DDX17 Promotes the Production of Infectious HIV-1 Particles Through Modulating Viral RNA Packaging and Translation Frameshift

Virology 443 (2013) 384–392

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Virology

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DDX17 promotes the production of infectious HIV-1 particles through modulating viral RNA packaging and translation frameshift

René-Pierre Lorgeoux a,c, Qinghua Pan a, Yann Le Duff a,b, Chen Liang a,b,c,n a Lady Davis Institute-Jewish General Hospital, Montreal, QC, Canada H3T 1E2 b Departments of Medicine and Microbiology & Immunology, McGill University, Montreal, QC, Canada H3A 2B4 c Microbiology & Immunology, McGill University, Montreal, QC, Canada H3A 2B4 article info abstract

Article history: RNA helicases are a large family of proteins that rearrange RNA structures and remodel ribonucleic Received 21 February 2013 protein complexes using energy derived from hydrolysis of nucleotide triphosphates. They have been Returned to author for revisions shown to participate in every step of RNA metabolism. In the past decade, an increasing number of 24 March 2013 helicases were shown to promote or inhibit the replication of different viruses, including human Accepted 18 May 2013 immunodeﬁciency virus type 1. Among these helicases, the DEAD-box RNA helicase DDX17 was recently Available online 12 June 2013 reported to modulate HIV-1 RNA stability and export. In this study, we further show that the helicase Keywords: activity of DDX17 is required for the production of infectious HIV-1 particles. Over expression of the HIV DDX17 mutant DQAD in HEK293 cells reduces the amount of packaged viral genomic RNA and DDX17 diminishes HIV-1 Gag-Pol frameshift. Altogether, these data demonstrate that DDX17 promotes the Helicase production of HIV-1 infectious particles by modulating HIV-1 RNA metabolism. RNA packaging & Infectivity 2013 Elsevier Inc. All rights reserved.

Introduction to promote or inhibit HIV-1 replication at distinct steps. These include RNA helicase A (RHA), Moloney leukemia virus 10 (MOV10), RNA helicases are a large family of proteins that are involved in XPB/XPD, Ku70/Ku80, Werner syndrome (WRN) helicase, DDX1, virtually all the steps of RNA metabolism (Cordin et al., 2006; DDX3, DDX17, DDX24, Upf1 and DHX30 (reviewed in (Jeang and Linder and Jankowsky, 2011). They utilize the energy derived from Yedavalli, 2006; Lorgeoux et al., 2012; Ranji and Boris-Lawrie, 2010; nucleotide triphosphates (NTPs) hydrolysis to unwind nucleic Steimer and Klostermeier, 2012)). RHA promotes HIV-1 reverse acids and remodel ribonucleic protein (RNP) complexes (Tanner transcription, transcription and translation (Bolinger et al., 2010; and Linder, 2001). RNA helicases are classified into five super Fujii et al., 2001; Roy et al., 2006; Xing et al., 2011). MOV10 inhibits families (SF1 to SF5) on the basis of their conserved motifs. They viral reverse transcription (Burdick et al., 2010; Furtak et al., 2010). are prevalent in eukaryotes, prokaryotes and viruses (Kwong et al., DDX1, DDX3 and DDX17 promote Rev-dependent HIV-1 RNA export 2005; Singleton et al., 2007). The SF2 has the most members, and (Fang et al., 2004; Naji et al., 2012; Yedavalli et al., 2004). In addition includes the DEAD-box helicases that harbor the Asp-Glu-Ala-Asp to its role in HIV-1 RNA export, DDX3 also enhances HIV-1 signature motif (Linder et al., 1989). In humans, malfunction of translation (Geissler et al., 2012; Lai et al., 2010, 2008; Soto-Rifo helicases causes various types of cancers (Clark et al., 2008; Fuller- et al., 2012, 2013). Upf1 and DDX17 are involved in HIV-1 RNA Pace and Moore, 2011; Rezazadeh, 2012). degradation (Ajamian et al., 2008; Zhu et al., 2011). DDX24 Viruses need helicases to successfully replicate their nucleic promotes HIV-1 RNA packaging (Ma et al., 2008), while DHX30 acid genome. Some viruses carry their own helicase, such as was reported to inhibit this step (Zhou et al., 2008). Recently, Hepatitis C virus that encodes the NS3 protein (Kanai et al., SLFN11 was shown to inhibit HIV-1 translation in a codon-usage- 1995; Kim et al., 1995), while others need to hijack cellular ones dependent manner (Li et al., 2012), adding a new layer of viral RNA to assist viral replication. Human immunodeficiency virus type 1 regulation by cellular helicases. (HIV-1), like all retroviruses, does not encode a viral helicase. Over DDX17, also known as RH70, has two isoforms, p72 and p82, as the past decade, many cellular RNA helicases have been reported a result of alternative translation of its messenger RNA (mRNA) (Uhlmann-Schiffler et al., 2002). DDX17 can form heterodimers with its paralog DDX5 (Ogilvie et al., 2003) and is involved in n Corresponding author at: Jewish General Hospital Lady Davis Institute McGill multiple aspects of RNA metabolism. Through interaction with AIDS Centre, Rm 326 3755 Cote Ste-Catherine Road Montreal, QC, Canada H3T 1E2. Fax: +1 514 3407535. other cellular factors, including HDAC1 (Wilson et al., 2004), E-mail address: [email protected] (C. Liang). estrogen receptor alpha (Wortham et al., 2009) and U1snRNP

(Lee, 2002), DDX17 regulates gene transcription and alternative with 10% Fetal Bovine Serum (FBS). Plasmids and short interfering splicing. Its dysfunction can lead to cancer development RNA (siRNA) were transfected into cells using lipofectamine2000 (Dardenne et al., 2012; Honig et al., 2002; Shin et al., 2007). according to the manufacturer's protocol. Recently, DDX17 was reported to act as a co-factor of the zinc ﬁ nger antiviral protein ZAP (Chen et al., 2008), which is involved Knockdown of DDX5 and DDX17 in HeLa cells in the degradation of the multiply spliced HIV-1 mRNA (Zhu et al., 2011). Another study by Naji et al., (2012) showed that DDX17 siRNA oligonucleotides targeting DDX17 (nt 1694–1713) were associates with HIV-1 Rev protein, and promotes the nuclear purchased from Qiagen (5′-AAT ACA CCT ATG GTC AAG GCA-3′) export of HIV-1 transcripts . We now further show that DDX17 (Cat. no. 1027423). siRNAs oligonucleotides targeting DDX5 and affects the production of infectious HIV-1 particles. Knockdown the DDX5-DDX17 subfamily were previously described (Jalal et al., and over expression of DDX17 change the ratios of unspliced vs 2007). Control siRNA was purchased from Qiagen (Cat. No. spliced HIV-1 RNA. Furthermore, over expression of the DDX17 1027281). shRNA targeting DDX17 (nt 1525–1545) was purchased DQAD mutant leads to a dramatic decrease in the amount of from SIGMA (5′-CCC AAT CTG ATG TAT CAG GAT-3′). Control shRNA ﬁ packaged viral RNA. Moreover, DDX17 is required for ef cient Gag was also purchased form SIGMA (Cat. No. SHC016). HeLa cells processing through its effect on HIV-1 Gag-Pol frameshift. Alto- (2 105 per well) were seeded in 6-well plates 1 day before siRNA gether, our results demonstrate new roles for DDX17 in promoting transfection. DDX5 and DDX17 were either knocked down indivi- the production of infectious HIV-1 particles through regulating dually, or simultaneously. 10 nM of each siRNA was used in HIV-1 Gag-Pol frameshift and genomic RNA packaging. transfection. After two sequential siRNA transfections, the cells were transfected with HIV-1 NL4-3 DNA. Cells and supernatants were harvested 40 h post HIV-1 NL4-3 transfection. Protein levels Materials and methods were analyzed by Western blotting, virus production was assessed by infecting the TZM-bl indicator cells. Plasmid DNA, viruses and antibodies

The cDNA clone of DDX17 gene was purchased from ATCC DDX17 overexpression in HEK293 cells (MGC-2030). Short and long isoforms of DDX17 cDNA were 5 amplified by PCR and a Flag tag was added at the N terminus using HEK293 cells (5 10 per well) were seeded in 6-well plates primer pairs DDX17-SS/DDX17-A, DDX17-SL/DDX17-A, respectively 1 day before transfection. 500 ng of DDX17 DNA constructs were (Table 1). The PCR products were digested with BamHI and MulI and co-transfected with 100 ng Tet-ON plasmid and 100 ng HIV-1 NL4-3. inserted into the pRetroX-Tight-Pur retroviral vector (Clontech) to Cells were washed in 1 phosphate buffered saline the next day and create DNA plasmids Tet-DDX17S and Tet-DDX17L that are expressed fresh DMEM containing 100 ng/mL doxycyclin was added to induce in response to doxycyclin. DDX17 mutants were generated by site- DDX17 expression. Cells and supernatants were harvested 40 h post directed mutagenesis using primers DQAD-S and DQAD-A (Table 1). HIV-1 NL4-3 transfection. Protein levels were analyzed by Western DDX5 cDNA clone was kindly provided by Dr Stahl (Rossler et al., blotting and virus production was measured by infecting TZM-bl 2000). The infectious HIV-1 proviral DNA clone NL4-3 was obtained indicator cells. from the NIH AIDS Research and Reference Reagent Program. Tat plasmid was kindly provided by Dr. Gatignol (Gatignol et al., 1991). Viral RNA analysis TheframeshiftluciferasereporterconstructsFS(0)andFS(-1)were previously described (Grentzmann et al., 1998)andprovidedby Levels of viral RNA in cells were assessed by Northern blots as Dr. Brakier-Gingras. HIV-1 viruses were generated by transfecting previously described (Roy et al., 2006; Zhou et al., 2008) (and HEK293T cells with the proviral DNA clone NL4-3, using lipofecta- RT-PCR followed by Southern blot, described below). Briefly, mine 2000 (Invitrogen) according to the manufacturer's instruction. transfected cells were washed with 1 phosphate buffered saline When indicated, the vesicular stomatitis virus (VSV) glycoprotein and lysed in Trizol (Invitrogen). RNA was extracted according to (G) was used to pseudotype HIV-1 particles. the manufacturer's instructions. Northern blot analysis was per- Anti-Flag and anti-tubulin antibodies were purchased from Sigma; formed as previously described (Zhou et al., 2008). Briefly, RNA anti-HIV-1 p24 antibody from ID Lab Inc.; anti-DDX5 and anti-DDX17 was subjected to electrophoresis on a 1% denaturing agarose gel. antibodies were purchased from Abnova (H00001655-B01) and RNA was transferred to nylon membranes (GE Healthcare UK Novus Biological (NB200–352), respectively. Limited) and incubated with [α-32P] labeled HIV-1 DNA. Mem- branes were washed with 1 saline-sodium citrate (SSC) buffer Cell culture and transfections containing 0.1% SDS and viral RNA signals were visualized by exposure to X-ray films. TZM-bl, HeLa and HEK293 cells were grown at 37 1C with 5% Levels of virion-associated viral RNA and total viral RNA were

CO2 in Dulbecco's Modiﬁed Eagle Medium (DMEM) supplemented determined by RT-PCR followed by Southern blots. RNA was ﬁrst

Table 1 List of primers used.

Primer Sequence

DDX17-SS 5′-GCACGGATCCATGGATTACAAGGATGACGACGATAAGCGCGGAGGAGGCTTTGGGGAC-3′ DDX17-SL 5′-GCACGGATCCATGGATTACAAGGATGACGACGATAAGCCCACCGGCTTTGTAGCCCCG-3′ DDX17-A 5′-GCAGACGCGTCATTTACGTGAAGGAGGAGG-3′ DQAD-S 5′-CTTGTATTGGACCAAGCTGACAGAATGC-3′ DQAD-L 5′-GCATTCTGTCAGCTTGGTCCAATACAAG-3′ FP 5′-AGTAAAGCCAGAGGAGATCTCTCG-3′ FL 5′-TACCTCTTGTGAAGCTTGCTCGGCTCT-3′ RP1 5′-ATTGGTATTAGTATCAATCTTCAAATC-3′ RP2 5′-GCCACCCATCTTATAGCAAAATCC-3′ 386 R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392 extracted from viruses using Trizol-LS (Invitrogen), followed by increase in the amount of HIV-1 virions and no change in the DNase I (Amp Grade) digestion at room temperature for 15 min to infectivity of HIV-1 particles (Fig. 1D). Interestingly, silencing remove any contaminating plasmid DNA. DNase I (Amp Grade) DDX5 moderately increased DDX17 expression (including both was inactivated at 65 1C for 10 min. The effectiveness of DNase I the p72 and p82 isoforms) (Fig. 1A). This suggests that cells (Amp Grade) treatment was tested by amplifying the treated RNA compensate for the loss of DDX5 by increasing DDX17 expression, samples by PCR with the primers FP and FL (Table 1). This primer as reported by others (Shin et al., 2007). Along this line, over pair amplifies HIV-1 cDNA sequence spanning nucleotides 665 to expression of DDX5 or DDX17 also moderately diminished the 1729. Reverse primers RP1 and RP2 were used for the amplifica- level of DDX17 or DDX5 (Fig. S1), which further indicates the tion of singly spliced and multiply spliced HIV-1 transcripts, mutual effects on their expressions. Knockdown of both DDX5 and respectively, and were previously described (Zhu et al., 2011). DDX17 severely diminished cell viability, thus was not tested on RNA equivalent to 25 ng of p24 was reverse transcribed and HIV-1 production (data not shown). In order to confirm the amplified with the primers FP and FL or RP1 or RP2, using the observed inhibitory effect of DDX17 knockdown on HIV-1 produc- Titan One Tube RT-PCR System (Roche) according to the manu- tion, we utilized an shRNA to deplete endogenous DDX17 in HeLa facturer's instruction and 20 PCR cycles (Reverse transcription at cells. A profound decrease in the generation of infectious HIV-1 50 1C for 1 h, followed by reverse transcriptase inactivation at particles was again observed (Fig. 1E). Collectively, we conclude 94 1C for 5 min; PCR: denaturation at 95 1C for 30 s, annealing at that DDX17 is required for HIV-1 production. 45 1C for 30 s and elongation at 68 1C for 2 min). RT-PCR products were subjected to electrophoresis on a 1% agarose gel (0.5 Tris HIV-1 production is inhibited by DDX17 mutant carrying the mutated Borate EDTA) for 2 h at 60 V. The gel was treated in 0.2 M HCl for DEAD box motif 13 min, NaOH 0.5 M for 30 min, NaCl 0.5 M (pH 7.5) for 30 min and double-distilled H2O for 10 min. The DNA was transferred to a As a typical RNA helicase, DDX17 carries seven conserved nylon Hybond-N membrane (GE Healthcare UK Limited) with 10 motifs including the DEAD box that is critical for helicase activity SSC overnight. The next day, the membrane was washed in 6 (Fig. 2A). To determine whether the helicase activity of DDX17 is SSC for 5 min at room temperature and RNA was fixed by UV required for its effect on HIV-1 production, we mutated the DEAD cross-linking. Next, the membrane was incubated with 10 mL motif to DQAD in the context of both the long (p82) and short pre-hybridization buffer (Ambion) at 42 1C for 2 h followed by (p72) forms of DDX17 (Fig. 2A). We then transfected HEK293 cells overnight hybridization at 42 1C with a [α-32P] labeled HIV-1 with various DDX17 constructs and the HIV-1 proviral DNA NL4-3 probe. The next day, the membrane was washed with 1 SSC, (Fig. 2B), and analyzed viral particles released in the supernatant. 0.1% SDS at room temperature for 15 min. Signals were quantified DDX17 DQAD (L) mutant expression led to a 3.5-fold decrease in using a phosphoscreen and the Phosphoimager (Amersham). the amount of viral particles as being determined by measuring viral reverse transcriptase activity in the supernatants (Fig. 2C). Virus production assays We further determined the levels of infectious HIV-1 particles by infecting the TZM-bl indicator cells, and observed that the DQAD The amount of infectious viruses in culture supernatants was (L) mutant caused a 10-fold reduction (Fig. 2E). Considering the determined by infecting the TZM-bl cells. Cells were seeded in 3.5-fold decrease in the viral RT level (Fig. 2C) and the diminution 24-well plate at a density of 4 104 cells per well 1 day before in p24 found in the supernatant (Fig. 2D), we conclude that DQAD infection. 50 μL of supernatant from transfected cells were used to (L) decreases the infectivity of HIV-1 particles by three-fold. For infect TZM-bl cells. Forty-eight hours after infection, cells were the DQAD (S) mutant, we observed a 50% decrease in the viral RT lysed in 1 passive lysis buffer (Promega), and levels of firefly (Fig. 2C) and a 70% decrease in virus production (Fig. 2E). We also luciferase activity in the cell lysates were measured using the ectopically expressed DDX17 and its DQAD mutants in HeLa cells Luciferase Assay kit and the Glomax luminometer (Promega). and observed a more than five-fold reduction in the production of Virus production was determined by measuring viral reverse infectious HIV-1 particles as a result of DQAD expression (Fig. S2). transcriptase (RT) activity in the supernatants (Lu et al., 2011). Taken together, these results suggest that the DQAD mutant of DDX17 impairs the production of infectious HIV-1 particles. Con- sidering the existence of endogenous DDX17 when DQAD mutant Results is ectopically expressed, we conclude that this mutant acts in a dominant negative fashion to prevent the endogenous DDX17 Knockdown of DDX17 but not DDX5 reduces HIV-1 production and from promoting HIV-1 production. infectivity DDX17 expression increases HIV-1 genomic RNA packaging DDX17 was recently identified as a co-factor of ZAP that causes the degradation of HIV-1 multiple spliced mRNAs (Chen et al., To understand how the DDX17 DQAD mutant reduces the 2008). DDX17 was also reported to associate with Rev, and production of infectious HIV-1 particles, we first measured the promote the export of incompletely spliced HIV-1 RNAs from the levels of viral RNA associated with HIV-1 particles. We thus nucleus to the cytoplasm (Naji et al., 2012). To determine the effect transfected 293 cells with the DDX17 and HIV-1 NL4-3 DNA clones. of DDX17 on HIV-1 production, we silenced DDX17 in HeLa cells Viral particles from culture supernatants were harvested 2 days using siRNAs, followed by transfection of HIV-1 NL4-3 DNA post transfection and amounts of particles were determined by (Fig. 1A). We observed a two-fold decrease in the amount of viral ELISA against HIV-1 p24. Viral RNA was amplified by RT-PCR and particles upon DDX17 knockdown (Fig. 1B), which correlates with further quantified in Southern blotting (Fig. 3). We observed that a diminution of the viral p24 levels in the supernatant (Fig. 1C). the short isoform of wild-type DDX17 over expression led to a 40% Furthermore, quantification of infectious viruses by infecting TZM- increase in the amount of RNA that is packaged into HIV-1 bl indicator cells revealed a four-fold decrease (Fig. 1D). Therefore, particles. In contrast, the DDX17 DQAD mutants resulted in a the infectivity of viral particles is decreased by two-fold. Together dramatic 5- to 10-fold decrease in the amount of packaged HIV-1 with the work from other studies, our data suggest that DDX17 genomic RNA (gRNA). The stronger inhibition of HIV-1 RNA affects the amount and the quality of HIV-1 particles made. When packaging by the longer DQAD mutant may result from its higher DDX5, a paralog of DDX17, was silenced, we observed a slight expression level than the shorter DQAD mutant. Alternatively, the R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392 387

Fig. 1. Effect of DDX5 and DDX17 knockdown on HIV-1 production. (A) HeLa cells (1.5 105) were transfected with 0.1 μg HIV-1 NL4-3 DNA and 20 pmol/mL siRNAs directed against DDX17 or DDX5. Levels of endogenous DDX5 and DDX17 were determined by Western blotting. (B) Culture supernatants were harvested 48 h post transfection and the amounts of virus particles were determined by measuring viral reverse transcriptase activity. (C) Levels of viral particles in the culture supernatants were analyzed by Western blotting with anti-HIV-1 p24 antibody. (D) Levels of infectious HIV-1 particles in the supernatants were determined by infecting the TZM-bl indicator cells. (E) HeLa cells (1.5 105) were transfected with 0.1 μg HIV-1 NL4-3 DNA and 1.0 μg shRNA directed against DDX17. Protein expression levels in cell lysate were analyzed by Western blotting with anti-DDX17 or DDX5 antibodies. Culture supernatants were harvested 48 h post-transfection and the levels of released viral particles were analyzed by the Western blotting with anti-p24 antibody. The amounts of infectious virus particles were further determined by infecting the TZM-bl indicator cells *** represents a P-valueo0.001, n¼6. The P value was calculated with reference to the control siRNA data. p82, DDX17 L (long) isoform; p72, DDX17 S (short) isoform; p68, DDX5. extra 79 N-terminal amino acids in the DDX17 long isoform (p82), The DDX17 DQAD mutant disturbs the balance of the unspliced vs which are absent in DDX17 short forms (p72) may be involved in spliced HIV-1 RNA pools preventing HIV-1 RNA packaging. These results suggest that the DQAD mutant impedes HIV-1 RNA packaging and thus reduces A decrease in the amount of packaged HIV-1 RNA can be the virus infectivity. result of multiple causes, including defects in viral RNA expression 388 R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392

Fig. 2. Effect of DDX17 overexpression on HIV-1 production.(A) Illustration of DDX17 DNA constructs. Conserved helicase Walker B motif, or DEAD-box, was mutated into DQAD and inserted into both short and long isoforms of DDX17, DQAD (S) and DQAD (L). (B) HEK293 cells (5 105) were transfected with 0.1 μg HIV-1 NL4-3 DNA and 0.5 μg DDX17. Levels of DDX17 and its mutants in cell lysate were analyzed by Western blot using either anti-Flag or anti-DDX17 antibodies. (C) Levels of viral reverse transcriptase activity in the culture supernatants. (D) The amounts of HIV-1 particles in the supernatants were determined by Western blotting using anti-HIV-1 p24 antibody. (E) Levels of infectious HIV-1 particles were determined by infecting the TZM-bl indicator cells. Levels of luciferase activity reflect the relative infectiousness of the virus samples tested. ** represents a P-valueo0.01, n¼3. p82, DDX17 L isoform; p72, DDX17 S isoform; Ctrl, control. in the cell. We first tested whether DDX17 affects HIV-1 Long DDX17 on HIV-1 RNA expression, we transfected HEK293 cells Terminal Repeat (LTR) promoter activity by transfecting DDX17 with DDX17 plasmids and HIV-1 NL4-3, and examined HIV-1 RNA DNA and Tat into the TZM-bl indicator cells that contain the HIV-1 levels by Northern blot (Fig. 4C). We found that overexpression of LTR-luciferase reporter (Fig. 4A). We observed no detectable effect wild-type DDX17 led to a slight increase in HIV-1 full length RNA of DDX17 on Tat-dependent HIV-1 transcription. Transfection of while over expression of DDX17 DQAD mutants induced an 40 ng HIV-1 Tat showed a further 10-fold increase in luciferase increase in HIV-1 multiply spliced mRNA species and a decrease activity, attesting that the assay was not saturated (data not in HIV-1 full length RNA (Fig. 4C). To confirm this observation, we shown). silenced DDX17 and DDX5 in HeLa cells and analyzed HIV-1 RNA Recently, DDX17 was reported to act as a cofactor of ZAP and by Northern blot (Fig. 4B). We observed that silencing DDX17 led contribute to the degradation of multiply spliced HIV-1 RNA (Zhu to an increase in HIV-1 spliced transcripts and a decrease in the et al., 2011). In addition, no noticeable effect was seen on the levels viral full length RNA (Fig. 4B). Interestingly, DDX5 knockdown of full length viral RNA upon silencing of ZAP (Zhu et al., 2011). In displayed an opposite effect, as we observed an increase in HIV-1 another recent study, Naji et al. (2012) reported that DDX17 full length RNA and a decrease in HIV-1 RNA spliced forms silencing led to a decrease in the levels of both multiply spliced (Fig. 4B). Consistent with the effect of DDX17 knockdown on viral and unspliced HIV-1 RNAs . Naji et al. (2012) also reported that particles shown in Fig. 2C, this pattern can be explained by the fact DDX17 associates with the viral protein Rev to promote the export that the cells compensate for the loss of DDX5 by upregulating the of RRE-containing viral RNAs. To further determine the effect of expression of DDX17. R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392 389

DDX17 on total Gag expression, we transfected HEK293 cells with DDX17 DNA and treated the cells with 100 nM duranavir, an HIV-1 PR inhibitor, and assessed the levels of Pr160Gag-Pol and Pr55Gag in the cell lysate (Fig. 5C). For wild-type DDX17, DRV treatment led to the accumulation of Pr160Gag-Pol, as a result of HIV-1 PR-dependent cleavage inhibition. No overall changes were observed for total Gag levels upon wild-type DDX17 expression (Fig. 5C). Inter- estingly, overexpression of DDX17 mutants led to a slight decrease in the amount of Pr160Gag-Pol but no signiﬁcant effect on Pr55Gag expression (Fig. 5C). In addition, as seen in Fig. 5A, p24 levels were dramatically diminished upon the expression of both DQAD (S) and DQAD (L) mutants. Our results suggest that the DDX17 DQAD mutant compromises Gag processing, and the decrease in the overall amount of Gag-Pol products suggests a possible defect in HIV-1 frameshift activity.

Effect of DDX17 on Gag-Pol frameshift

To regulate the ratio of Gag and Gag-Pol expression, HIV-1 has evolved a slippery RNA sequence that causes -1 frameshift, which allows producing HIV-1 enzymes (Jacks et al., 1988; Vaishnav and Fig. 3. Effect of DDX17 on HIV-1 gRNA packaging. HEK293 cells (5 105) were Wong-Staal, 1991). Therefore, a decrease in HIV-1 frameshift μ μ transfected in 6-well plates with 0.5 g DDX17 DNA and 0.1 g HIV-1 NL4-3. Culture efficiency implies a defect in the production of viral enzymes, supernatants were harvested 48 h post transfection and the amounts of particles were quantified by ELISA against HIV-1 p24. Virus particles corresponding to 55 ng including HIV-1 PR that is responsible for Gag cleavage, thus of p24 were spun at 35,000 rpm for 1 h and RNA equivalent to 10 ng of p24 were reduces virus particle infectivity. To test this hypothesis, we used used for RT-PCR assay. RT-PCR products corresponding to 6 ng of p24 were two dual-luciferase reporter plasmids FS (0) and FS (−1) previously analyzed by Southern blot, and radioactive signal intensity was quantified by described by Grentzmann to measure HIV-1 Gag-Pol frameshift Phosphoimager (Amersham). Results of one of the three independent transfection (Grentzmann et al., 1998)(Fig. 6A). We transfected HEK293 cells experiments are shown. **Represents a P-valueo0.01. with DDX17 DNA with either FS (0) or FS (−1) reporter and quantified luciferase expression levels. We observed that over To distinguish the different spliced mRNA species and to expression of DDX17 DQAD mutant led to up to 40% decrease in quantify their relative amounts, we analyzed viral transcripts by frameshift efficiency (Fig. 6B). No significant effect was observed RT-PCR followed by Southern blotting as described in (Zhu et al., for the wild type DDX17. These results suggest that the helicase 2011)(Fig. 4D–F). We observed that over expression of DDX17 activity of DDX17 is involved in maintaining the proper ratio of DQAD (L) led to a 3.5-fold increase in the amount of multiply Gag/Pol gene expression required for optimal infectivity. spliced RNAs, and up to a 50% decrease in the full length RNA (Fig. 4D and E), which results in 6.5-fold increase for the multiply spliced/full length HIV-1 RNA ratio (Fig. 4F). Over expression of Discussion wild-type DDX17 had no significant effect on the relative amounts of different HIV-1 RNA species. Supporting the work from other In this study, we found that DDX17 promotes HIV-1 particle groups (Chen et al., 2008; Naji et al., 2012; Zhu et al., 2011), our infectivity by modulating HIV-1 RNA packaging and Gag-Pol results suggest that DDX17 affects the balance of spliced and frameshift. Viral RNA analysis also demonstrates that the over unspliced HIV-1 RNA populations. expression of the helicase negative DDX17 DQAD mutant increases the multiply spliced/unspliced HIV-1 RNA ratio by up to six-fold DDX17 modulates HIV-1 Gag processing (Fig. 4F). Recently, Chen et al., (2008) reported that the zinc finger antiviral protein ZAP interacts with DDX17 that acts as a cofactor Expression of DDX17 DQAD mutant in HEK293 cells decreases to potentiate the activity of ZAP in inhibiting the replication of a the amount of viral p24 in the supernatant (Fig. 1D). This defect few viruses, including murine leukemia virus. The same group may result from diminished HIV-1 protein expression in cells. To also showed that ZAP targets HIV-1 multiply spliced mRNA to test this, we transfected HEK293 cells with DDX17 constructs and exosomes for degradation (Zhu et al., 2011). The crystal structure HIV-1 NL4-3 DNA and analyzed viral protein expression by of the N-terminal domain of ZAP reveals the RNA binding region Western blot (Fig. 5A). We observed that expression of DDX17 that is likely involved in HIV-1 RNA recognition (Chen et al., 2012). DQAD decreased the amount of viral p24. However, the level of In addition to its role in ZAP-mediated degradation of multiply Pr55Gag was not affected, suggesting that the DDX17 mutant likely spliced HIV-1 mRNA, DDX17 also has ZAP-independent functions inhibits Gag processing rather than impeding Gag production in HIV-1 replication. Naji et al. (2012) found that DDX17 interacts (Fig. 5A). Similarly, DDX17 knockdown in HeLa cells reduced the with HIV-1 Rev and that DDX17 silencing reduced the amount of amount of viral p24 but not Pr55Gag, suggesting the same defect in viral transcripts and inhibited the export of both unspliced and Gag cleavage (Fig. 5B). spliced HIV-1 mRNAs to the cytoplasm. HIV-1 genome encodes two polyproteins Pr160Gag-Pol and In our study, we also found that DDX17 silencing led to a gp160Env. While gp160Env precursor is cleaved in gp120 and decrease in full length HIV-1 RNA (Fig. 4C). Interestingly, this gp41 by the cellular protease Furin (Hallenberger et al., 1992), decrease in full length RNA correlates with an increase in multiply the Gag-Pol polyprotein is self-cleaved by the viral protease (PR) spliced viral transcripts (Fig. 4). We propose that several mechan- (Darke et al., 1988). HIV-1 Pol gene encodes the three viral isms may account for the accumulation of multiply spliced HIV-1 enzymes, including protease (PR), reverse transcriptase (RT) and RNA upon DDX17 silencing or DDX17 DQAD over expression. The integrase (IN) among which PR is responsible for the processing of first mechanism is simply the inefficient ZAP-mediated degrada- Pr160Gag-Pol and Pr55Gag precursors. In order to assess the effect of tion of HIV-1 multiply spliced RNA (Zhu et al., 2011). A second 390 R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392

Fig. 4. Effect of DDX17 on HIV-1 RNA expression. (A) HeLa TZM-bl cells (4 104) were transfected in 24-well plates with 0.1 μg DDX17 DNA and either 10 ng of HIV-1 Tat or 10 ng cDNA3.1. Luciferase activity was measured 24 h post-transfection (n¼4). (B) HeLa cells (1.5 105) were transfected with 0.1 μg HIV-1 NL4-3 DNA and 20 pmol/ml siRNAs against DDX17 or DDX5. Cells were lysed in Trizol (Invitrogen) 48 h post transfection and HIV-1 RNA profile was analyzed by Northern blot. (C) HEK293 cells (5 105) were transfected with 0.1 μg HIV-1 NL4-3 DNA and 0.5 μg DDX17. Cells were lysed in Trizol (Invitrogen) 48 h post-transfection and HIV-1 RNA profile was analyzed by Northern blot. (D) HIV-1 RNA samples from the co-transfected HEK293 cells were first amplified by RT-PCR using different primer pairs, then subject to Southern blot analysis. (E, F) Radioactive signals from the distinct viral RNA species were quantified from the Southern blot using phosphorescreen and Phosphoimager (Amersham) and splicing ratios were calculated. FL, full length; SS, singly spliced; MS, multiply spliced. mechanism involves excessive splicing due to sequestration of sites for distinct members of the spliceosome machinery, thereby HIV-1 RNA in the nucleus, which leads to decreased amount of influencing exon definition (Chen and Manley, 2009; Stoltzfus, unspliced viral mRNA and increased amount of multiply spliced 2009). One hypothesis would be the involvement of DDX17 in HIV-1 mRNAs. Lastly, DDX17 may directly modulate HIV-1 RNA remodeling HIV-1 RNA structure, leading to a change in exon splicing, as previously reported for DDX17 to affect alternative definition, resulting in an increase of full length and a decrease in splicing of cellular genes (Honig et al., 2002). HIV-1 splicing is a spliced HIV-1 RNAs, as seen in Fig. 4. Generally, moderate effects highly regulated process, which involves stem-loops regulatory were seen for the over expression of wild type DDX17, which can elements called splicing silencers and splicing enhancers and be due to the fact that endogenous levels are sufficiently high to generates more than 30 HIV-1 RNA species (Stoltzfus, 2009). promote the production of infectious HIV-1 particles. During splicing, RNA is remodeled in order to expose splicing We found that expression of DDX17 mutants induce a dramatic silencers and splicing enhancer stem-loops that act as docking decrease in the amount of packaged gRNA (Fig. 3), which leads to R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392 391

Fig. 6. Effect of DDX17 on Gag-Pol frameshift. (A) Depiction of the HIV-1 frameshift dual-luciferase reporter constructs FS (0) and FS (-1), described in (Grentzmann et al., 1998). (B) HEK293 cells (5 105) were transfected with 0.5 μg DDX17 DNA and 75 ng of either dual luciferase reporter FS(0) or FS (-1). FL/RL ratios were calculated and the FL/RL FS(-1)/ FL/RL FS(0) was plotted to indicate frameshift efficiency. **Represents a P-valueo0.01, n¼3. (C) Levels of the ectopically Fig. 5. DDX17 modulates HIV-1 Gag processing. (A) HEK293 cells (5 105) were expressed DDX17 and its DQAD mutants as determined by Western blotting. transfected with 0.1 μg HIV-1 NL4-3 DNA and 0.5 μg DDX17. Protein expression levels in cell lysate were analyzed by Western blot using anti-p24, anti-FLAG and anti-tubulin antibodies. (B) HeLa cells (1.5 105) were transfected with 0.1 μg HIV- Interestingly, although no noticeable change in the level of 1 NL4-3 DNA and 20 pmol/mL siRNAs directed against DDX17 or DDX5. Protein Pr55Gag was observed, over expression of DDX17 DQAD mutants in expression levels in cell lysate were analyzed by Western blot using anti-p24, anti- HEK293 cells and knockdown of DDX17 in HeLa cells lead to a DDX5, anti-DDX17 and anti-tubulin antibodies. (C) HEK293 cells (5 105) were transfected with 0.1 μg HIV-1 NL4-3 clone and 0.5 μg DDX17 DNA and treated with dramatic decrease in viral p24 levels, which suggests that DDX17 100 nM protease inhibitor duranavir (DRV). Cells were lysed 48 h post-transfection DQAD mutants inhibit Gag processing. This effect on Gag proces- and protein levels were analyzed by Western blot using anti-p24 and anti-tubulin sing correlated with a slight diminution of Pr160Gag-Pol (Fig. 5C), antibodies. suggesting that the helicase activity of DDX17 is required for frameshift to occur and for producing optimal ratios of Gag vs Gag-Pol. Indeed, results of experiments with the frameshift the production of less infectious viral particles. Considering that reporter constructs showed that DDX17 DQAD (L) led to a the amount of full-length HIV-1 RNA decreased by only two-fold in significant 40% decrease in frameshift efficiency (Fig. 6B), which the cells for DQAD (L), we conclude that the packaging of gRNA is expected to result in less production of Pr160Gag-Pol. The was diminished by five-fold. Similarly, we observed a 1.5 fold decrease in Pr160Gag-Pol synthesis is expected to reduce viral decrease in full length HIV-1 RNA for DQAD (S), but a 3.5-fold protease concentration in the progeny virus particles and conse- diminution in packaged gRNA (Fig. 3). This data suggests that the quently, diminish viral Gag processing. helicase activity of DDX17 is necessary to promote HIV-1 gRNA packaging. HIV-1 RNA packaging is a complicated process that involves interactions of cellular and viral proteins with specific Competing interests viral RNA motifs. HIV-1 5′UTR contains a number of stem-loop (SL) structures that act as regulatory sites for multiple processes, The authors declare they have no competing interests. including splicing and packaging. Being a RNA helicase, it is possible that DDX17 remodels the viral RNP complex within the nucleus, therefore modifying its composition through the recruit- Acknowledgments ment of various factors and changes the fate of the viral RNA. We do not lose sight of the possibility that perturbing the levels of We thank Vicky Cheng and Zhenlong Liu for technical assistance DDX17 in cells may modulate cellular processes such as RNA in this study, Drs Hans Stahl, Anne Gatignol and Léa Brakier-Gingras transcription and splicing which in turn affects the process of for providing valuable reagents. This work was supported by funding HIV-1 packaging. from the Canadian Institutes of Health Research. 392 R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392

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