helicases are enzymes that separate in an energy - dependent manner stretches of duplexed dna and/or rna into single - stranded components . currently , based on characteristic motifs and the sequence comparisons , three superfamilies ( sf1 through 3 ) and two smaller families ( f4 , f5 ) of helicases have been identified ( 1 ) . superfamily 1 and 2 contain helicases which share seven or more recognized signature amino acid motifs while sf3 and f4 and f5 helicases are characterized generally by three conserved motifs ( 2 ) ; the f4 and f5 proteins are largely bacterial and bacteriophage proteins . currently , it should be cautioned that many  helicases are not bona fide helicases , but may only function as rna translocases , perhaps to fulfill functions in the remodeling of ribonucleoprotein complexes ( rnp ) . dead - box and the related deah , dexh and dexd ( 3 ) helicases are the most numerous members of sf2 and are ubiquitously present in eukaryotic genomes . these helicases share eight conserved motifs and are commonly refered to as the dexh / d family of helicases . humans , arabidopsis and saccharomyces have 38 , 55 and 25 such entities , respectively ( 4 ) . differing from dna helicases and dexh proteins , dead helicases are poor in unwinding long nucleic acid duplexes and are best suited for separating short rna hybrids . this suggests that the specificity determinants for dead helicases may be through the recognition of protein factors . in this regard , a better understanding of the roles for dead proteins depends on the clear characterization of their respective interacting proteins . although the precise substrate for most helicases awaits definition , dead helicases are generally thought to participate pleiotropically in many aspects of rna metabolism including transcription , mrna splicing , mrna export , translation , rna stability and mitochondrial gene expression ( 58 ) . uap56 , brr2 , prp16 , prp22 and prp43 play roles in rna - splicing ( 4,9 ) , while dbp5 ( 10,11 ) and ddx3 ( 12 ) chaperone rnas from the nucleus into the cytoplasm . eif4a and ded1 serve for translation of mrnas while rh1b , ski2 , dob1 , dhh1 helicases contribute to mrna stability ( 4 ) . other dead helicases act in ribosome biogenesis through regulation of small nucleolar rnas and ribosomal rnas ( rrnas ) interactions ( 13,14 ) . finally , neurospora and trypanosoma dead proteins contribute to mitochondrial gene expression ( 15,16 ) ; a cryptococcus dead helicase is required for cryptococcosis pathogenesis ( 17 ) , and the dipteran chironomus tentans uses a hrp84 dead helicase to regulate mrna transport from the nucleus into the cytoplasm onto polyribosomes ( 18 ) . given that helicases significantly contribute to normal cellular metabolism , are they similarly essential to viruses ? the operational answer appears to be a qualified  yes. indeed , when dead / deah - box helicase motif ( interpro ipr001410 ) was used to search the embl - ebi database , 1561 matches to individual viral sequence entries were found ( ) , suggesting that many viruses have evolved to encode directly helicase or helicase - like proteins . the strongest biological evidence which supports the importance of a helicase in the virus life cycle comes from those viruses with an rna genome . hence , all positive - strand rna viruses encode one or more helicase / helicase - like open reading frame ( orf ) which , aside from the rna - dependant rna polymerase , is the most highly conserved viral sequence . although less ubiquitous , helicases are also found in other types of viruses ( see some examples listed in table 1 ) . direct mutagenesis studies have confirmed that a helicase function is biologically required for the replication of many viruses including vaccinia virus ( 19 ) , poliovirus ( 20 ) , alphaviruses ( 21 ) , brome mosaic virus ( 22 ) , nidoviruses ( 23,24 ) and flaviviruses ( 2527 ) . in 1981 , the first cases of acquired immunodeficiency syndrome ( aids ) were described in american homosexual men . thereafter , within three short years , french and american scientists confirmed that the human immunodeficiency virus ( hiv ) is the causative agent for aids . in the ensuing 20 years , > 20 million individuals have died from aids ; and currently , in 2006 , 50 million people worldwide are infected by hiv-1 with 3 million incremental aids deaths and 45 million new infections occurring annually . hiv-1 is a retrovirus of the lentivirus genus with an rna genome of 9 kilobases which encodes nine polypeptides . the major hiv-1 structural proteins are encoded by three genes , gag ( group - specific antigen ) , pol ( polymerase ) and env ( envelope ) , while the accessory proteins , vif , vpu , vpr and nef , and the regulatory proteins , tat and rev , are the primary translation products of multiply - spliced mrna . hiv-1 infects cd4 + human t - cells and macrophages and integrates as a provirus into the host cell 's dna . gene expression of hiv-1 is governed transcriptionally by a viral protein , tat ( 28,29 ) , via its binding to a nascent viral tar rna ( 30 ) , and post - transcriptionally by a second viral protein rev ( 31,32 ) through its association with the viral rre rna . both tat and rev interact with several host cell proteins in their transcriptional and post - transcriptional functions ( 33 ) hiv-1 does not encode for any rna helicase ; however , findings suggest that host cell rna helicases may be involved in the reverse transcription of hiv-1 rna , in hiv-1 mrna transcription and in the nucleus - to - cytoplasm transport of viral mrna . a recent unexpected finding revealed the possibility that an rna helicase may potentially contribute roles in hiv-1 particle assembly and reverse transcription ( 34 ) . using proteomic analyses , roy et al . ( 34 ) reported that the deah protein rna helicase a ( rha ) was found associated with hiv-1 gag and packaged into hiv-1 virions in an rna - dependent manner . when rha was knocked down in cells , hiv-1 particles which were produced from these cells were significantly less infectious . first , it is conceivable that rha participates in the formation of infectious virus particles either by shaping gag second , roy et al . ( 34 ) reported evidence that hiv-1 particles that do not contain rha showed reduced virion - endogenous reverse transcriptase activity . in this respect , it may be that rha assists hiv-1 reverse transcriptase to more efficiently copy rna by unwinding rna secondary structure or by promoting the interaction of viral rna with the nucleocapsid protein in order to assemble a better reverse transcription complex . separate from reverse transcription , the unwinding of highly structured rnas might also be reasoned to be important for transcription ( 35 ) . however , direct evidence for an rna helicase role has been somewhat elusive . first , in vaccinia virus , it has been postulated that the nph - ii helicase assists transcription by strand - separating duplexed rna structures to prevent r - loop formation behind the elongating rna polymerase ( 36 ) . second , rha has been invoked to provide a factor - recruitment role , bridging at the promoter the creb - binding protein ( 37 ) and rna polymerase ii ( 37 ) . third , the p68 dead - box helicase was shown recently to be a novel transcriptional co - activator for p53 's transcriptional function ( 38 ) . interestingly , in the latter two instances , neither the atpase nor the helicase activity of rha and p68 is apparently required for their attributed transcriptional roles . for hiv-1 , two recent studies provide clues that rna helicases may also serve co - factor function for transcription from the viral long terminal repeat ( ltr ) . fujii et al . ( 39 ) observed that rha conserves in its n - terminus two double - stranded rna - binding ( dsrbd ) domains characterized previously for the tar rna - binding protein , trbp ( 40,41 ) . these investigators found in both reporter and virus replication assays that rha activated , in a tar rna - binding dependant manner , hiv-1 ltr - directed transcription ( 39 ) . next , cocude et al . ( 42 ) separately described that the expression of a dexh rna helicase , rh116 , was significantly induced after infection of hela - cd4 cells by hiv-1 . rh116 was found localized in the nucleus of hiv-1 infected cells and was observed to augment the transcription of unspliced hiv-1 transcripts . while both studies offer tantalizing evidence for roles by two different rna helicases in hiv-1 transcription , critical details on how the helicases function by binding to tar rna and how this binding might cooperate with the activities of the viral transcriptional activator , tat , pending more direct experimental tests , it remains unclear whether rha and rh116 provide direct roles in transcription or indirectly influence the milieu of polymerase ii initiation / elongation at the ltr . downstream from transcription , the fate of hiv-1 encoded rna is regulated at the step of export of unspliced / partially spliced moieties from the nucleus into the cytoplasm . unspliced and partially spliced viral rnas code for genomic rnas that are packaged into progeny virions and structural proteins . hence , the egress of these rnas from the nucleus into the cytoplasm is critical to the life cycle of the virus . exit of hiv rnas from the nucleus is a significant issue because unspliced / partially spliced cellular mrnas are routinely retained in and not permitted export from the nucleus ( 4346 ) . thus , it was established that the hiv-1 encoded rev protein binds a highly secondary structured element ( rev responsive element ; rre ) present in all unspliced and partially spliced hiv transcripts ( 4757 ) ; and this binding specifically distinguishes , for purposes of nuclear export , viral transcripts from cellular rnas . new evidence now suggests that rna helicases are also co - factors for rev - directed export of hiv-1 mrnas ( 58 ) . in its role of transporting unspliced and incompletely spliced viral rnas from the nucleus , rev directly interacts with nuclear export receptor crm1 ( 59,60 ) , and crm1 is required for rev - mediated export of hiv rnas ( 59,61,62 ) . a recent report provides data that an rna helicase , ddx3 , is an additional player in the rev thus , it was shown that ddx3 over - expression enhanced rev - dependent , but not other export , pathway ; and that ddx3 is a nucleo - cytoplasmic shuttling protein which binds crm1 and rev . moreover , ddx3 's necessity for rev / rre / crm1 function was demonstrated by knock - down of cell endogenous ddx3 . finally , because ddx3 locates to nuclear pore complexes ( npc ) , yedavalli et al . ( 12 ) further proposed that this human helicase , like the analogous yeast dbp5p ( 11 ) , may function with rev / crm1 to remodel and  thread large unspliced hiv-1 rnas through the nuclear pore , facilitating their final release to the cytoplasmic side of the npc . the above ddx3 results are consistent with two additional papers which described similar findings for a related rna helicase , ddx1 . thus , pomerantz and co - workers ( 63 ) showed that ddx1 binds directly to the n - terminus of rev and to the rre - rna motif and participates in the export of unspliced hiv-1 rna from the nucleus to the cytoplasm . additionally , they illustrated that reduced expression of ddx1 in astrocytes explains the previously observed tissue restricted function of hiv-1 rev ( 64 ) . fully spliced viral mrnas encoding for viral tat , rev and nef , proteins have been shown previously to exit the nucleus using the cellular mrna export pathway . as yet , the involvement of dbp5 in export of spliced hiv-1 viral rna has not been fully clarified . the story of hiv-1 and rna helicases is , however , likely to be more complex than and unlikely to conclude simply with rha , rh116 , ddx1 and ddx3 ( figure 1 ) . splicing is a multiple - step process requiring the recognition of splice sites by spliceosomes . it is generally believed that remodeling of rna  rna and rna  protein interactions within the spliceosome is catalyzed by a family of dead / dexh box rna helicases . to date , seven mammalian proteins that are rna helicases have been implicated in mrna splicing ( 67,68 ) . whether there is specific preference by subclasses of rna helicases for viral mrna splicing remains to be clarified . moreover , how cellular rna helicases might contribute to the translation of viral mrnas also require further investigation . ( 69 ) and krishnan and zeichner ( 70 ) have provided evidence that the expression of several cellular rna helicases including ddx24 , ddx21 , ddx18 , ddx11 and ddx9 is modulated during hiv-1 infection ; however , the precise cellular role and significance of these helicases for hiv-1 pathogenesis have not be characterized . interestingly , krishnan and zeichner reported microarray data which examined the transition of hiv-1 infection from latency to productive replication , and found that several cellular rna helicases were upregulated ( 71 ) . for future understanding of functions , it will be important to design experiments which can segregate helicases which serve direct , although perhaps overlapping and redundant , roles on hiv-1 from those that might participate indirectly in the viral life cycle . nevertheless , the convergence of evidence would support that several discrete cellular rna helicases contribute importantly to the efficient execution of several steps in the hiv-1 replicative cycle . given that the hiv-1/aids disease burden has reached pandemic global proportions , new antiviral strategies that target molecularly delineated mechanisms used by this virus are urgently needed ( 72 ) . is there a possibility that host cell helicases can be therapeutic targets for anti - hiv-1 chemotherapy ? implicit within this question is the concept that one could attack a host cell protein in order to treat an infecting pathogen . although targeting a cellular protein involved in a viral pathway risks obvious cytotoxicity , this approach avoids the inherent problem posed by rapid hiv-1 mutation to all currently utilized chemotherapeutics targeted to virus - encoded proteins . we note that inhibition of cell - encoded enzymes in medical therapy is not an unprecedented strategy . suppression of angiotensin - converting enzyme ( ace ) is widely used to treat hypertension , congestive heart failure , myocardial infarction , endothelial dysfunction and renal disease ( 73 ) . elsewhere , aromatase inhibitors have been used to treat hormone - dependent breast cancer ( 74 ) , and inhibitors of cellular secretory proteases are contemplated for alzheimer 's disease ( 75 ) . we recently inhibited the cellular polyprotein convertase , furin , at minimal toxicity to the cell in order to block hiv-1 replication ( 76 ) . thus , a priori exclusion of cellular helicase as an antiviral target is not warranted . guarded optimism that small molecule helicase inhibitors can be developed against viruses arises from encouraging progress in non - retroviral systems . unlike hiv-1 , human herpesviruses physically encode helicases . the herpes simplex virus ul5 and ul9 genes are helicases in superfamily 1 and 2 , respectively ( 77 ) . two recent studies provide proof - of - concept that the hsv helicase  primase can be targeted at low host cell toxicity by two new classes of drugs , amino - thiazolyphenyl - molecules ( 78 ) and thiazole amide derivatives ( 79 ) . in addition , other studies suggest that the ns3 protein , a rna helicase encoded by hepatitis c virus and related west nile virus and japanese encephalitis virus can be targeted to inhibit viral replication ( 8082 ) . this conceptual break through in drug development is important because it indicates that target discrimination between different helicases by small molecule inhibitors is possible . of relevance to hiv-1 , a synthetic immunomodulator murabutide was shown recently to suppress hiv-1 replication in macrophages and t cells . murabutide was shown to inhibit the activity of rna helicase rh116 , blocking its positive transcriptional activity for hiv-1 gene expression ( 42 ) . if one looks beyond the signature motifs conserved amongst helicases , then it becomes clear that the different proteins are widely divergent in their coding sequences . in principle , this suggests that individual helicases can be abrogated with specificity in a knowledge - directed manner . in theory , a helicase can be attacked by ( i ) inhibition of ntpase activity through direct competition for ntp binding , ( ii ) inhibition of substrate binding through direct competition at active site , ( iii ) allosteric mechanisms to affect ntp - binding / ntp hydrolysis and/or polynucleotide binding , and ( iv ) inhibition of unwinding activity by steric hinderance of helicase translocation along the polynucleotide substrate ( 83,84 ) . because the ntp - binding and substrate - binding pockets may be sufficiently similar between various helicases , specificity of inhibition through these sites on the other hand , the tremendous variations in sequence and sizes of helicases , in their oligomerization states , in their discrete domains responsible for protein  protein interactions and/or for targeting to specific nucleic acids ( 85 ) , and in their differential localizations within cells ( 86 ) offer interventional possibilities outside of the ntp- or polynucleotide - binding sites . we are in the preliminary stages of screening ring - expanded nucleoside analogs found previously to be successful ntpase / helicase inhibitors of west nile virus , hepatitis c virus and japanese encephalitis virus ( 81,82 ) . we have observed that a few of these candidate inhibitors have substantial anti - hiv-1 activity at doses that do not incur cytotoxicity to cells treated in tissue culture for 1 week . further studies are needed before concluding that these compounds exert specific inhibition of ddx3 , one of the other cellular helicases , or some other target altogether . there is another area where a cellular helicase activity and hiv-1 are likely to intersect . an emerging research focus is the role of small interfering rnas ( 87 ) and micrornas ( mirna ) as innate cell defenses against viruses including hiv-1 ( 8790 ) . in human cells , the precursor for mirna ( pre - mirna ) is processed by dicer ( figure 2a ) which is a ribonuclease with a bona fide rna helicase domain ( 91,92 ) . a surprising recent finding revealed that the human trbp , which has been shown to be a potent binder of the hiv-1 tar rna rna ( 40,41 ) , is an indispensable dsrna - binding partner of dicer which allows the latter to associate with pre - mirna ( 91,92 ) . without trbp , dicer 's mirna processing activity is lost . helicase protein dicer requires trbp to process duplex - structured mirnas in order that the cell can use such matured mirnas for antiviral defense , it could be speculated that hiv-1 has evolved to restrict this defense by the ability to transcribe viral tar rna to squelch trbp away from dicer ( y. bennasser , m. l. yeung and k. t. jeang , manuscript submitted ) ( figure 2b ) . if this thinking is correct , then hiv-1 has developed mechanisms not only to co - opt the active functions of a virus - propitious cellular helicase ( i.e. ddx3 ) but also to inactivate the role of a second virus - pernicious helicase ( i.e. dicer ) for purposes of selfish gain . in a separate perspective , virus infection can trigger through double - stranded viral rnas an innate antiviral immune response . thus viral dsrnas can be recognized by cellular proteins [ pattern - recognition receptors ( prrs ) ] which initiate antiviral responses by inducing the production of a variety of cytokines including type i interferons ( ifn- and ifn- ) and initiating additional inflammatory and adaptive immune responses . recently , dexd / h rna helicases such as rig-1 ( retinoic acid inducible gene-1 ) ( 93 ) and mda5 ( melanoma differentiation - associated gene 5 ) ( 94 ) have been identified as suppressors of viral replication by binding to virus associated dsrna and activating type i interferon - dependent antiviral immunity . over - expression of rig-1 and mda5 currently , it remains speculative whether helicases like rig-1 and mda5 may recognize hiv-1 dsrna and trigger an innate immune response . intriguingly , several reports exist in the literature that hiv-1 infection does induce activation of type 1 interferons ( 95,96 ) . in conclusion , by studying helicase proteins one can gain insights into normal cellular metabolic processes , abnormal inherited human diseases ( e.g. bloom syndrome , werner syndrome , cockayne 's syndrome and xeroderma pigmentosum ; all diseases with mutations in cellular helicases ) , and remarkably also the biology of viruses . schematic representation of the involvement of cellular rna helicases in reverse transcription , transcription and post - transcriptional regulation of hiv-1 rnas . rha has been found to be a part of the hiv-1 virion and may participate in hiv-1 reverse transcription as well as particle assembly . rha and rh116 have been reported to participate in hiv-1 transcription through interaction with the viral leader rna , tar . ddx1 and ddx3 act with rev and rre - containing rnas for the transport of unspliced and partially spliced hiv-1 rna from the nucleus to the cytoplasm . involvement of dicer rna helicase , trbp and hiv-1 tar rna in the biology of the cell 's micro rna pathway . ( b ) a schematic representation of a potential mechanism for tar rna - dependent suppression of dicer activity . tar rna is suggested to bind to trbp and decoy it from dicer preventing dicer - dependent mirna processing activity .