1 Laboratoire d'Immunologie Moléculaire de l'Infection et de l'Inflammation, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59019 Lille Cedex, France
2 INSERM Unité 547, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59019 Lille Cedex, France
3 Service des Maladies Infectieuses, Hopital Dron, 59208 Tourcoing Cedex, France
Correspondence
Georges Bahr
georges.bahr{at}pasteur-lille.fr
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ABSTRACT |
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The nucleotide sequence data reported in this paper have been submitted to GenBank under accession number AY017378.
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INTRODUCTION |
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In an effort to further define cellular factors that could mediate HIV-1 suppression by Murabutide, we had carried out a differential display RT-PCR (DD-RT-PCR) analysis on CD8-depleted PBMCs, stimulated or not with Murabutide, from a patient infected with HIV-1 (Bahr, 2003b; Billaut-Mulot et al., 2001a
). The Murabutide-regulated genes of known functions belonged to families encoding factors implicated in transcription, splicing, translation, proteolysis and protein translocation (Bahr, 2003b
). However, we were particularly interested in two of the Murabutide-downregulated genes whose sequences are still unknown. In a previous report, one of the two genes in question was cloned and was named SS56, since it was revealed as a new member of the Sjögren's syndrome (SS) family of autoantigens (Billaut-Mulot et al., 2001a
). In the present study, we have cloned the full-length cDNA of the second Murabutide-downregulated gene, which initially showed no identity with published gene sequences. The corresponding amino acid sequence revealed a protein with a predicted molecular mass of 116 kDa and presented similarity with members of the DExH/D family of RNA helicases (Jankowsky & Jankowsky, 2000
; Luking et al., 1998
). This protein, named RH116 (RNA helicase 116 kDa), is shown to play a role in regulating cell growth and HIV-1 replication.
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METHODS |
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Cloning of RH116 cDNA.
A cDNA fragment of 164 bp was isolated from differential display gels and which was underexpressed in Murabutide-treated PBMCs from HIV-1-infected patient. The SMART Rapid Amplification of cDNA Ends (RACE) kit (Clontech) was used to synthesize the 5' and 3' cDNA ends. To generate the 5' end, three successive and specific oligonucleotides primers were used: (1) 5'-CACAATACTCATCATCACCACCCTCATCA-3'; (2) 5'-GTAGGGCCTTATTGTACTTCCTCAAAT-3'; and (3) 5'-CTAAGCAGCTGACACTTCCTTCTGCCAAACTTGTGTCTG-3'. The 3' end of RH116 cDNA was amplified with the following primer: 5'-TGATGAGGGTGGTGATGATGAGTATTGTG-3'. The full-length cDNA encoding RH116 was then obtained by RT-PCR using two synthetic oligonucleotides that included the start codon for the 5' end and the stop codon for the 3' end. All PCR amplification products were cloned into the pCR2.1 vector (Life Technologies) and nucleotide sequences were determined in both strands using dye terminator sequencing and the ABI 377 DNA sequencer equipped with ABI Prism Model version 2.1.1 software for data recording and analysis (Applied Biosystems). Both nucleotide and deduced amino acid sequences were analysed for similarity with known sequences using BLAST search (Altschul et al., 1997) and ExPASy proteomics tools (http://www.expasy.ch/tools/). The initial differential display cDNA fragment corresponded to nt 19252089 in the complete cDNA sequence of RH116. The 2·5kb EcoRI fragment of RH116 cDNA was radiolabelled with [
-32P]dCTP, employing the Megaprime labelling kit (Amersham Pharmacia Biotech) and was used to screen the
TriplEx spleen cDNA library, according to the manufacturer's instructions (Clontech).
Expression of His-tagged protein and production of mouse polyclonal antibodies.
Efforts to generate recombinant RH116 following expression in Escherichia coli were repeatedly unsuccessful due to the high toxicity of the protein in bacteria. Therefore, a partial fragment corresponding to the first 335 aa of RH116 cDNA (RH1161335) was amplified using the following oligonucleotides: 5'-TGAGAGGATCCGATGTCGAATGGGTATTCC-3' (sense) and 5'-GTGGTCGACGGCAATGTAAACAGCCACTCTGG-3' (antisense). The partial cDNA was subcloned into the pQE-81 vector (Qiagen) and was used to transform TOP 10F' E. coli (Life Technologies). Purification of the recombinant protein RH1161335 fused to six histidine residues was performed under denaturing conditions using Ni2+ affinity chromatography and following the manufacturer's instructions (Qiagen). To prepare polyclonal antibodies, 6-week-old female BALB/c mice (Iffa-Credo) were immunized with 50 µg recombinant protein, as described elsewhere (Billaut-Mulot et al., 2001a). Prior to immunization, sera from the same mice were collected and used as antibody negative controls.
Western blot.
Total cellular extracts, prepared in lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 1 % Triton X-100, 0·5 mM DTT, 100 mM PMSF, 1 µg pepstatin ml-1 and 0·1 % aprotinin), were fractionated by SDS-PAGE on 620 % gels, electroblotted onto nitrocellulose membranes and incubated for 1 h at room temperature with polyclonal mouse anti-RH1161335 or with preimmune mouse serum at a 1 : 50 dilution. After washing, the membranes were probed with a 1 : 500 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulins and incubated for 1 h at room temperature. Reactive bands were then revealed using either the 4 CN peroxidase substrate system (KPL) or ECL reagents (Amersham Pharmacia Biotech). To control for equal quantity of loaded proteins, the amount of actin in cell extracts was quantified using the monoclonal anti-actin antibody.
Indirect immunofluorescence analysis.
This was performed as described previously (Billaut-Mulot et al., 2001a) and nuclei were stained using 0·1 µg DAPI ml-1 prior to examination of slides with fluorescence microscopy (Axioskop, Zeiss).
DNA transfection and HIV-1 infection.
All of the plasmids used were prepared using endotoxin-free materials (EndoFree, Giga Kit, Qiagen). The RH116 cDNA was subcloned as a XhoIBamHI fragment into the pEGFP-N1 vector (Clontech), which permits the expression of RH116 fused to the N terminus of the green fluorescent protein (pEGFP/RH116). The RH116 cDNA was also subcloned as a BamHIXhoI fragment into the pcDNA6/V5-His vector (Invitrogen). The correct in-frame fusion of the cDNA was controlled by sequencing. The native plasmids, pEGFP-N1 (pEGFP) and pcDNA6/V5-His (pcDNA6), as well as plasmids containing the SS56 gene (pEGFP/SS56 and pcDNA6/SS56) or the partial cDNA fragment of RH116 (pEGFP/RH1161335), were used as controls. In addition, Tat cDNA was cloned into the pCR3 plasmid, as described previously (Billaut-Mulot et al., 2001b). At 1 day prior to transfection, 5x104 HeLa-CD4 cells were seeded per well in 12-well plates and were then transfected using 500 ng DNA and 5 µl Effectene (Qiagen). In all experiments, transfection with each plasmid was done in triplicate wells and 24 h after transfection cells were infected with the T-tropic HIV-1LAI strain obtained from the Central Virology Laboratory, Lille, France. Transfected cells were exposed to 2·5x105 c.p.m. of virus reverse transcriptase activity and incubated overnight at 37 °C. Free virus was then removed and cells were washed and maintained in fresh medium. Starting 1 day after the infection period and for the following 4 days, supernatants were collected from each well and cells were recovered and counted using trypan blue. Virus replication was evaluated by the detection of HIV-1 DNA, 24 h after infection, and HIV-1 RNA or p24 protein from days 2 to 5 post-infection (p.i.).
Detection of HIV-1 DNA and RNA.
Total cellular DNA was extracted from HIV-1-infected cells and subjected to 35 repeated rounds of amplification with AmpliTaq Gold DNA polymerase, as described previously (Truong et al., 1999). PCR amplification of
-actin sequences was performed to standardize for cell equivalence and HIV-1 proviral DNA was amplified using the GAG06/GAG04 primer pair (Piatak et al., 1993
). To measure levels of HIV-1 RNA, total cellular RNA was extracted with RNAplus (Q-BIOgene) and was amplified using rTth polymerase (Applied Biosystems) in the presence of the GAG06/GAG04 primer pair to detect the HIV-1 unspliced GagPol mRNA and the BSS/KPNA primer pair to detect the intermediate-size, singly spliced mRNA, as reported previously (Amiel et al., 1999
). All PCR products were separated on acrylamide gels and visualized by ethidium bromide staining. Using imaging systems (Image Master 1D prime, Amersham Pharmacia Biotech), HIV-1 DNA and RNA expression was deduced after normalization to the levels of the corresponding internal standards GAPDH and
-actin, respectively (Amiel et al., 1999
; Truong et al., 1999
). The change in HIV expression in RH116-, SS56- or Tat-transfected cells was calculated relative to the expression level detected in cells transfected with the corresponding native plasmid.
p24 assay.
Virus replication was evaluated by measuring p24 antigen levels in culture supernatants using the HIV-1 p24 Antigen Assay kit (Coulter), following the manufacturer's instructions.
Cell proliferation assay.
HeLa-CD4 cells, transfected with pEGFP/RH116, pEGFP/RH1161335, pEGFP/SS56 or with the native plasmid, were seeded at 5x103 cells per well in 96-well microtitre plates (Falcon). Following 1, 2 and 3 days in culture, the level of DNA synthesis was measured after a 6 h pulse with 0·5 µCi [3H]thymidine per well (Amersham Pharmacia Biotech). Cells were harvested on a filter mat for scintillation counting (Skatron). Radioactivity was read using a Tricard 1600LR liquid scintillation -counter (Packard).
Measurement of apoptosis by flow cytometry.
HeLa-CD4 cells transfected with pcDNA6 constructs were evaluated using Annexin V and propidium iodide (PI) double staining (Pharmingen), according to the manufacturer's instructions. Stained cells were analysed on a FACSCalibur flow cytometer using CELLQuest software (Becton Dickinson).
Semi-quantitative RT-PCR for the detection of gene expression.
To determine the level of mRNA expression of a transfected gene, total cellular RNA was extracted using RNAplus and was treated with DNase I. First-strand cDNA was synthesized using a poly(dT)15 primer (Roche) and Moloney murine leukaemia virus reverse transcriptase (Promega) following the manufacturer's instructions. The resulting cDNA was subjected to 2535 repeated rounds of amplification with AmpliTaq Gold DNA polymerase. PCR amplification of different concentrations of RH116, SS56, Tat and GAPDH cDNAs was performed using the following oligonucleotide primers: RH116 sense, 5'-GGAAGTACAATGAGGGCCTACAAA-3', and RH116 antisense, 5'-TCCTCAGCCCTAGTATATTGCTCC-3'; SS56 sense, 5'-GAAAGAGAGGTCGCAGAGGCCTGT-3', and SS56 antisense, 5'-TGATAAGGCTGAGGAAGGGAAATG-3'; Tat sense, 5'-CTAGACCCCTGGAAGCATCCA-3', and Tat antisense, 5'-TCGGGCCTGTCGGGTCCCCTC-3'; GAPDH sense, 5'-GCCATCAATGACCCCTTCATTGAC-3', and GAPDH antisense, 5'-TGACGAACATGGGGGCATCAGCAG-3'. All PCR products were separated on a 2 % agarose gel and visualized by ethidium bromide staining.
Analysis of RH116 expression following HIV-1 infection.
To evaluate the effect of HIV-1 infection on RH116 expression, HeLa-CD4 cells were infected with HIV-1LAI strain or were mock-infected using the same virus inactivated by a 2 h treatment at 56 °C. Starting 4 h after infection and for the following 72 h, total RNA was extracted and subjected to RT-PCR amplification using RH116-specific primers, as described above. To determine the effect of HIV-1 infection on RH116 protein levels, total cell lysates and purified cytoplasmic and nuclear fractions were prepared as described elsewhere (You et al., 1999) and were subjected to Western blot analysis using anti-RH116 antibodies. To control for equal quantity of loaded proteins, the amount of
-actin in total cell extracts,
-tubulin in cytoplasmic fractions and histone H1 in nuclear fractions were quantified. The absence of cross-contaminants between the cytoplasmic and nuclear preparations was verified by reprobing the blots, respectively, with anti-histone and anti-
-tubulin antibodies.
Statistical analysis.
Student's t-test was used to determine significance. SE values were also determined.
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RESULTS |
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Comparison of the inferred amino acid sequence with proteins in the current databases revealed a striking homology of the RH116 protein sequence with that of a newly identified human interferon-inducible putative RNA helicase, encoded by the melanoma differentiation-associated gene-5, termed MDA-5 (Kang et al., 2002). The overall amino acid sequence identity between the two proteins was 99·5 % and the similarity, including conservative changes, was even 99·9 %. The minor differences between RH116 and MDA-5 may reflect natural variations in alleles; however, the pattern of helicase motifs in the two sequences was the same.
Detection and localization of RH116
To verify the size of the native protein and to determine its intracellular localization under steady state, we performed Western blotting on total cell extracts and immunofluorescence on HeLa-CD4 cell monolayers. Results from Western blots, using a mouse antiserum raised against the partial recombinant protein RH1161335 (Fig. 1A), revealed a single band of 120130 kDa, either in HeLa-CD4 or in U937 total cell extracts. Analysis by immunofluorescence of HeLa-CD4 cells indicated a cytoplasmic localization of the RH116 protein with no evident presence in the nucleus (Fig. 1B
). The specificity of detection was verified using preimmune mouse serum as antibody control and identical results were observed in U937 cells (data not shown).
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HIV-1 infection of HeLa-CD4 cells upregulates endogenous RH116 expression
The question as to whether HIV-1 infection of HeLa-CD4 cells could regulate endogenous RH116 expression was then addressed. Thus, HeLa-CD4 cells were either infected with HIV-1 or mock-infected with heat-killed virus and the level of RH116 mRNA expression in total RNA extracts, taken at different time points after infection, was evaluated by RT-PCR. Representative results from one of two identical experiments (Fig. 7A) demonstrated increased RH116 gene expression following 4, 8 and 24 h of infection with HIV-1, as compared with the levels observed in mock-infected cultures. This increase in RH116 mRNA expression was also noted 48 h after infection (data not shown). To ensure that the observed HIV-1-induced increase in RH116 transcription correlated with an increase in protein levels, we performed Western blot analysis on total cell lysates extracted before (0 h) and after HIV-1 infection. Results shown in Fig. 7(B)
indicated a clear increase in RH116 protein levels that was detectable after 8, 24 and 48 h of infection.
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DISCUSSION |
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Although we did not address the potential implication of RH116 in different biochemical events, we were able to confirm and extend the recently reported growth-suppressive properties of MDA-5 (Kang et al., 2002). Thus, overexpression of RH116 in HeLa-CD4 cells resulted in the inhibition of cellular proliferation; however, this effect did not correlate with increased cell death. In this respect, the activity of RH116/MDA-5 is very similar to that reported with another RNA Helicase, CHAMP, capable of inhibiting cell proliferation by upregulating cyclin-dependent kinase (CDK) inhibitors (Liu & Olson, 2002
). Therefore, it would be of interest to address in future studies the potential role of RH116 in regulating the expression of, or the interaction between, factors implicated in different cell cycle checkpoints, including cyclins, CDKs and CDK inhibitors (Balomenos & Martinez-A, 2000
; Morgan, 1997
). On the other hand, a putative role of MDA-5/RH116 in programmed cell death has been suggested due to the presence of a potential CARD domain in the N-terminal part of the protein (Kang et al., 2002
). However, our findings on the absence of increased apoptosis following ectopic expression of RH116 and the similar results that have been observed with Helicard (Kovacsovics et al., 2002
), strongly suggest the lack of a critical role for the helicase in driving programmed cell death.
Binding of cellular helicases to viruses or to viral proteins has long been known to lead to the regulation of viral and/or of cellular gene expression (Mamiya & Worman, 1999; You et al., 1999
). Moreover, the implication of human RNA helicase A in multiple steps of the HIV-1 life cycle has been described previously (Li et al., 1999
; Reddy et al., 2000
). Based on these finding, we have addressed the potential role of RH116 in HIV-1 replication. Our results clearly indicate that ectopic expression of RH116 induces a dramatic upregulation of viral p24 release. This increase in virus replication could not be linked to an effect on the early process of proviral DNA formation but correlated with increased levels of viral mRNA transcripts. At this stage, the mechanism by which RH116 upregulates HIV-1 expression is still unclear, although few possibilities are accessible for verification. For instance, the effect of RH116 on cell growth and a potential regulation of cyclin levels may result in optimal Tat transactivation and in increased long terminal repeat (LTR)-directed gene expression (Hrimech et al., 1999
; Liou et al., 2002
). Furthermore, a possible binding of RH116 to HIV-1 LTR, to viral proteins or to cellular factors necessary for LTR activation (Al-Harthi & Roebuck, 1998
; Flores et al., 1999
) may be a key element in the RH116-induced upregulation of HIV-1 replication. Nevertheless, our findings of a nuclear presence of RH116 following HIV-1 infection support a potential and direct role for the helicase in HIV transcription. Although analysis of the amino acid sequence of RH116 did not reveal a classical NLS, the translocation of the helicase or one of its cleaved fragments (Kovacsovics et al., 2002
) to the nucleus might occur either via the endoplasmic reticulum or complexed to other proteins (Bickmore & Sutherland, 2002
). Additional studies would be needed to dissect the mechanism of nuclear transport of RH116 following HIV-1 infection and the molecular events leading to enhanced virus expression.
The implication of RH116 in HIV-1 replication was substantiated further by the finding that following infection of HeLa-CD4 cells, a marked upregulation of endogenous RH116 gene and protein expression could be detected. This suggests a mutual cross regulation between RH116 and HIV-1 and the potential requirement of RH116 for virus replication. A similar cross regulation between a porcine virus and the RNA helicase induced by virus (RHIV-1) has been reported also in alveolar macrophages (Zhang et al., 2000). It is interesting to note that porcine RHIV-1 is closely related to human RH116/MDA-5 and that both proteins share a strong similarity with another human RNA helicase, RIG-1 (retinoic acid-induced gene-1, accession number NP055129). Although the role of RIG-1 as a virus cofactor has not been studied, it is tempting to suggest that the three inducible DExH helicases, RH116, RHIV-1 and RIG-1, which share a strong amino acid similarity, could constitute a subfamily of RNA helicases that are upregulated by virus infections and are themselves necessary cofactors for virus replication.
Finally, based on our findings implicating RH116 in the regulation of HIV-1 replication, it would be highly pertinent to determine, through the use of interference RNA, whether or not this novel cellular RNA helicase is a valid therapeutic target for blocking virus replication.
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ACKNOWLEDGEMENTS |
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Received 17 April 2003;
accepted 21 August 2003.