RNA helicases

Modular domain structure of RNA helicases.

Helicases can be classified as RNA or DNA helicases based on the substrate they bind and remodel. The helicase core is comprised of eight conserved motifs (motifs I, Ia, Ib, II, III, IV, V and VI).^2,5,11,12 Along with DNA helicases, RNA helicases can be grouped into 5 superfamilies (SF1-SF5) based on conservation of sequence motifs.¹³ Most RNA helicases fall into SF2 and encode the DEAD (amino acid residues Asp-Glu-Ala-Asp), DExH, DExD or DEAH motif,¹⁴ which are named for the residues of their Walker B motif (motif II) that contains the ATP-binding domain.^5,15 SF2 comprises eleven families, five of which are DExH/D helicases (DEAD box, SKI2-like DExH, RIG-I-like DExH, DEAH/RHA and viral DExH).¹⁴ Identified in all eukaryotes examined, the DExH/D box family members are divergent in the amino acid sequences that flank the helicase core. These flanking residues represent interchangeable domains (Fig. 1) that reoccur amongst proteins involved in the full scope of gene expression, and determine interaction with protein cofactors or target RNA.¹⁶ The RNA helicases may exhibit processive activity that affects long stretches of RNA or rearrangement of short oligonucleotides. They may catalyze RNP remodeling or act as nonenzymatic inducers of the antiviral state, as described below.

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Figure 1

Domain structure of DExH/D helicases involved in viral replication. Upper rectangle: RNA helicases of the RIG-I-like receptor family are components of the innate antiviral response that attenuates viral replication. Lower rectangle: RNA helicases that promote viral replication. Protein sequences were retrieved from the NCBI conserved domains database and domains determined by annotation and query of the Uniprot database. The predicted molecular weight (kDa) and number of amino acid residues (aa) are indicated. Jagged edges indicate interruption by additional sequence or domains. Domains are: CARD, Caspase-activation and recruitment domain; DExDc, DEAD-like helicase superfamily ATP binding domain; HELICc, Helicase superfamily C-term domain associated with DExH/D box proteins; RIG-I_CRD, Regulatory domain of RIG-I; RS/GYR, Arginine/serine, glycine tyrosine-rich domain; dsRBD, Double-stranded RNA binding domain; HA2, Helicase-associated domain of unknown function; DUF, Domain of unknown function; RG-rich, arginine and glycine-rich domain; SPRY, SP1a and RYanodine receptor domain; p68HR, Characteristic of p68-like RNA helicase; Peptidase_S29, serine protease domain with trypsin-like fold; Poty_PP, Observed in polyproteins of the Potyviridae.

Induction of the antiviral state.

RNA helicases play a prominent role in the cellular response to viral infection. Viral infections are recognized by membrane-bound receptors and cytoplasmic sensors that trigger signaling pathways that operate by cascades of protein interactions. The outcome is the ubiquitinylation, phosphorylation and nuclear translocation of transcription factors that activate expression of interferons, interferon-stimulated genes (ISGs)¹⁷ and inflammatory cytokines.¹⁸ One family of cytosolic sensor is the RIG-I-like receptor (RLR) family, which includes retinoic acid-inducible gene 1 (RIG-I/DDX58) and melanoma differentiation-associated gene 5 (MDA-5).¹⁷ Classified as RIG-I-like DExH family members (DECH variants), these RNA helicases contain amino-terminal caspase activation and recruitment domains (CARD) (Fig. 1). Their interaction with viral double-stranded RNA (dsRNA) induces conformational changes that reveal CARD domains for interaction with another CARD-containing adaptor protein, mitochondria antiviral signaling (MAVS; also called interferonbeta promoter stimulator 1, IPS-1) (Fig. 2). This leads to activation of the TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) complex that phosphorylates transcription factors IRF-3 and IRF-7.¹⁹ The phosphorylated IRF-3 and IRF-7 are translocated to the nucleus and activate transcription of antiviral and immunological genes, including interferons (IFNs); IFN-α and IFN-β. Even though RIG-I and MDA-5 encode similar DECH and CARD domains, they recognize different subsets of viral RNA by their divergent structural features or nucleotide composition. RIG-I recognizes free 5′ triphosphate structures and short dsRNAs (<1 kb) whereas MDA-5 recognizes longer RNAs (>1 kb in length), such as encephalomyocarditis virus (EMCV) RNA and synthetic poly(I:C) RNA.^17,20 RIG-I and MDA-5 signaling converges at the MAVS/IPS-1 adapter protein, which integrates response to a variety of viral infections.¹⁷ The complete characterization of the ligands of RIG-I and MDA-5 may identify additional ligands and cofactor interactions involved in IRF signaling. For instance, the activity of RIG-I and MDA-5 can be downregulated by another cytoplasmic DExH helicase, LGP2 (Laboratory of Genetics and Physiology 2). LGP2 shares the domain structure of RIG-I and MDA-5 with the exception of the CARD domains (Fig. 1). Preferentially-expressed in mammary tissues and tumors,²¹ under some circumstances, LGP2 acts to sequester viral double-stranded RNA from RIG-I and MDA-5 and attenuate induction of the antiviral state (Fig. 2).²² In other cases, LGP2 facilitates the induction of the antiviral state, and residues of the ATPase domain of LGP2 are involved in the viral RNA recognition.²⁰ Results in LGP-2-deficient mice revealed differences in response to two viral infections.²³ Compared to control mice, LGP2-deficient mice exhibited reduced sensitivity to lethal vesicular stomatitis virus (VSV) infection, but increased sensitivity to lethal EMCV infection, which are sensed by RIG-I and MDA-5, respectively. A possible explanation is that LGP2 in mice contributes to regulation of RIG-I or MDA-5 activity.²⁴ In sum, recognition of viral RNA by RIG-I and MDA-5 induces the innate immune system, whereas LGP2 interferes with the viral RNA recognition and attenuates induction of the antiviral state, at least in some circumstances. The apparent counterbalance of RIG-1 and MDA-5 by LGP2 sets the precedent that RNA helicases may be adapted to attenuate the antiviral state. The results posit that LGP2 participates in a quality control system to monitor proper induction of the antiviral state.

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Figure 2

RNA helicases of the RIG-I-like receptor (RLR) family play a crucial role in the innate antiviral response. RLR family members, retinoic acid-inducible gene 1 (RIG-I/DDX58) and melanoma differentiation-associated gene 5 (MDA-5) are cytoplasmic sensors that detect viral RNA and trigger induction of the antiviral state. RIG-I and MDA-1 recognize viral RNA as foreign by features including free 5′ triphosphate ends, short double-stranded RNA (dsRNA) and long dsRNA (≥1 kb), respectively. RLR family member, laboratory of genetics and physiology 2 (LGP2) can antagonize this activity (dashed line) by regulation that is poorly understood. Interaction with their RNA ligands induces conformational changes in RIG-I and MDA-5 that favor interaction with mitochondria antiviral signaling (MAVS), also called interferon-beta promoter stimulator 1 (IPS-1). MAVS/IPS-1 activates the TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) complex, which phosphorylate IFN-regulatory factor (IRF)-3, IRF-7 and NFκB (not shown). The IRFs dimerize, translocate to the nucleus and bind to target genes. Transcriptional activation of interferon (IFN) genes leads to production of IFNs (α and β), which are secreted from the cell. IFN binding to the IFN receptor activates the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. The JAK and tyrosine kinase 2 (TYK2) phosphorylate one another, the receptor and STAT2. STAT2 binds STAT1 and triggers phosphorylation and STAT1/2 dimerization. The STAT heterodimer translocates to the nucleus with IRF-9. They activate transcription of interferon stimulated genes (ISG), which generate the anti-viral state that attenuates viral replication.

DDX3.

DDX3 is a 73 kD nucleo-cytoplasmic DEAD helicase that is ubiquitously expressed in a wide range of tissues.^31–33 DDX3 has multiple activities in the cell, which involve ATPase activity and ability to act as a scaffold for alternative protein-protein interactions. The central helicase and DEAD domains are flanked at the N-terminus by a protein-protein interaction domain and a putative nuclear export signal and a glycine-rich C-terminus (Fig. 1). DDX3 is highly conserved in metazoans (Table 1). Ded1 is the yeast homolog and is essential for general translation initiation.³⁴ Ded1 was identified in a yeast genetic screen and the ded1-deficiency can be rescued by human DDX3, implicating DDX3 in translation of metazoan RNAs.^34,35 Since then, metazoan DDX3 has been observed to modulate gene expression at the transcriptional and post-transcriptional levels, both of which affect the broader processes of cell growth regulation, apoptosis and the innate response to virus infection.^32,36,37 Members of four virus families use DDX3 to promote their life cycle (Table 2).

Table 1

Significant conservation is observed amongst RNA helicases involved in virus biology

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Retrovirus. Jeang and colleagues identified DDX3 affects HIV-1 posttranscriptional gene expression.³³ The exogenous expression of DDX3 increases production of HIV-1 Gag virion structural protein (5 to 12-fold), while suppression of endogenous DDX3 by antisense RNA decreases Gag production (factor of 13).³³ Northern analysis determined that the overexpression of DDX3 did not change the abundance of the unspliced gag transcript or the ratio of unspliced to spliced viral transcripts. The results indicated that DDX3 did not affect HIV-1 transcription or pre-mRNA processing and is likely to enhance Gag production by increasing the Rev-dependent nuclear export of the unspliced gag transcripts. Consistent with this activity, DDX3 is a binding partner of the CRM1 nuclear export receptor and the HIV-1 viral posttranscriptional trans-activator, Rev.³³

DDX3 expression is enhanced in a HeLa cell line that expresses HIV-1 Tat.³³ Two possible explanations are that ddx3 expression is upregulated by the transcriptional trans-activation activity of Tat or the post-transcriptional RNA silencing suppressor activity of Tat, which may attenuate miRNA activity against ddx3.^38,39 Yedavalli et al. explored DDX3 as a target to inhibit HIV-1 replication.⁴⁰ Two-ring expanded nucleoside analogs were identified that inhibit DDX3 activity, suppress HIV-1 replication and exhibit insignificant toxicity in cell culture or in mice. These data document that cellular helicases are a feasible target for the development of novel antiretroviral drugs.

Poxvirus. DDX3 promotes the innate response to virus infection by its ability to act as a scaffold for protein-protein interactions.^41–44 DDX3 augments the activity of TBK1/IKKε, which activates IRF-3, IRF-7 and the NFκB transcription factor to stimulate interferon and interferon response genes that induce the antiviral state.^41,42 While the helicase activity of DDX3 is not required, the N-terminal domain of DDX3 is required and associates with IKKε. Notably the same domain is targeted by vaccinia virus K7 protein.⁴¹ The crystal structure of K7 in complex with the N-terminal DDX3 peptide reveals that a hydrophobic cleft of K7 residues engage a thumb-like projection of tandem phenylalanine residues.⁴⁵ Presumably this protein-protein interaction prevents DDX3 from engaging IKKe to promote interferon induction. The findings suggest that DDX3 is a prime target for viral manipulation of interferon induction.^41,42

Flavivirus. Yeast two-hybrid studies initially identified an interaction between DDX3 and the HCV core structural protein.^31,35,46 Subsequently RNA interference verified that DDX3 is important for viral RNA replication in HuH-7 liver cells.⁴⁷ The N-terminal residues of DDX3 are important for interaction with the HCV core and DDX3 interaction is significantly reduced by a single alanine substitution in HCV core.⁴⁸ However it remains to be determined whether or not the HCV core blocks interaction between DDX3 and IKKε, which would attenuate IRF signalling. Alternatively, HCV core may block interaction between DDX3 and another cellular protein that bolsters replication by another mechanism.

Hepadnavirus. Whereas DDX3 enhances replication of HIV-1 and HCV, DDX3 restricts hepatitis B virus (HBV) replication in both hepatoma and non-hepatoma cells. DDX3 binds to HBV polymerase (Pol), which is responsible for reverse transcription of viral RNA to DNA⁴⁹ and is incorporated into nucleocapsid with the pre-genome RNA.⁵⁰ DDX3 ATPase activity, but not helicase activity, is necessary for the inhibitory effect on HBV reverse transcription, which posits that inhibition of viral DNA synthesis occurs at a step following ATP hydrolysis, but prior to RNA unwinding.⁵⁰ In addition to catalyzing reverse transcription, HBV Pol exhibits an immune modulatory role that is attributed to disrupted IRF signaling.⁵⁰ Similar to vaccinia virus K7, HBV Pol disrupts the interaction between DDX3 and IKKε. The results suggest that DDX3 is manipulated to evade IFN induction at the expense of viral reverse transcription. The identification of DDX3 residues essential for the interaction with HBV Pol may provide another selective target for small molecule inhibitors to impair virus replication.

RNA helicase A (RHA)/DHX9.

Human RHA/DHX9 is an SF2 DEIH helicase that is the homolog of bovine nuclear DNA helicase II and Drosophila melanogaster Maleless.^51–53 RHA is the largest and most complex of the helicases involved in viral replication (140 kDa and six domains) (Fig. 1). RHA executes protein-protein interactions that impact the continuum of gene expression, beginning at transcription and culminating in mRNA translation and influence cell growth and apoptosis, and the innate response to virus infection.^3,54–59 RHA contains two double-stranded RNA binding domains (dsRBDs) of conserved α-β-β-β-α topology; the DEIH helicase core; the domain of unknown function (DUF), which is conserved in Ago2; the helicase-associated domain 2 (HA2) of a diversity of RNA helicases; a nuclear transport domain that is recognized by importin-α; and the carboxyl-terminal arginine and glycine-rich residues that are recognized by PRMT1 methylase.^60,61 RHA is prominently nuclear⁶² and interacts with RNA polymerase II and transcription factors and coactivators that modulate gene transcription.^63–65 In the cytoplasm, RHA interacts with structural features of the 5′ untranslated region (UTR) of the junD mRNA to facilitate efficient cap-dependent translation of this protooncogene.^57,66,67 In sum, virus interaction with RHA may be advantageous for efficient expression of viral genes. Furthermore, the impact of RHA on cellular gene expression may produce cell growth conditions that are advantageous for the viral life cycle. Members of three virus families use RHA to promote their replicative cycle.

Retrovirus. RHA overexpression has been suggested to increase retrovirus gene transcription, the ratio of spliced to unspliced viral transcripts^68,69 and nuclear export of unspliced RNA.^62,70 However downregulation experiments with siRNAs and quantitative RNA analysis determined RHA is not required for retrovirus transcription, RNA splicing or nuclear export, but that RHA is necessary for retrovirus translation.^57,71–73 RHA selectively interacts with structural features of the 5′ UTR of many retroviruses and overexpression of RHA increases their translation.^57,71–73 RHA interaction with the 5′ UTR increases polysome association and requires the ATP-binding activity of the DEIH helicase domain.^57,73 The molecular basis of RHA translation activity is catalytic rearrangement of the RNP to facilitate ribosome scanning and translation initiation, and possibly and protein-protein interaction that secures a circular polysome for efficient translation reinitiation.⁵⁷ In sum, RHA is necessary for efficient translation of many retroviruses including HIV-1 (Fig. 3).

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Figure 3

Cellular helicases involved in HIV-1 replication. DExH/D helicases with a known function in HIV-1 replication are indicated beside the corresponding step in the replication cycle of HIV-1: (1) Receptor-mediated entry; (2) Reverse transcription of viral genomic RNA to DNA and integration into host chromosome to render the provirus; (3) Transcription of HIV -1 provirus and accumulation of viral mRNA; (4) Post-transcription includes RNA processing and export from the nucleus; (5) Translation of viral transcripts on polysomes; (6) Assembly of viral structural and enzymatic proteins and unspliced viral transcript into progeny virions that bud from the plasma membrane.

In the case of the simian Mason-Pfizer monkey virus (MPMV), RHA interacts with the 5′ UTR to facilitate translation.⁷⁴ Additionally, RHA interacts with the 3′ UTR of MPMV in a region necessary for nuclear export of the unspliced viral transcript.⁷⁰ This region is designated the constitutive transport element (CTE)⁶² and facilitates nuclear export by recruiting the TAP nuclear export receptor.⁷⁵ Conceivably RHA interacts with MPMV RNA in the nucleus and facilitates consecutive remodeling of the viral RNP necessary for nuclear export and subsequent translation in the cytoplasm.⁷⁶

Studies on HIV-1 determined that RHA promotes the infectivity of progeny virions⁷⁷ (Fig. 3). Molecular analysis of the defect determined that RHA in the virus producer cells fosters the reverse transcription activity of the progeny virions⁷⁷ (Fig. 3). Kleiman and colleagues determined the defect is attributable to reduced annealing of tRNA to viral RNA (40% of control) during the virion assembly process.¹³³ Furthermore, RHA is incorporated into the progeny virions, possibly through interaction with the nucleocapsid domain of Gag,⁷⁷ the HIV-1 RNA or an adapter protein.⁷³ RHA-deficient virions are less infectious to Hela-based TZM-bl reporter cells⁷⁷ and primary T lymphocytes.⁷³ The defect is attributed, in part, to reduced reverse transcription⁷⁷ but may also involve replication steps that are downstream of reverse transcription. In sum, RHA appears to be involved in multiple steps in the retroviral life cycle.

Flavivirus. Behrens and colleagues identified that RHA binds to the 5′ and 3′ UTRs of bovine viral diarrhea virus RNA and affects viral replication.⁷⁸ Two other proteins that contain the conserved dsRBD (NF-45 and NF-90/NFAR-1) facilitate viral replication. These findings suggest the dsRBD may be acting as a scaffold to secure the viral RNA in a configuration helpful to replication of the bovine diarrheal virus.

To investigate possible importance of RHA in a human flavivirus, He et al. studied HCV replicon cells using RHA downregulation strategies. A lentivector-based inducible RNA interference system administered partial RHA downregulation. Partial RHA downregulation produced a gradual reduction of HCV RNA and protein over 21 days, as measured by RT-PCR and immunoblot, respectively.⁷⁹ The results suggest RHA is a necessary host factor for HCV in replicon cells.

Picornavirus. Recent study of foot and mouth disease virus (FMDV) determined that RHA downregulation decreases viral replication.⁸⁰ In infected cells, RHA co-precipitates FMDV replication proteins, 2C and 3A.⁸⁰ Furthermore, FMDV infection changes the subcellular localization of RHA from prominently nuclear and a uniform distribution, to prominently cytoplasmic and a punctuate distribution. Similar to results in retroviruses and bovine diarrheal virus,^57,73,78 RHA interacts with terminal regions of FMDV RNA. Together the results posit the model that FMDV uses RHA to promote viral RNA replication and to facilitate viral protein synthesis. The authors speculate that FMDV uses RHA to connect the 5′ and 3′ UTRs to switch between viral RNA replication to viral protein synthesis, which may occur on a circular polysome.⁸⁰

In summary, RHA interacts with the terminal regions of many viral RNAs. Future analysis is warranted to determine the domain(s) of RHA required for interaction with viral RNA and test the possibility that similar residues are required across several virus families. This fundamental investigation will be informative to development of antiviral therapy that selectively discriminates between the cellular and viral RNA targets of RHA.

DHX30.

To date a single virus, HIV-1, has been shown to interact with DHX30 (isoform 2).⁸¹ DHX30 is a 131 kDa protein with the conserved ATP-dependent helicase domain (DEVH variant) followed by the DUF domain and HA2 domain and one dsRBD at the amino-terminus (Fig. 1). The DHX30 domain structure is highly similar to RHA. Similar to results with RHA, overexpression of DHX30 increases expression of the HIV-1 provirus (2 to 3-fold) and an HIV-1 LTR luciferase reporter (1.5–4 fold), and this activity is eliminated by a DEVH mutation.⁸¹ In distinction from RHA, DHX30 overexpression decreased viral RNA packaging into progeny virions (factor of ∼10 as measured by RT-real time PCR on equivalent particle-associated Gag), suggesting proper trafficking of HIV-1 RNA between the cytoplasm and virions was disrupted (Fig. 3). The authors speculated that DHX30 might cause a modification of the packaging signal by binding to the 5′ UTR. As expected, the reduction in viral RNA packaging correlated with reduced infectivity of progeny virions (by a factor of 6). Downregulation of DHX30 did not produce an effect on HIV-1 gene expression,⁸¹ which is likely attributable to redundant activity by RHA. RHA is abundantly expressed in many types of animal cells and displays high sequence identity with DHX30 in basic residues that direct RHA interaction with retrovirus RNA.¹³⁴ Domain exchange experiments between DHX30 and RHA will be instrumental to understand the specificity and potential functional redundancy of these RNA helicases.

DDX1.

Members of three virus families exhibit interaction with DDX1 (Table 2). DDX1 is an 82 kDa ATP-dependent DEAD box family member that contains a SPRY domain (Fig. 1). DDX1 exhibits affinity for poly(A) RNA, heterogenous nuclear ribonucleoprotein K and cleavage stimulatory CSTF-64, which suggests DDX1 contributes to proper 3′ end maturation of mRNAs.^82,83

Retrovirus. In common with DDX3, RHA and DHX30, DDX1 activity affects HIV-1 replication (Fig. 3). Yeast and mammalian two hybrid systems identified DDX1 interacts with HIV-1 Rev.⁸⁴ DDX1 downregulation reduces the activity of an HIV-1 Rev/RRE-dependent luciferase reporter gene, and RT-PCR determined that nuclear export of unspliced reporter mRNA was impaired. The reduced Rev/RRE activity correlated with disordered Rev subcellular distribution in nucleus and nucleolus,⁸⁵ consistent with an important role for this host factor in HIV-1 biology. While DDX1 is upregulated in neuroblastoma and retinoblastoma cell lines and tumors,⁸⁶ expression in astrocytes is low. Astrocytes are nonpermissive for HIV-1 replication and ectopic expression of DDX1 is sufficient to complement the defect in HIV-1 replication.⁸⁵ The findings that DDX1 facilitates Rev/RRE activity and is a rate-limiting factor for HIV-1 replication in astrocytes indicates that DDX1 is a required host factor for HIV-1 replication.⁸⁷

Polyoma virus. DDX1 also modulates cell specificity of the human polyoma virus, JC virus.⁸⁸ DDX1 expression is high in cells susceptible to JC virus infection and low in nonpermissive cells; exogenous DDX1 expression renders nonpermissive cells susceptible to JC virus infection. DDX1 and CSTF64 bind the JC virus transcriptional control region and the outcome is productive expression and accumulation of early and late JC virus transcripts. By contrast, downregulation of DDX1 impairs JC virus gene expression. The results are likely attributable to impaired 3′ end processing that culminates in the decay of viral transcripts.

Coronavirus. Yeast 2-hybrid screens determined DDX1 interacts with coronavirus nonstructural protein 14.⁸⁹ Vero cells infected with infectious bronchititis virus exhibited transit of DDX1 from the nucleus to the cytoplasm and colocalization with the coronaviral RNA.⁸⁹ Mutation of DDX1 DEAD motif to AAAA eliminated the colocalization and the replication of coronavirus, indicating a necessary role for the ATPase-dependent helicase activity of DDX1.⁸⁹ The results suggest that DDX1 is important for efficient coronavirus replication.

RH116.

RH116 has been observed to affect replication of one virus to date, HIV-1 (Table 2). RH116 is a 116 kDa amino acid interferon-induced helicase (DECH variant). Assigned to the RIG-I like DExH box helicase family, RH116 contains two CARD domains (Fig. 1) and exhibits nuclear accumulation. RH116 is 99.5% identical to MDA-5 and recapitulates its inhibition of cellular proliferation.⁹⁰ RH116 was identified by a differential display RT-PCR screen for cofactors required for suppression of HIV-1 infection by the synthetic immunomodulator, Murabutide.⁹⁰ The overexpression of RH116 increases HIV-1 replication in HeLa-CD4 cells coincident with nuclear accumulation of endogenous RH116.⁹⁰ A possible explanation for this observation is squelching of a cofactor needed for the antiviral activity of RH116. The remarkable amino acid identity with MDA-5 suggests that RH116 is a cytosolic sensor for HIV-1 under some circumstances. These findings warrant new investigations into the control of RH116 gene expression, the cofactors that control the subcellular localization of RH116 and the effect of RH116 on IRF signaling activity during HIV-1 infection of lymphocytes.

DDX24.

DDX24 is a 96 kDa DEAD box helicase that lacks other conserved motifs (Fig. 1) and exhibits amino acid content that is dissimilar to typical DEAD box family members.⁹¹ Proteomic analysis identified DDX24 coprecipitates with HIV-1 Gag. This interaction is mediated by the Gag nucleocapsid domain and is disrupted by RNase, indicating that the interaction is RNA-dependent.⁷⁷ DDX24 also coprecipitates with HIV-1 Rev.²⁵ The downregulation of DDX24 changes the subcellular localization of Rev from nucleolar to nuclear and moderately increases Rev/RRE-dependent nuclear export of unspliced HIV-1 mRNA (2–3 fold).²⁵ Despite the increase of viral transcript in the cytoplasm, the infectivity of HIV-1 progeny virions was reduced on Hela-based TZM-bl reporter cells. Real time PCR determined reduced abundance of virion-associated RNA, which is sufficient to account for the reduction in titer.²⁵ In sum, DDX24 appears to be important for proper trafficking of the viral RNA for assembly into virions (Table 2 and Fig. 3).

P68/DDX5.

p68 is a DEAD box helicase that contains a p68HR domain that is characteristic of p68-like RNA helicases (Fig. 1). Named for its molecular weight of 68 kDa, p68 cross reacts with monoclonal antibody to SV40 large-T antigen (pAB204 epitope), which suggests these proteins display a similar epitope.^92–94 In common with SV40 large-T antigen, p68 affects cellular proliferation and tumor development, and mechanistically p68 intersects the processes of transcriptional regulation, RNA splicing and the processing of pre-ribosomal RNA.^95–100 Using results from a yeast two-hybrid screen, Goh et. al. determined that p68 interacts with HCV NS5B RNA-dependent RNA polymerase.¹⁰¹ Expression of NS5B of HCV changes the subcellular localization of endogenous p68 from the nucleus to the cytoplasm,¹⁰¹ similar to the effect of FMDV on RHA.⁸⁰ Downregulation of p68 reduces the transcription of negative strand HCV RNA. Taken together, p68 is an important host factor for HCV at the level of HCV gene expression (Table 2).

HCV nonstructural protein 3 (NS3).

HCV encodes a single open reading frame that is translated to a 3,000 amino acid polyprotein and cleaved into 10 mature proteins. NS3 is a 70 kDa DExH box protein (DECH variant) that resembles SF2 family helicases.^10,109 NS3 contains five domains: two N-terminal domains that comprise a protease and three distal domains that comprise the helicase^10,110 (Fig. 1). Until recently, the NS3 helicase and protease domains were considered functionally independent, however, new data indicate the protease domain exhibits a positive charge that enhances the interaction of the helicase domain with RNA.^111,112 NS3 RNA helicase activity is essential for HCV replication and is not replaced by cellular helicases.^112,113 NS3 RNA helicase affects two steps in the HCV replication cycle. During RNA-dependent RNA replication, NS3 is required to unwind the double-stranded RNA intermediate, which may enable movement of HCV NS5b polymerase.^110,114,115 Second, NS3 assists in virus assembly and is likely attributable to ability to act as a scaffold for interaction with viral or cellular cofactors. Point mutation of the NS3 helicase domain (both subdomains) demonstrated a role for NS3 helicase in infectious virus assembly, independent of a role in HCV RNA replication.¹¹⁶ Homologous NS3 proteins are encoded by two other Flaviviruses, yellow fever virus and dengue virus.¹⁴ In sum, NS3 helicase activity is essential for Flavivirus replication and is a potential therapeutic target against the significant human pathogen, HCV.

HCV affects nearly 180 million people worldwide and is responsible for about 50–75% of liver cancer cases.^110,117 Current therapy against HCV is ineffective in about 20% and 60% of patients chronically infected with HCV genotypes 2 and 3 and genotype 1, respectively.¹¹⁸ HCV helicase has been actively pursued as a target to inhibit HCV.^119,120 Several studies to identify small molecule, nucleic acid and antibody inhibitors of HCV NS3 have been described in detail^10,110 and clinical trials have involved combinations to target HCV protease and polymerase.¹¹⁰ The continued mechanistic investigation of NS3 and ready availability of a cache of candidate compounds are essential tools to develop an efficacious treatment for HCV infection.

Vaccinia virus nucleoside triphosphate phosphohydrolase II (NPH-II).

Vaccinia virus encodes a 74 kDa helicase that contains a DEVH ATP binding domain and a DUF domain, and is designated nucleoside triphosphate phosphohydrolase II (NPH-II) (Fig. 1). NPH-II is an RNA-dependent NTPase that is activated by single-stranded RNA, and can hydrolyze all four ribonucleoside triphosphates.¹²¹ Homology is observed between the helicase domain of NPH-II, three yeast PRP splicing proteins (PRP16, PRP2 and PRP22), RHA/DHX9 and the Drosophila homolog Maleless.¹²² NPH-II unwinds RNA substrates or RNA-DNA hybrids in a processive and directional fashion with a small step size of around 6 base pairs.¹²³

Essential for vaccinia virus replication, NPH-II is incorporated into the vaccinia virion.¹²² Fathi and Condit uncovered several temperature sensitive mutations of NPH-II that generate non-infectious progeny virions at non-permissive temperature.^124,125 Gross and colleagues determined the temperature sensitive mutations produced defects in early transcription of the DNA template.¹²⁶ DNA-mediated marker rescue of NPH-II temperature sensitive viruses revealed the ATPase and helicase activity of NPH-II are essential for vaccinia virus replication.¹²⁷ Mutations that block RNA unwinding without significantly affecting the ATPase activity are lethal, possibly by promoting genomic instability. The authors posited that NPH-II facilitates productive viral transcription by preventing R loop formation behind the elongating RNA polymerase, which otherwise may result in genomic instability.¹²⁸ Homologous NPHII proteins are encoded by Poxviridae members variola virus and fowlpox virus.¹⁴ In sum, NPH-II provides essential function(s) in the viral life cycle, which apparently are lacking from cellular RNA helicases or cannot be provided in an accessible configuration. NPH-2 viral RNA helicase activity is a potential therapeutic target to attenuate poxvirus replication and pathogenesis.

We thank Mr. Tim Vojt for illustration, Dr. Paula Pitha and Reviewers for their helpful comments. The authors appreciate the support of Glenn Barber Predoctoral Fellowship, College of Veterinary Medicine, Ohio State University to A.R. and NIH NCI P30CA100730 and RO1CA108882 to K.B.L.