. A nonspecific siRNA targeted to EGFP was used as a negative control.

FIG. 2.

Modulation of HIV-1 virion production in U251MG astrocytoma cells by PKR inhibitors and RNA binding proteins. U251MG astrocytoma cells were cotransfected with expression plasmids encoding either PKR inhibitors or RNA binding proteins with pNL4-3 proviral plasmid. The amount of expression plasmid was titrated and virion production was assessed by reverse transcriptase assay on culture supernatants collected 72 h posttransfection. The cotransfected plasmids expressed either TRBP (⧫), human Staufen (huStauf, ▵), NF-90 (×), the C-terminal domain of NF-90 (NF-90c, ○), polypyrimidine tract binding protein (PTB, ⋄), or PRBP (□).

FIG. 3.

TRBP relieves the effects of PKR on HIV-1 expression in 293T cells. Increasing amounts of a PKR expression plasmid (pcDNA3-PKR) were cotransfected with pNL4-3 proviral plasmid (NL4-3) in 293T cells to show the effects of PKR activation on HIV-1 replication. A TRBP expression plasmid (pCMV-TRBP) was cotransfected to demonstrate countering of the effects of PKR on HIV-1 replication. (A) Immunoblotting of transfected 293T cells using an antibody against HIV-1 Gag protein. Equal amounts of total protein from cell lysates were loaded from the 293T cells transfected with pNL4-3 provirus and pcDNA3-PKR and pCMV-TRBP plasmids. (B) Expression of HIV-1 Gag from the transfected 293T cells in panel A was quantified by densitometry analysis of the Pr55^Gag band and graphically displayed to demonstrate the potent effects of PKR and TRBP on HIV-1 expression. (C) Virion production was assessed by RT assay of culture supernatants.

FIG. 4.

TRBP expression rescues efficient virus expression in astrocytes. (A) The fold increase in virion production, as detected by RT activity in culture supernatants, from HeLa (open bar) and U251MG astrocytoma cells (black bar) following cotransfection with 5 μg of a TRBP expression plasmid (pCMV-TRBP) and pNL4-3 provirus relative to an empty vector cotransfection control with pEN1 and pNL4-3 (n = 5). Representative experiments comparing the level of virion production, measured as supernatant RT activity (B) or as supernatant p24 capsid protein level (C), by HeLa cells (open bar) and astrocytes (black bar) with and without cotransfected pCMV-TRBP. (D) Impact of TRBP on PKR phosphorylation. U251MG astrocytoma cells transfected with pNL4-3 provirus and either pEN1 (− TRBP) or pCMV-TRBP (+ TRBP) plasmids. Transfected U251MG cell lysate, immunoprecipitated with PKR 71/10 monoclonal antibody, was probed with either the PKR F9 mouse monoclonal antibody (PKR) or a phosphorylation-specific PKR polyclonal antibody (P-PKR). (E) Immunoblot analysis of HIV-1 proteins by HIV-1-positive patient serum using equal amounts of total protein from lysates of U251MG cells transfected with pNL4-3 provirus and either pEN1 empty vector (− TRBP) or pCMV-TRBP (+ TRBP) plasmids. (F) Infection of CEM-12D7 cells by U251MG astrocytoma-derived progeny virus from cells that were transfected with pNL4-3 provirus (▪), pNL-TRBP plasmids (✖), or supernatant from mock transfection (⧫), using input standardized by RT activity level (see Materials and Methods).

FIG. 5.

Basal TRBP expression in astrocytes. (A) Comparison of TRBP protein expression in U251MG astrocytoma cells and primary fetal astrocytes with HeLa cells. Equal amounts of total protein from cell lysates were loaded onto a 10% polyacrylamide gel and, following transfer to membrane, probed with a TRBP antiserum (TRBP pep672). (B) Comparison of TRBP mRNA expression in U251MG astrocytoma cells with HeLa and Jurkat cells by Northern blot of TRBP mRNA using equal amounts of polyadenylated RNA.

FIG. 6.

TRBP increases expression of HIV-1 structural proteins. U251MG astrocytoma cells were transfected with an EGFP-TRBP expression reporter plasmid pCMV-TRBP and stably transduced cells were selected using geneticin. (A) Four distinct cell populations of antibiotic-selected U251MG cells (designated EGFP-TRBP 1, 2, 3, and 4) expressing different levels of EGFP-TRBP were sorted by flow cytometry. (B) Expression of the EGFP-TRBP chimera in the stably transfected U251MG astrocytoma cells was confirmed by immunoblotting with TRBP pep672 serum. (C) HIV-1 protein expression profile by immunoblot of transfected cell lysates using HIV-1-positive patient serum. Equal amounts of total protein were loaded from the U251MG stable cell lysates transfected with pNL4-3 proviral plasmids. Cell lysates from HeLa cells transfected with pNL4-3 were used as a positive control.