Critical role of hnRNP A1 in HTLV-1 replication in human transformed T lymphocytes

Abstract

Background: In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1.

Results: We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown in C91PL cells increased viral production by these cells. Thus, hnRNP A1 is implicated in the modulation of the level of HTLV-1 gene expression in T cells transformed by this human retrovirus.

Conclusions: These observations provide an insight into a new cellular control of HTLV-1 replication and suggest that hnRNP A1 is likely part of the regulatory mechanisms of the life cycle of this human retrovirus in T cells.

Figure 1

Functional characterization of HTLV-1 mutated XRE sequence. (A) Schematic representation of the HTLV-1 XRE. On the left, the XRE corresponds to U3 and R sequences within the HTLV-1 long terminal repeat, and consists of four stem-loops. On the right, the predicted secondary structure of the stem-loopD (SLD) with the minimal Rex binding site and the mutations introduced within the putative hnRNP A1 binding site are indicated. (B) Schematic view of the reporter plasmid CMV/XRE. (C) Effect of mutations within the XRE sequence on the Rex trans-activation capacity. Jurkat cells were transfected with 1 μg of the indicated reporter plasmid in the presence or not of Rex expression plasmid (200 ng) and the constitutive internal control tk-renilla luciferase vector (10 ng). Data are expressed as normalized luciferase activity and the error bars represent the standard deviations from three independent experiments.

Figure 2

Expression of a dominant negative mutant of hnRNP A1 in HTLV-1 producing C91PL cells. Confocal microscopy of untransduced (a) or NLS-A1-HA transduced (b)-C91PL cells after dual immunofluorescence staining with anti-HA (green) and anti-hnRNP A1 (red) antibodies; the right panels show the overlay of the green and red staining;

Figure 3

Effect of ectopic expression of a dominant negative mutant of hnRNP A1 in HTLV-1 producing C91PL cells. (A) Primer location on HTLV-1 mRNA; (B) Analysis of the nucleo-cytoplasmic distribution of viral gene expression in NLS-A1- and LXSP- transduced cells. Four days after transduction, mRNAs were extracted from the nuclear and cytoplasmic compartments of each cell type and levels of unspliced (gag/pol), singly spliced (env) and doubly spliced (tax/rex) mRNAs were reverse transcribed and quantified by real-time quantitative PCR (RQ-PCR), by using specific primers. Results are expressed as the amount of nuclear (grey bar) and cytoplasmic (black bar) indicated mRNA relative to β-actin. (C) Evaluation of the nuclear export rate (NER) of Rex-dependent (gag/pol plus env) mRNA and of Rex-independent (tax/rex) mRNA in NLS-A1- or LXSP- transduced C91PL cells. Numbers are the ratio between cytoplasmic (C) to total (T) RNA and nuclear (N) to total RNA.

Figure 4

RNAi-mediated reduction of hnRNP A1 expression in Jurkat cells. (A) hnRNP A1 mRNA levels in cells transduced with the indicated retroviruses were determined by RQ-PCR. Levels in knockdown cells are given as percent mRNA reduction relative to the level in control cells transduced with empty pRS virus. Standard deviations are from at least three determinations performed in duplicate. (B) Equal amounts of protein from either nontransduced (lane1) or transduced with the indicated virus (lanes 2 to 4) were analyzed by immunoblotting. Actin and ASF/SF2 were used as control. Note that hnRNP A1 was significantly depleted in cells transduced with siRNA+548, whereas ASF/SF2 was not affected.

Figure 5

Analysis of hnRNP A1 depletion in HTLV-1 producing C91PL cells. (A) Analysis of hnRNP A1 mRNA levels in cells transduced with the indicated retroviruses. Four days after transduction, cytoplasmic RNA were extracted, reverse transcribed with oligo-dT, and levels of hnRNP A1 mRNA were determined by RQ-PCR. (B) Expression of hnRNP A1, Rex and hnRNP C1/C2 was monitored by immunoblotting of total protein extract from C91PL cells transduced with the indicated virus. Equivalent protein loading was confirmed by immunoblotting with an anti-actin antibody. (C) Detection of hnRNP A1 and p19gag expression in C91PL cells transduced with the indicated virus. Dot plots showing both hnRNP A1 and HTLV-1 gag expressions in one representative experiment. The percentage of cells in each quadrant is indicated.

Figure 6

Effect of hnRNP A1 depletion on viral gene expression. (A) Quantification of total viral gene expression in siRNA-transduced C91PL cells by quantitative PCR. Nuclear and cytoplasmic mRNAs were extracted from siRNA (black bars)- or control PRS (white bars)- transduced C91PL cells. Equal amounts of mRNA were reverse transcribed with oligo-dT and subjected to RQ- PCR. Results are expressed as the relative levels of total viral mRNA to cellular β-actin. Error bars indicate standard deviations. (B) Analysis of the nucleo-cytoplasmic expression of viral genes. Four days after transduction, mRNAs were extracted and analyzed as in Fig. 3B. Results are expressed as the amount of nuclear (grey bar) and cytoplasmic (black bar) indicated mRNA relative to β-actin. (C) Evaluation of the nuclear export rate (NER) of Rex-dependent (gag/pol plus env) mRNA and of Rex-independent (tax/rex) mRNA in PRS- or siRNA- transduced C91 PL cells.