A novel cellular RNA helicase, RH116, differentially regulates cell growth, programmed cell death and human immunodeficiency virus type 1 replication

C. Cocude1, M.-J. Truong1, O. Billaut-Mulot1, V. Delsart1, E. Darcissac1, A. Capron2, Y. Mouton3 and G. M. Bahr1

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


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In an effort to define novel cellular factors regulating human immunodeficiency virus type 1 (HIV-1) replication, a differential display analysis has been performed on endogenously infected cells stimulated with the HIV-suppressive immunomodulator Murabutide. In this study, the cloning and identification of a Murabutide-downregulated gene, named RH116, bearing classical motifs that are characteristic of the DExH family of RNA helicases, are reported. The 116 kDa encoded protein shares 99·9 % similarity with MDA-5, an inducible RNA helicase described recently. Ectopic expression of RH116 in HeLa-CD4 cells inhibited cell growth and cell proliferation but had no measurable effect on programmed cell death. RH116 presented steady state cytoplasmic localization and could translocate to the nucleus following HIV-1 infection. Moreover, the endogenous expression of RH116, at both the transcript and protein levels, was found to be considerably upregulated after infection. Overexpression of RH116 in HIV-1-infected HeLa-CD4 cells also resulted in a dramatic increase in the level of secreted viral p24 protein. This enhancement in virus replication did not stem from upregulated proviral DNA levels but correlated with increased unspliced and singly spliced viral mRNA transcripts. These findings implicate RH116 in the regulation of HIV-1 replication and point to an apoptosis-independent role for this novel helicase in inducing cell growth arrest.

The nucleotide sequence data reported in this paper have been submitted to GenBank under accession number AY017378.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although highly active antiretroviral therapy (HAART) has considerably improved the management of patients infected with human immunodeficiency virus type 1 (HIV-1), several issues continue to limit the efficacy of HAART, including incomplete immune reconstitution and the emergence of drug-resistant escape variants (Angel, 2001; Bélec et al., 2000; Boden et al., 1999; Connick et al., 2000). Therefore, approaches aimed at correcting these virus-induced immune deficits are being sought and, in particular, immunotherapies that could also contribute to virus inhibition by targeting the expression of cellular cofactors needed at different steps in the virus life cycle (Bahr, 2003a). The synthetic immunomodulator Murabutide, long known to enhance the host's resistance against microbial infections (Bahr et al., 1995; Chedid et al., 1982; Chomel et al., 1988), has been observed recently to induce dramatic inhibition of virus replication in acutely infected macrophages or dendritic cells in endogenously infected T lymphocytes and in a mouse model of HIV-1 infection (Bahr et al., 2001; Darcissac et al., 2000). These HIV-suppressive activities of Murabutide were not linked to a direct effect on the virus and rather correlated with a regulated expression of cellular factors needed for the nuclear transport of virus preintegration complexes and virus transcription (Bahr et al., 2001; Darcissac et al., 2000). The potential use of this immunomodulator, as adjunct to HAART, in the management of HIV-1 patients has been examined recently (Amiel et al., 2002; Bahr, 2003a).

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.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell lines and reagents.
HeLa-CD4 and U937 cell lines were maintained in RPMI 1640 medium supplemented with 10 % FCS, 8 µg gentamicin sulfate ml-1 (Schering-Plough) and 2 mM glutamine (Life Technologies). Anti-mouse immunoglobulins, conjugated to FITC or horseradish peroxidase, and monoclonal anti-actin (AC-40) were purchased from Sigma. Mouse monoclonal anti-histone H1 and anti-{beta}-tubulin antibodies were obtained from Santa Cruz Biotechnology.

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 1925–2089 in the complete cDNA sequence of RH116. The 2·5kb EcoRI fragment of RH116 cDNA was radiolabelled with [{alpha}-32P]dCTP, employing the Megaprime labelling kit (Amersham Pharmacia Biotech) and was used to screen the {lambda}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 (RH1161–335) 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 RH1161–335 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 6–20 % gels, electroblotted onto nitrocellulose membranes and incubated for 1 h at room temperature with polyclonal mouse anti-RH1161–335 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 XhoI–BamHI 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 BamHI–XhoI 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/RH1161–335), 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 {beta}-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 Gag–Pol 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 {beta}-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/RH1161–335, 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 {beta}-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 25–35 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 {beta}-actin in total cell extracts, {beta}-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-{beta}-tubulin antibodies.

Statistical analysis.
Student's t-test was used to determine significance. SE values were also determined.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Molecular characterization of RH116 cDNA
Based upon the cDNA fragment obtained by DD-RT-PCR, a first round of 5' and 3' RACEs allowed us to characterize the poly(A)+ tail and to extend the cDNA sequence at the 5' end. The complete 5' end of the cDNA was obtained following three steps of 5' RACE. This attempt produced a complete cDNA of 3373 bp. To confirm this sequence, we then screened a human spleen cDNA library and the complete nucleotide sequence was obtained by determining overlapping sequences of two different clones. The RH116 cDNA sequence contained an initiation codon at position 155, an ORF of 3075 bp (nt 155–3232) and a 3' untranslated region of 141 bp with a consensus AATAAAA polyadenylation signal 24 bp upstream of the poly(A)+ tail of 21 bp. The ORF encoded a polypeptide of 1025 aa, with a calculated molecular mass of 116 kDa and an isoelectric point of 5·2. Sequence analysis of the cDNA in the Prosite database identified conserved domains: a caspase recruitment domain (CARD) (aa 11–200), a DExD/H-box RNA helicase (aa 298–531) and a helicase-conserved C-terminal domain (aa 748–826). The latter two domains are the signatures of the DEAD-box protein family of RNA helicases, which comprises distinct subgroups, the DEAD, DEAH and DExH, named according to their ATPase B conserved motif (Jankowsky & Jankowsky, 2000; Luking et al., 1998). No bipartite motif of nuclear localization signals (NLS) could be identified and eight sites of N-glycosylation were detected. The family of RNA helicases shares at least eight characteristic sequence motifs, and despite some minor differences, all of them are present in the RH116 amino acid sequence and are very closely related to the DExH subgroup. Therefore, we considered RH116 as a putative member of the DExH subgroup of the DEAD-box protein family of RNA helicases.

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 RH1161–335 (Fig. 1A), revealed a single band of 120–130 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).



View larger version (100K):
[in this window]
[in a new window]
 
Fig. 1. Detection and intracellular localization of RH116 protein. (A) Western blot analysis of the presence of RH116 protein in human cell lines. Total cell extracts (150 µg per lane) were immunoblotted either with preimmune mouse serum as control antibody (control-Ab) or with mouse anti-RH1161–335 serum. (B) Cytoplasmic localization of RH116 in HeLa-CD4 cells, as demonstrated by immunofluorescence. Cells were incubated with control-Ab or with anti-RH1161–335 serum and were stained with DAPI (blue nuclei in 2) after incubation with FITC-labelled anti-mouse immunoglobulins. Reactivity was visualized for FITC (green fluorescence in 1) or for FITC and DAPI (green and blue fluorescence in 3).

 
RH116 inhibits proliferation of HeLa-CD4 cells
Because mda-5 was cloned in the context of growth-suppressive properties (Kang et al., 2002), we have investigated whether overexpression of the RH116 protein in pEGFP/RH116-transfected HeLa-CD4 cells could affect the level of [3H]thymidine incorporation. Results from one of three identical experiments (Fig. 2) clearly demonstrated that overexpression of RH116 induced >50 % inhibition of the level of [3H]thymidine uptake compared to that detectable in cells transfected with the native pEGFP plasmid. This effect could not be detected in cells transfected with either the partial RH116 cDNA (pEGFP/RH1161–335) or the SS56 cDNA (Fig. 2). Similar results were also obtained using HeLa-CD4 cells transfected with the pcDNA6 plasmid constructs (data not shown).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2. Transfection of HeLa-CD4 cells with pEGFP/RH116 inhibits cell proliferation. HeLa-CD4 cells were cultured at 5x103 cells per well and were transfected with pEGFP/RH116, pEGFP/RH1161–335, pEGFP/SS56 or with the native plasmid. The level of DNA synthesis, on each of the 3 days following transfection, was measured after a 6 h pulse with 0·5 µCi [3H]thymidine per well. Values shown are the means±SE of the mean of quadruplicate readings from one representative experiment.

 
Overexpression of RH116 reduces viable cell numbers without increasing apoptosis
To gain insight into the effect of RH116 on cell growth, HeLa-CD4 cells were transfected with plasmids containing, or not, RH116 cDNA, and the number of viable cells per culture as well as the percentages of dead cells were enumerated (Fig. 3A). Transfection of pcDNA6/RH116 was found to reduce by nearly 50 % the number of viable cells without increasing the percentages of trypan blue-positive dead cells. Moreover, when the level of apoptotic cells was determined by FACS analysis, no difference could be observed between cultures transfected with the native pcDNA6 plasmid or with the pcDNA6/RH116 plasmids. This was the case whether apoptosis levels were measured at 48 h after transfection (Fig. 3B, C) or at 24 and 72 h after transfection (data not shown).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. RH116 reduces viable cell numbers without a concomitant increase in cell death. (A) Following 1 and 2 days of transfection of Hela-CD4 cells with either pcDNA6 or pcDNA6/RH116, the number of viable cells (left panel) and the percentage of dead cells (right panel) per well were enumerated using trypan blue dye. Results shown are the means from five independent experiments. (B) Analysis of the percentage of apoptotic cells by flow cytometry using Annexin V and PI staining. Results from one representative experiment, 2 days following transfection of HeLa-CD4 cells, are shown. (C) The mean percentage of Annexin V- or/and PI-positive cells from four independent experiments is presented. Bars reflect the SE of the mean.

 
Overexpression of RH116 increases HIV-1 replication
The identification of RH116 and SS56 (Billaut-Mulot et al., 2001a) originated from a study using HIV-1-infected cells and both genes were initially selected on the basis of a downregulated expression following stimulation of cells with an HIV-suppressive immunomodulator. Therefore, the potential role of the two genes in regulating HIV replication was addressed. In a first series of experiments, HeLa-CD4 cells were transfected with different pcDNA6 constructs and were then infected with HIV-1LAI. Following 2, 3 and 4 days p.i., overexpression of the RH116 protein was determined by Western blots on total cell lysates. Moreover, cells were also recovered from triplicate wells and counted, and supernatants were collected for evaluation of p24 content. Representative results from one of three identical experiments are shown in Fig. 4. At 2 days after infection, overexpression of RH116 protein was easily detectable in pcDNA6/RH116-transfected cells but not in cells transfected with the SS56 cDNA (Fig. 4A); this profile was also evident on days 3 and 4 p.i. The overexpression of SS56 protein in pcDNA6/SS56-transfected cells was verified in a similar manner using immunoblots probed with anti-SS56 antibodies (data not shown). Cultures from HIV-1-infected HeLa-CD4 cells that were transfected with pcDNA6/RH116, but not with pcDNA6/SS56, presented between 48 and 74 % reduction in the number of viable cells (Fig. 4B). Because of this major effect of RH116 overexpression on cell growth, the level of virus replication was quantified by calculating the ng levels of secreted viral p24 protein per 5x105 viable cells. Results shown in Fig. 4(C) clearly demonstrate a dramatic potentiation of HIV-1 replication in pcDNA6/RH116-transfected cultures, ranging from 6-fold on day 2 to 27-fold on day 4 p.i. No measurable effect on virus replication could be noticed in pcDNA6/SS56-transfected cultures (Fig. 4C). Moreover, increased virus replication in pcDNA6/RH116-transfected cells was also accompanied by an increase in cell death (12·4 % on day 3 p.i. as compared with 4·8 and 4·6 % in cultures transfected, respectively, with the empty plasmid and with the SS56 cDNA), a phenomenon widely attributed to HIV-induced apoptosis (Gandhi et al., 1998; Glynn et al., 1996).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. Overexpression of RH116, but not of SS56, in HeLa-CD4 cells inhibits cell growth and upregulates HIV-1 replication. Cells were transiently transfected with the native pcDNA6 plasmid, pcDNA6/RH116 or pcDNA6/SS56 and were infected after a 24 h period with HIV-1LAI. (A) Overexpression of RH116 protein in transfected cells was controlled by Western blot analysis using total cell extracts (100 µg per lane) and mouse polyclonal antiserum against RH1161–335. (B) Number of viable cells per well was evaluated on days 2, 3 and 4 p.i. using trypan blue exclusion. (C) HIV-1 replication, assessed by the level of p24 protein in supernatants of cell cultures, is represented as ng protein per 5x105 viable cells. Results shown are from one of three identical experiments.

 
To confirm and analyse further the observed effects of RH116 overexpression on cell growth and HIV-1 replication, a second series of four experiments were performed using pEGFP plasmid constructs. Overexpression of each transfected cDNA-encoded protein was verified consistently (data not shown) and no differences could be observed, within a single experiment, in the efficiency of transfection between any of the four constructs used (pEGFP, pEGFP/SS56, pEGFP/RH1161–335 and pEGFP/RH116). Results shown in Fig. 5(A) represent the mean number of viable cells per well, in four separate experiments, calculated as percentages of detectable viable cells in cultures transfected with the control pEGFP native plasmid. Although transfection with SS56 had no significant effect on the number of viable cells, transfection of HeLa-CD4 cells with RH116 cDNA induced a significant inhibition of cell growth. Moreover, when HIV-1 replication was evaluated and normalized to ng p24 protein per 5x105 viable cells, a significant increase was observed, ranging between a mean of 3·8- and 9·8-fold on different days p.i., in pEGFP/RH116-transfected cultures. This effect could be detected neither in pEGFP/SS56-transfected cells (Fig. 5B) nor following transfection with the partial RH116 cDNA (pEGFP/RH1161–335), tested only in two separate experiments (data not shown). In addition, higher levels of virus replication in pEGFP/RH116-transfected cultures were accompanied with a 1·5- to 2-fold increase in the percentages of dead cells detected on different days p.i.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Transfection of HeLa-CD4 cells with pEGFP/RH116 reduces cell growth and upregulates HIV-1 replication. HeLa-CD4 cells were transiently transfected with the native pEGFP plasmid, pEGFP/SS56 or pEGFP/RH116. After a 24 h period, cells were infected with HIV-1LAI and were then maintained in culture for up to 5 days p.i. The number of viable cells per well (A), evaluated by trypan blue exclusion, and the level of HIV-1 replication (B), assessed by quantification of p24 antigen release, were examined daily between days 2 and 5 after infection. Values obtained from cultures transfected with pEGFP/SS56 or pEGFP/RH116 are presented as percentages of the corresponding values detected in cultures transfected with the native plasmid (indicated by broken lines as 100 %). Results shown are the means ±SE of the means from four independent experiments.

 
RH116 regulates viral mRNA expression
To determine the mechanism of enhanced p24 release in response to RH116 overexpression, we examined further viral mRNA and proviral DNA levels in transfected and HIV-1LAI-infected HeLa-CD4 cells. We also included in these experiments, as a positive control for the upregulation of viral transcription, cultures transfected with Tat cDNA cloned into the pCR3 (pCR3/Tat) plasmid. First, total RNA from transfected and infected HeLa-CD4 cells were isolated and the mRNA overexpression of each transfected cDNA was verified by RT-PCR (Fig. 6A). Then, RNA samples were subjected to a second RT-PCR analysis aimed at evaluating the levels of unspliced and intermediate-size, singly spliced viral transcripts. Representative results shown in Fig. 6(B) on samples taken 3 days p.i. indicate an upregulation of both forms of HIV-1 mRNA in HeLa-CD4 cells overexpressing either RH116 or Tat but not in cells overexpressing SS56. The mean fold increase, obtained from three independent experiments, in the level of unspliced viral mRNA following RH116 or Tat overexpression was 3·14±1·5 or 4·75±1·25, respectively. The mean fold increase in the levels of intermediate-size, singly spliced viral transcripts was 2·81±0·73 in RH116-transfected and 4·18±0·31 in Tat-transfected cells. The ratio of viral mRNA expression in pEGFP/SS56-transfected cells to that detected in cells transfected with the native plasmid was 0·81±0·12 for the unspliced and 0·91±0·14 for the singly spliced forms of HIV-1 mRNA.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 6. Overexpression of RH116 increases the level of viral transcripts in HIV-1-infected HeLa-CD4 cells. Following transfection with different plasmids, HeLa-CD4 cells were infected with HIV-1LAI and total cellular RNA was extracted, 48 h p.i, and was subjected to semi-quantitative RT-PCR analysis. (A) Different concentrations of RNA (500, 100, 20 and 4 ng) were analysed for the expression level of RH116 and SS56 in extracts from cells transfected with pEGFP, pEGFP/RH116 and pEGFP/SS56. In parallel, RNA samples from cells transfected with pCR3 or pCR3/Tat were analysed for Tat mRNA expression. (B) RNA samples were subjected to RT-PCR amplifications to detect viral transcripts using the primer pair GAG04/GAG06 to detect unspliced Gag–Pol mRNA and primer pair BSS/KPNA to detect intermediate-size, singly spliced viral transcripts. These mRNAs were named on the basis of the exons they contain and the proteins they produce (Neumann et al., 1994): 1.4E Tat (exons 1 and 4E), 1.2.4BE Vpu/Env (exons 1, 2 and 4BE), 1.2.5E Vpu/Env (exons 1, 2 and 5E), 1.4BE Vpu/Env (exons 1 and 4BE) and 1.5E Vpu/Env (exons 1 and 5E). (C) Total DNA was extracted 24 h p.i. and various concentrations (150, 30, 6 and 1·2 ng) were subjected to PCR amplification with primer pair GAG04/GAG06 to detect the HIV-1 gag gene. Cell equivalence was determined by amplification of the {beta}-actin or GAPDH housekeeping genes. Presented results are from one of three identical experiments.

 
To address the question of whether the mechanism of HIV-1 activation by RH116 could target the process of proviral DNA formation, we evaluated by PCR the proviral DNA content in total cell DNA extracts. Results shown from one representative experiment (Fig. 6C) indicated no difference whatsoever in the proviral DNA content, detected 1 day p.i., between extracts obtained from cells transfected with different constructs. This suggested that overexpression of RH116 could upregulate HIV-1 transcription without affecting the earlier process of proviral DNA formation.

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.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7. HIV-1 infection of HeLa-CD4 cells upregulates RH116 expression. Cells were infected with HIV-1LAI or were mock-infected with the virus inactivated by treatment at 56 °C for 2 h. (A) Total RNA samples (3–100 ng), extracted at different time points after infection, were subjected to RT-PCR amplification using RH116 or GAPDH primer pairs. (B) Total cell lysates (150 µg per lane) obtained before (0 h) and after (8, 24 and 48 h) HIV-1 infection were analysed by Western blotting for the level of RH116 protein using mouse polyclonal antiserum against RH1161–335. Equivalent levels of loaded proteins were verified using the anti-actin antibody. (C) Western blot analysis of nuclear or cytoplasmic extracts (50 µg per lane) from mock-infected and HIV-1-infected HeLa-CD4 cells tested at different time points (24–72 h) after infection. The level of RH116 protein was revealed by the use of polyclonal mouse anti-RH1161–335 antiserum. Total protein equivalence and the absence of contaminants in each purified preparation were determined by immunoblotting with anti-histone H1 and anti-{beta}-tubulin antibodies.

 
To answer the question of whether HIV-1 infection could alter the intracellular localization of RH116 protein and promote its translocation to the nucleus, HeLa-CD4 cells were either mock-infected or infected with HIV-1, and nuclear as well as cytoplasmic extracts were prepared at different time points after infection. Analysis by Western blotting repeatedly revealed the absence of detectable RH116 protein in nuclear extracts of HeLa-CD4 cells that were either mock-infected or HIV-1-infected for periods of 4 and 8 h (data not shown). In contrast, the presence of RH116 protein in nuclear extracts of HIV-1-infected cells became evident as early as 24 h and peaked between 48 and 72 h p.i. Equal loading of nuclear proteins was verified by immunoblotting with anti-histone H1 antibody, and the absence of cytoplasmic contaminants was revealed by the lack of detectable bands following hybridization with the anti-{beta}-tubulin monoclonal antibody (Fig. 7C). On the other hand, whereas cytoplasmic extracts from mock-infected cultures showed stable levels of RH116 protein at all time points tested, comparable extracts from HIV-1-infected cells presented increased helicase levels at the 48 and 72 h periods (Fig. 7C). The absence of nuclear contaminants in all cytoplasmic preparations was verified by the lack of reactivity with anti-histone H1 antibody and equivalent protein loading was confirmed by immunoblotting with an antibody against {beta}-tubulin (Fig. 7C).


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present study, a DD-RT-PCR approach performed on cells from an HIV-1 patient allowed us to characterize a gene, named RH116, which is a putative member of the DExH subgroup of RNA helicases (Jankowsky & Jankowsky, 2000; Luking et al., 1998). RH116 is identical (99·9 % similarity) to the newly characterized putative RNA helicase MDA-5 (Kang et al., 2002) and presents the four conserved elements accounting for the major RNA helicase activities, namely ATP hydrolysis and RNA unwinding (Luking et al., 1998). Furthermore, RH116/MDA-5 has also been identified recently as Helicard, a cytoplasmic helicase cleaved during apoptosis and which accelerates DNA fragmentation (Kovacsovics et al., 2002). Thus, three different names have been given to the same helicase (RH116, MDA-5 and Helicard), reflecting its multiple biological activities in human cells.

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.


   ACKNOWLEDGEMENTS
 
This work was supported by the Fonds Européen du Développement Régional in France and by the association Stop SIDA, Lille, France. We acknowledge the technical assistance of S. Caby and K. Mondon and the secretarial assistance of J. Ruzicka.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Al-Harthi, L. & Roebuck, K. A. (1998). Human immunodeficiency virus type-1 transcription: role of the 5'-untranslated leader region. Int J Mol Med 1, 875–881.[Medline]

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[Abstract/Free Full Text]

Amiel, C., Darcissac, E., Truong, M. J., Dewulf, J., Loyens, M., Mouton, Y., Capron, A. & Bahr, G. M. (1999). Interleukin-16 (IL-16) inhibits human immunodeficiency virus replication in cells from infected subjects, and serum IL-16 levels drop with disease progression. J Infect Dis 179, 83–91.[CrossRef][Medline]

Amiel, C., de la Tribonnière, X., Vidal, V., Darcissac, E., Mouton, Y. & Bahr, G. M. (2002). Clinical tolerance and immunological effects after single or repeated administrations of the synthetic immunomodulator Murabutide in HIV-1-infected patients. J Acquir Immune Defic Syndr 30, 294–305.[Medline]

Angel, J. B. (2001). Improving immune function and controlling viral replication in HIV-1-infected patients with immune-based therapies. AIDS Read 11, 209–221.[Medline]

Bahr, G. M. (2003a). Non-specific immunotherapy of HIV-1 infection: potential use of the synthetic immunomodulator murabutide. J Antimicrob Chemother 51, 5–8.[Free Full Text]

Bahr, G. M. (2003b). Immune and antiviral effects of the synthetic immunomodulator Murabutide: molecular basis and clinical potential. In Vaccine Adjuvants: Immunological and Clinical Principles. Edited by C. Hackett & D. Harn. Totowa: Humana Press (in press).

Bahr, G. M., Darcissac, E., Bevec, D., Dukor, P. & Chedid, L. (1995). Immunopharmacological activities and clinical development of muramyl peptides with particular emphasis on murabutide. Int J Immunopharmacol 17, 117–131.[CrossRef][Medline]

Bahr, G. M., Darcissac, E. C., Casteran, N., Amiel, C., Cocude, C., Truong, M. J., Dewulf, J., Capron, A. & Mouton, Y. (2001). Selective regulation of human immunodeficiency virus-infected CD4+ lymphocytes by a synthetic immunomodulator leads to potent virus suppression in vitro and in hu-PBL-SCID mice. J Virol 75, 6941–6952.[Abstract/Free Full Text]

Balomenos, D. & Martinez, A. C. (2000). Cell-cycle regulation in immunity, tolerance and autoimmunity. Immunol Today 21, 551–555.[CrossRef][Medline]

Bélec, L., Piketty, C., Si-Mohamed, A., Goujon, C., Hallouin, M. C., Cotigny, S., Weiss, L. & Kazatchkine, M. D. (2000). High levels of drug-resistant human immunodeficiency virus variants in patients exhibiting increasing CD4+ T cell counts despite virologic failure of protease inhibitor-containing antiretroviral combination therapy. J Infect Dis 181, 1808–1812.[CrossRef][Medline]

Bickmore, W. A. & Sutherland, H. G. (2002). Addressing protein localization within the nucleus. EMBO J 21, 1248–1254; erratum 21, 25.

Billaut-Mulot, O., Cocude, C., Kolesnitchenko, V., Truong, M. J., Chan, E. K., Hachula, E., de la Tribonnière, X., Capron, A. & Bahr, G. M. (2001a). SS-56, a novel cellular target of autoantibody responses in Sjogren syndrome and systemic lupus erythematosus. J Clin Invest 108, 861–869.[Abstract/Free Full Text]

Billaut-Mulot, O., Idziorek, T., Loyens, M., Capron, A. & Bahr, G. M. (2001b). Modulation of cellular and humoral immune responses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost. Vaccine 19, 2803–2811.[CrossRef][Medline]

Boden, D., Hurley, A., Zhang, L. & 9 other authors (1999). HIV-1 drug resistance in newly infected individuals. JAMA 282, 1135–1141.[Abstract/Free Full Text]

Chedid, L. A., Parant, M. A., Audibert, F. M., Riveau, G. J., Parant, F. J., Lederer, E., Choay, J. P. & Lefrancier, P. L. (1982). Biological activity of a new synthetic muramyl peptide adjuvant devoid of pyrogenicity. Infect Immun 35, 417–424.[Medline]

Chomel, J. J., Simon-Lavoine, N., Thouvenot, D., Valette, M., Choay, J., Chedid, L. & Aymard, M. (1988). Prophylactic and therapeutic effects of murabutide in OF1 mice infected with influenza A/H3N2 (A/Texas/1/77) virus. J Biol Response Mod 7, 581–586.[Medline]

Connick, E., Lederman, M. M., Kotzin, B. L. & 12 other authors (2000). Immune reconstitution in the first year of potent antiretroviral therapy and its relationship to virologic response. J Infect Dis 181, 358–363.[CrossRef][Medline]

Darcissac, E. C., Truong, M. J., Dewulf, J., Mouton, Y., Capron, A. & Bahr, G. M. (2000). The synthetic immunomodulator murabutide controls human immunodeficiency virus type 1 replication at multiple levels in macrophages and dendritic cells. J Virol 74, 7794–7802.[Abstract/Free Full Text]

Flores, O., Lee, G., Kessler, J., Miller, M., Schlief, W., Tomassini, J. & Hazuda, D. (1999). Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication. Proc Natl Acad Sci U S A 96, 7208–7213.[Abstract/Free Full Text]

Gandhi, R. T., Chen, B. K., Straus, S. E., Dale, J. K., Lenardo, M. J. & Baltimore, D. (1998). HIV-1 directly kills CD4+ T cells by a Fas-independent mechanism. J Exp Med 187, 1113–1122.[Abstract/Free Full Text]

Glynn, J. M., McElligott, D. L. & Mosier, D. E. (1996). Apoptosis induced by HIV infection in H9 T cells is blocked by ICE-family protease inhibition but not by a Fas (CD95) antagonist. J Immunol 157, 2754–2758.[Abstract]

Hrimech, M., Yao, X. J., Bachand, F., Rougeau, N. & Cohen, E. A. (1999). Human immunodeficiency virus type 1 (HIV-1) Vpr functions as an immediate–early protein during HIV-1 infection. J Virol 73, 4101–4109.[Abstract/Free Full Text]

Jankowsky, E. & Jankowsky, A. (2000). The DExH/D protein family database. Nucleic Acids Res 28, 333–334.[Abstract/Free Full Text]

Kang, D. C., Gopalkrishnan, R. V., Wu, Q., Jankowsky, E., Pyle, A. M. & Fisher, P. B. (2002). mda-5: an interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties. Proc Natl Acad Sci U S A 99, 637–642.[Abstract/Free Full Text]

Kovacsovics, M., Martinon, F., Micheau, O., Bodmer, J. L., Hofmann, K. & Tschopp, J. (2002). Overexpression of Helicard, a CARD-containing helicase cleaved during apoptosis, accelerates DNA degradation. Curr Biol 12, 838–843; erratum 12, 1633.

Li, J., Tang, H., Mullen, T. M., Westberg, C., Reddy, T. R., Rose, D. W. & Wong-Staal, F. (1999). A role for RNA helicase A in post-transcriptional regulation of HIV type 1. Proc Natl Acad Sci U S A 96, 709–714.[Abstract/Free Full Text]

Liou, L. Y., Herrmann, C. H. & Rice, A. P. (2002). Transient induction of cyclin T1 during human macrophage differentiation regulates human immunodeficiency virus type 1 Tat transactivation function. J Virol 76, 10579–10587.[Abstract/Free Full Text]

Liu, Z. P. & Olson, E. N. (2002). Suppression of proliferation and cardiomyocyte hypertrophy by CHAMP, a cardiac-specific RNA helicase. Proc Natl Acad Sci U S A 99, 2043–2048.[Abstract/Free Full Text]

Luking, A., Stahl, U. & Schmidt, U. (1998). The protein family of RNA helicases. Crit Rev Biochem Mol Biol 33, 259–296.[Abstract]

Mamiya, N. & Worman, H. J. (1999). Hepatitis C virus core protein binds to a DEAD box RNA helicase. J Biol Chem 274, 15751–15756.[Abstract/Free Full Text]

Morgan, D. O. (1997). Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 13, 261–291.[CrossRef][Medline]

Neumann, M., Harrison, J., Saltarelli, M., Hadziyannis, E., Erfle, V., Felber, B. K. & Pavlakis, G. N. (1994). Splicing variability in HIV type 1 revealed by quantitative RNA polymerase chain reaction. AIDS Res Hum Retroviruses 10, 1531–1542.[Medline]

Piatak, M., Jr, Luk, K. C., Williams, B. & Lifson, J. D. (1993). Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. Biotechniques 14, 70–81.[Medline]

Reddy, T. R., Tang, H., Xu, W. & Wong-Staal, F. (2000). Sam68, RNA helicase A and Tap cooperate in the post-transcriptional regulation of human immunodeficiency virus and type D retroviral mRNA. Oncogene 19, 3570–3575.[CrossRef][Medline]

Truong, M. J., Darcissac, E. C., Hermann, E., Dewulf, J., Capron, A. & Bahr, G. M. (1999). Interleukin-16 inhibits human immunodeficiency virus type 1 entry and replication in macrophages and in dendritic cells. J Virol 73, 7008–7013.[Abstract/Free Full Text]

You, L. R., Chen, C. M., Yeh, T. S., Tsai, T. Y., Mai, R. T., Lin, C. H. & Lee, Y. H. (1999). Hepatitis C virus core protein interacts with cellular putative RNA helicase. J Virol 73, 2841–2853.[Abstract/Free Full Text]

Zhang, X., Wang, C., Schook, L. B., Hawken, R. J. & Rutherford, M. S. (2000). An RNA helicase, RHIV-1, induced by porcine reproductive and respiratory syndrome virus (PRRSV) is mapped on porcine chromosome 10q13. Microb Pathog 28, 267–278.[CrossRef][Medline]

Received 17 April 2003; accepted 21 August 2003.