Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Dec;85(23):12614-21.
doi: 10.1128/JVI.05240-11. Epub 2011 Sep 21.

The cellular TAR RNA binding protein, TRBP, promotes HIV-1 replication primarily by inhibiting the activation of double-stranded RNA-dependent kinase PKR

Viraj R Sanghvi  1 , Laura F Steel
Affiliations

The cellular TAR RNA binding protein, TRBP, promotes HIV-1 replication primarily by inhibiting the activation of double-stranded RNA-dependent kinase PKR

Viraj R Sanghvi et al. J Virol. 2011 Dec.

Abstract

The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5'-cap or by activating the dsRNA-dependent kinase, PKR. Several mechanisms by which TRBP can facilitate translation of viral transcripts have been proposed, including the binding and unwinding of TAR and the suppression of PKR activation. Further, TRBP has been identified as a cofactor of Dicer in the processing of microRNAs (miRNAs), and sequestration of TRBP by TAR in infected cells has been proposed as a viral countermeasure to potential host cell RNA interference-based antiviral activities. Here, we have addressed the relative importance of these various roles for TRBP in HIV-1 replication. Using Jurkat T cells, primary human CD4(+) T cells, and additional cultured cell lines, we show that depletion of TRBP has no effect on viral replication when PKR activation is otherwise blocked. Moreover, the presence of TAR-containing mRNAs does not affect the efficacy of cellular miRNA silencing pathways. These results establish that TRBP, when expressed at physiological levels, promotes HIV-1 replication mainly by suppressing the PKR-mediated antiviral response, while its contribution to HIV-1 replication through PKR-independent pathways is minimal.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Inhibition of PKR activation prevents the suppression of HIV-1 replication by TRBP knockdown in 293T cells. (A) 293T cells were transfected with NS siRNA, siTRBP-1, siTRBP-2, or siTRBP-3, together with a constant amount of pLAI and different amounts of pPKR-Δ6, as indicated. Two days posttransfection, infectious virus released into the supernatant was determined in a P4R5 infection assay. Data are represented as β-galactosidase specific activity in the P4R5 indicator cells and shown as a percentage of control (NS siRNA, no pPKR-Δ6). Error bars represent standard deviations, derived from a total of 4 replicates. (B, upper panel) Total protein extracts prepared from transfected cells shown in panel A were immunoblotted for detection of P-eIF2α, total eIF2α, and actin. (Lower panel) Relative expression of P-eIF2α to total eIF2α as determined by densitometry analysis of the immunoblot shown in the upper panel. (C) Total RNA isolated from transfected cells as in panel A was analyzed by RT-PCR using primers specific for HIV-1 Tat mRNA, TRBP mRNA, and β-actin mRNA, with a limited number of PCR cycles to enable detection of quantitative differences in input RNA. Replicate samples are shown for each experimental condition.
Fig. 2.
Fig. 2.
TRBP depletion does not reduce HIV-1 replication in Jurkat cells. (A) Jurkat cells (upper panels) or 293T cells (lower panels) were transfected with poly(I:C) (2.5 μg/ml) or treated with DTT (10 mM for 1 h), as indicated. Protein extracts were analyzed by 12% PAGE–SDS and immunoblotting with antibodies specific to P-eIF2α, total eIF2α, and β-actin. Numbers show the relative ratio of P-eIF2α to eIF2α, with the value for untreated cells set to 1.0. (B) Jurkat cells were transfected with NS or TRBP-specific siRNAs together with pLAI and different amounts of pPKR-Δ6, as indicated. The release of infectious virus into the supernatant was measured 2 days posttransfection by P4R5 infection assays. β-Galactosidase specific activity is shown as a percentage of the NS siRNA/no-PKR-Δ6 control. Error bars represent standard deviations from four replicates. (C) Protein lysates from 293T cells and Jurkat cells untreated or treated for 24 h with 1,000 U/ml IFN-α, as indicated, were immunoblotted using antibodies to detect PKR and GAPDH. (D) Jurkat cells were transfected as described for panel B, and after 18 h, cells were treated with 1,000 U/ml IFN-α for 24 h. Supernatant was collected, and P4R5 infection assays were performed and analyzed as for panel B. Error bars represent standard deviations from four replicates. In panels A and C, replicate samples are shown for each experimental condition.
Fig. 3.
Fig. 3.
TRBP promotes HIV-1 replication in primary CD4+ T cells only when the cells express functional PKR. (A) RNA isolated from 293T and Jurkat cells was subjected to RT-PCR using primers specific for either TRBP mRNA or actin mRNA. (B) TRBP expression was analyzed by quantitative RT-PCR in nonactivated primary CD4+ T cells isolated from two different donors. Data were normalized to actin and are represented as the fold change relative to TRBP mRNA expression in Jurkat cells. (C) Total RNA was isolated from nonactivated and activated CD4+ T cells, treated or not treated with 1,000 U/ml IFN-α for 24 h, and subjected to quantitative RT-PCR analysis to examine TRBP expression. Data were normalized to actin and are represented as the fold change relative to nonactivated, untreated T cells. (D) Activated primary CD4+ T cells were transfected with the indicated siRNAs, and 24 h posttransfection, cells were infected with HIV-LAI (4 ng p24). Production of infectious virus was determined by P4R5 infection assays 2 days postinfection. β-Galactosidase specific activity is shown as a percentage of the NS siRNA/no-siPKR control. Error bars represent standard deviations from four replicates. **, P < 0.005 (relative to NS siRNA/no-siPKR control); *, P < 0.05.
Fig. 4.
Fig. 4.
The presence of TAR-containing mRNA does not reduce the silencing capacity of cells. (A) Schematic depicting the plasmid constructs pTAR-Rluc and pRluc and the corresponding Renilla luciferase transcripts. (B) Total protein extracts from HeLa cells transfected with the indicated plasmids were immunoblotted and analyzed for EGFP expression. Actin served as a loading control. Replicate samples are shown for each experimental condition. (C) RT-PCR was performed on total RNA obtained from either TNF-α-stimulated ACH-2 cells or transfected HeLa cells, as indicated, using primers specific for the HIV-1 TAR-containing 5′-UTR (upper panel) or actin mRNA (lower panel). (D) HeLa cells were transfected with plasmids encoding EGFP and miEGFP along with siRNAs, as indicated, and EGFP silencing was assayed by immunoblotting of total protein lysates obtained 2 days posttransfection using antibodies against EGFP and GAPDH. Numbers indicate silencing relative to the no-miEGFP control, which was set at 1.0.
Fig. 5.
Fig. 5.
Overexpression of TRBP prevents the TAR-dependent phosphorylation of eIF2α. P4R5 cells were transfected with 150 ng of p-ds-EGFP along with 50 ng p-miEGFP and 50 ng pTat, as specified, and total protein extracts prepared 2 days posttransfection were separated on a 12% PAGE–SDS gel and immunoblotted with the indicated antibodies. Paired lanes are loaded with protein from replicate samples, as indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bannwarth S., et al. 2001. Organization of the human tarbp2 gene reveals two promoters that are repressed in an astrocytic cell line. J. Biol. Chem. 276:48803–48813 - PubMed
    1. Benkirane M., et al. 1997. Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR. EMBO J. 16:611–624 - PMC - PubMed
    1. Bennasser Y., Yeung M. L., Jeang K. T. 2006. HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP. J. Biol. Chem. 281:27674–27678 - PubMed
    1. Boo K. H., Yang J. S. 2010. Intrinsic cellular defenses against virus infection by antiviral type I interferon. Yonsei Med. J. 51:9–17 - PMC - PubMed
    1. Brand S. R., Kobayashi R., Mathews M. B. 1997. The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR. J. Biol. Chem. 272:8388–8395 - PubMed

Publication types

MeSH terms