A novel class III endogenous retrovirus with a class I envelope gene in African frogs with an intact genome and developmentally regulated transcripts in Xenopus tropicalis.

BACKGROUND: Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses. RESULTS: Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag, pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env-less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5' and 3' LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution. CONCLUSIONS: XtERV-S represents a genera-spanning member of the largely env-less class III ERV that has ancient and modern copies in Anurans. This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years.

Recombinant Origins of Pathogenic and Nonpathogenic Mouse Gammaretroviruses with Polytropic Host Range.

Ecotropic, xenotropic, and polytropic mouse leukemia viruses (E-, X-, and P-MLVs) exist in mice as infectious viruses and endogenous retroviruses (ERVs) inserted into mouse chromosomes. All three MLV subgroups are linked to leukemogenesis, which involves generation of recombinants with polytropic host range. Although P-MLVs are deemed to be the proximal agents of disease induction, few biologically characterized infectious P-MLVs have been sequenced for comparative analysis. We analyzed the complete genomes of 16 naturally occurring infectious P-MLVs, 12 of which were typed for pathogenic potential. We sought to identify ERV progenitors, recombinational hot spots, and segments that are always replaced, never replaced, or linked to pathogenesis or host range. Each P-MLV has an E-MLV backbone with P- or X-ERV replacements that together cover 100% of the recombinant genomes, with different substitution patterns for X- and P-ERVs. Two segments are always replaced, both coding for envelope (Env) protein segments: the N terminus of the surface subunit and the cytoplasmic tail R peptide. Viral gag gene replacements are influenced by host restriction genes Fv1 and Apobec3 Pathogenic potential maps to the env transmembrane subunit segment encoding the N-heptad repeat (HR1). Molecular dynamics simulations identified three novel interdomain salt bridges in the lymphomagenic virus HR1 that could affect structural stability, entry or sensitivity to host immune responses. The long terminal repeats of lymphomagenic P-MLVs are differentially altered by recombinations, duplications, or mutations. This analysis of the naturally occurring, sometimes pathogenic P-MLV recombinants defines the limits and extent of intersubgroup recombination and identifies specific sequence changes linked to pathogenesis and host interactions.IMPORTANCE During virus-induced leukemogenesis, ecotropic mouse leukemia viruses (MLVs) recombine with nonecotropic endogenous retroviruses (ERVs) to produce polytropic MLVs (P-MLVs). Analysis of 16 P-MLV genomes identified two segments consistently replaced: one at the envelope N terminus that alters receptor choice and one in the R peptide at the envelope C terminus, which is removed during virus assembly. Genome-wide analysis shows that nonecotropic replacements in the progenitor ecotropic MLV genome are more extensive than previously appreciated, covering 100% of the genome; contributions from xenotropic and polytropic ERVs differentially alter the regions responsible for receptor determination or subject to APOBEC3 and Fv1 restriction. All pathogenic viruses had modifications in the regulatory elements in their long terminal repeats and differed in a helical segment of envelope involved in entry and targeted by the host immune system. Virus-induced leukemogenesis thus involves generation of complex recombinants, and specific replacements are linked to pathogenesis and host restrictions.

Activation of HIV-1 from latent infection via synergy of RUNX1 inhibitor Ro5-3335 and SAHA.

A major barrier to the elimination of HIV-1 infection is the presence of a pool of long-lived, latently infected CD4+ memory T-cells. The search for treatments to re-activate latent HIV to aid in clearance is hindered by the incomplete understanding of the mechanisms that lead to transcriptional silencing of viral gene expression in host cells. Here we identify a previously unknown role for RUNX1 in HIV-1 transcriptional latency. The RUNX proteins, in combination with the co-factor CBF-ß, are critical transcriptional regulators in T-cells. RUNX1 strongly modulates CD4 expression and contributes to CD4+ T-cell function. We show that RUNX1 can bind DNA sequences within the HIV-1 LTR and that this binding represses transcription. Using patient samples we show a negative correlation between RUNX1 expression and viral load. Furthermore, we find that pharmacologic inhibition of RUNX1 by a small molecule inhibitor, Ro5-3335, synergizes with the histone deacetylase (HDAC) inhibitor SAHA (Vorinostat) to enhance the activation of latent HIV-1 in both cell lines and PBMCs from patients. Our findings indicate that RUNX1 and CBF-ß cooperate in cells to modulate HIV-1 replication, identifying for the first time RUNX1 as a cellular factor involved in HIV-1 latency. This work highlights the therapeutic potential of inhibitors of RUNX1 to re-activate virus and aid in clearance of HIV-1.

CBFß enhances de novo protein biosynthesis of its binding partners HIV-1 Vif and RUNX1 and potentiates the Vif-induced degradation of APOBEC3G.

UNLABELLED: Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFß was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFß remains unclear. Several studies noted reduced Vif expression in CBFß knockdown cells while others saw no significant impact of CBFß on Vif stability. Here, we confirmed that CBFß increases Vif steady-state levels. CBFß affected expression of neither viral Gag nor Vpu protein, indicating that CBFß regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFß on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFß to interact with Vif. Importantly, at comparable Vif levels, CBFß further enhanced A3G degradation, suggesting that CBFß facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFß also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFß had no detectable effect on RUNX1 metabolic stability. We propose that CBFß acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. IMPORTANCE: In this study, we show that CBFß has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFß. Kinetic studies revealed that CBFß increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFß were nonresponsive to CBFß. Our data suggest that CBFß exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.

Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1-CBFß interaction.

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ~24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ~50%. We hypothesize that the interaction between RUNX1 and CBFß is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFß interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFß directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFß interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.

Trimethylguanosine capping selectively promotes expression of Rev-dependent HIV-1 RNAs.

5'-mRNA capping is an early modification that affects pre-mRNA synthesis/splicing, RNA cytoplasmic transport, and mRNA translation and turnover. In eukaryotes, a 7-methylguanosine (m7G) cap is added to newly transcribed RNA polymerase II (RNAP II) transcripts. A subset of RNAP II-transcribed cellular RNAs, including small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and telomerase RNA, is further hypermethylated at the exocyclic N2 of the guanosine to create a trimethylguanosine (TMG)-capped RNA. Some of these TMG-capped RNAs are transported within the nucleus and from the nucleus to the cytoplasm by the CRM-1 (required for chromosome region maintenance) protein. CRM-1 is also used to export Rev/RRE-dependent unspliced/ partially spliced HIV-1 RNAs. Here we report that like snRNAs and snoRNAs, some Rev/RRE-dependent HIV-1 RNAs are TMG-capped. The methyltransferase responsible for TMG modification of HIV-1 RNAs is the human PIMT (peroxisome proliferator-activated receptor-interacting protein with methyltransferase) protein. TMG capping of unspliced/partially spliced HIV-1 RNAs represents a new regulatory mechanism for selective expression.

Expression of a protein phosphatase 1 inhibitor, cdNIPP1, increases CDK9 threonine 186 phosphorylation and inhibits HIV-1 transcription.

CDK9/cyclin T1, a key enzyme in HIV-1 transcription, is negatively regulated by 7SK RNA and the HEXIM1 protein. Dephosphorylation of CDK9 on Thr(186) by protein phosphatase 1 (PP1) in stress-induced cells or by protein phosphatase M1A in normally growing cells activates CDK9. Our previous studies showed that HIV-1 Tat protein binds to PP1 through the Tat Q(35)VCF(38) sequence, which is similar to the PP1-binding RVXF motif and that this interaction facilitates HIV-1 transcription. In the present study, we analyzed the effect of expression of the central domain of nuclear inhibitor of PP1 (cdNIPP1) in an engineered cell line and also when cdNIPP1 was expressed as part of HIV-1 pNL4-3 in place of nef. Stable expression of cdNIPP1 increased CDK9 phosphorylation on Thr(186) and the association of CDK9 with 7SK RNA. The stable expression of cdNIPP1 disrupted the interaction of Tat and PP1 and inhibited HIV-1 transcription. Expression of cdNIPP1 as a part of the HIV-1 genome inhibited HIV-1 replication. Our study provides a proof-of-concept for the future development of PP1-targeting compounds as inhibitors of HIV-1 replication.

Wip1 and p53 contribute to HTLV-1 Tax-induced tumorigenesis.

BACKGROUND: Human T-cell Leukemia Virus type 1 (HTLV-1) infects 20 million individuals world-wide and causes Adult T-cell Leukemia/Lymphoma (ATLL), a highly aggressive T-cell cancer. ATLL is refractory to treatment with conventional chemotherapy and fewer than 10% of afflicted individuals survive more than 5 years after diagnosis. HTLV-1 encodes a viral oncoprotein, Tax, that functions in transforming virus-infected T-cells into leukemic cells. All ATLL cases are believed to have reduced p53 activity although only a minority of ATLLs have genetic mutations in their p53 gene. It has been suggested that p53 function is inactivated by the Tax protein. RESULTS: Using genetically altered mice, we report here that Tax expression does not achieve a functional equivalence of p53 inactivation as that seen with genetic mutation of p53 (i.e. a p53 -/- genotype). Thus, we find statistically significant differences in tumorigenesis between Tax+p53 +/+ versus Tax+p53 -/- mice. We also find a role contributed by the cellular Wip1 phosphatase protein in tumor formation in Tax transgenic mice. Notably, Tax+Wip1 -/- mice show statistically significant reduced prevalence of tumorigenesis compared to Tax+Wip1 +/+ counterparts. CONCLUSIONS: Our findings provide new insights into contributions by p53 and Wip1 in the in vivo oncogenesis of Tax-induced tumors in mice.

Inflammatory cardiac valvulitis in TAX1BP1-deficient mice through selective NF-kappaB activation.

Nuclear factor kappa B (NF-kappaB) is a key mediator of inflammation. Unchecked NF-kappaB signalling can engender autoimmune pathologies and cancers. Here, we show that Tax1-binding protein 1 (TAX1BP1) is a negative regulator of TNF-alpha- and IL-1beta-induced NF-kappaB activation and that binding to mono- and polyubiquitin by a ubiquitin-binding Zn finger domain in TAX1BP1 is needed for TRAF6 association and NF-kappaB inhibition. Mice genetically knocked out for TAX1BP1 are born normal, but develop age-dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF-alpha and IL-1beta. TAX1BP1-/- cells are more highly activated for NF-kappaB than control cells when stimulated with TNF-alpha or IL-1beta. Mechanistically, TAX1BP1 acts in NF-kappaB signalling as an essential adaptor between A20 and its targets.

Matrin 3 is a co-factor for HIV-1 Rev in regulating post-transcriptional viral gene expression.

Post-transcriptional regulation of HIV-1 gene expression is mediated by interactions between viral transcripts and viral/cellular proteins. For HIV-1, post-transcriptional nuclear control allows for the export of intron-containing RNAs which are normally retained in the nucleus. Specific signals on the viral RNAs, such as instability sequences (INS) and Rev responsive element (RRE), are binding sites for viral and cellular factors that serve to regulate RNA-export. The HIV-1 encoded viral Rev protein binds to the RRE found on unspliced and incompletely spliced viral RNAs. Binding by Rev directs the export of these RNAs from the nucleus to the cytoplasm. Previously, Rev co-factors have been found to include cellular factors such as CRM1, DDX3, PIMT and others. In this work, the nuclear matrix protein Matrin 3 is shown to bind Rev/RRE-containing viral RNA. This binding interaction stabilizes unspliced and partially spliced HIV-1 transcripts leading to increased cytoplasmic expression of these viral RNAs.

Rev-ing up post-transcriptional HIV-1 RNA expression.

The post-transcriptional export of spliced and unspliced HIV-1 (human immunodeficiency virus type 1) RNAs from the nucleus to the cytoplasm is a complex process. Part of the complexity arises from the fact that eukaryotic cells normally retain unspliced RNAs in the nucleus preventing their exit into the cytoplasm. HIV-1 has evolved a protein, Rev, that participates in the export of unspliced / partially spliced viral RNAs from the nucleus. It has been documented that several cellular factors cooperate in trans with Rev, and certain cis-RNA motifs / features are important for transcripts to be recognized by Rev and its co-factors. Here, the post-transcriptional activities of Rev are discussed in the context of a recent finding that an RNA cap methyltransferase contributes to the expression of unspliced / partially spliced HIV-1 transcripts.

Human immunodeficiency virus type 1 replication and regulation of APOBEC3G by peptidyl prolyl isomerase Pin1.

APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) replication. HIV-1 synthesizes a viral infectivity factor (Vif) to counter A3G restriction. Currently, it is poorly understood how A3G expression/activity is regulated by cellular factors. Here, we show that the prolyl isomerase Pin1 protein modulates A3G expression. Pin1 was found to be an A3G-interacting protein that reduces A3G expression and its incorporation into HIV-1 virion, thereby limiting A3G-mediated restriction of HIV-1. Intriguingly, HIV-1 infection modulates the phosphorylation state of Pin1, enhancing its ability to moderate A3G activity. These new findings suggest a potential Vif-independent way for HIV-1 to moderate the cellular action of A3G.

Multivalent binding oligomers inhibit HIV Tat-TAR interaction critical for viral replication.

We describe the development of a new type of scaffold to target RNA structures. Multivalent binding oligomers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that favorable RNA binding occurs. We have used this strategy to develop MBO-based inhibitors to prevent the association of a protein-RNA complex, Tat-TAR, that is essential for HIV replication. In vitro binding assays combined with model cell-based assays demonstrate that the optimal MBOs inhibit Tat-TAR binding at low micromolar concentrations. Antiviral studies are also consistent with the in vitro and cell-based assays. MBOs provide a framework for the development of future RNA-targeting molecules.

Role of RNA helicases in HIV-1 replication.

Viruses are replication competent genomes which are relatively gene-poor. Even the largest viruses (i.e. Herpesviruses) encode only slightly >200 open reading frames (ORFs). However, because viruses replicate obligatorily inside cells, and considering that evolution may be driven by a principle of economy of scale, it is reasonable to surmise that many viruses have evolved the ability to co-opt cell-encoded proteins to provide needed surrogate functions. An in silico survey of viral sequence databases reveals that most positive-strand and double-stranded RNA viruses have ORFs for RNA helicases. On the other hand, the genomes of retroviruses are devoid of virally-encoded helicase. Here, we review in brief the notion that the human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle.

Methylation: a regulator of HIV-1 replication?

Recent characterizations of methyl transferases as regulators of cellular processes have spurred investigations into how methylation events might influence the HIV-1 life cycle. Emerging evidence suggests that protein-methylation can positively and negatively regulate HIV-1 replication. How DNA- and RNA- methylation might impact HIV-1 is also discussed.

Activation of HIV-1 expression and replication by cGMP dependent protein kinase type 1-beta (PKG1beta).

The effect of cGMP (cyclic GMP) dependent protein kinase 1-beta (PKG1-beta) and cGMP analogues on transcriptional activity and replication of human immunodeficiency virus type 1 (HIV-1) was investigated. Transfection of PKG1beta expression plasmid increased expression from an HIV-1 LTR-reporter as well as from an infectious HIV-1 molecular clone, pNL4-3. Treatment of HIV-1 AD8-infected monocyte derived macrophages (MDMs) with cGMP agonists and cGMP antagonists caused respectively increased and decreased virus replication. These findings provide evidence that cGMP and PKG serve to regulate HIV-1 infection in human cells.

Segregation of NF-kappaB activation through NEMO/IKKgamma by Tax and TNFalpha: implications for stimulus-specific interruption of oncogenic signaling.

Nuclear factor-kappaB essential modulator (NEMO), also called IKKgamma, has been proposed as a 'universal' adaptor of the I-kappaB kinase (IKK) complex for stimuli such as proinflammatory cytokines, microbes, and the HTLV-I Tax oncoprotein. Currently, it remains unclear whether the many signals that activate NF-kappaB through NEMO converge identically or differently. We have adopted two approaches to answer this question. First, we generated and targeted intracellularly three NEMO-specific monoclonal antibodies (mAbs). These mAbs produced two distinct intracellular NF-kappaB inhibition profiles segregating TNFalpha from Tax activation. Second, using NEMO knockout mouse fibroblasts and 10 NEMO mutants, we found that different regions function in trans either to complement or to inhibit dominantly TNFalpha, IL-1beta, or Tax activation of NF-kappaB. For instance, NEMO (1-245 amino acids) supported Tax-mediated NF-kappaB activation, but did not serve TNFalpha- or IL-1beta signaling. Altogether, our findings indicate that while NEMO 'universally' adapts numerous NF-kappaB activators, it may do so through separable domains. We provide the first evidence that selective targeting of NEMO can abrogate oncogenic Tax signaling without affecting signals used for normal cellular metabolism.

NEAT1 long noncoding RNA and paraspeckle bodies modulate HIV-1 posttranscriptional expression.

Most of the human genome is transcribed into protein-noncoding RNAs (ncRNAs), including small ncRNAs and long ncRNAs (lncRNAs). Over the past decade, rapidly emerging evidence has increasingly supported the view that lncRNAs serve key regulatory and functional roles in mammal cells. HIV-1 replication relies on various cell functions. To date, while the involvement of host protein factors and microRNAs (miRNAs) in the HIV-1 life cycle has been extensively studied, the relationship between lncRNAs and HIV-1 remains uncharacterized. Here, we have profiled 83 disease-related lncRNAs in HIV-1-infected T cells. We found NEAT1 to be one of several lncRNAs whose expression is changed by HIV-1 infection, and we have characterized its role in HIV-1 replication. We In the abstract, added definition of INS OK, or should "cis-acting" be added?report here that the knockdown of NEAT1 enhances virus production through increased nucleus-to-cytoplasm export of Rev-dependent instability element (INS)-containing HIV-1 mRNAs. IMPORTANCE Long protein-noncoding RNAs (lncRNAs) play roles in regulating gene expression and modulating protein activities. There is emerging evidence that lncRNAs are involved in the replication of viruses. To our knowledge, this report is the first to characterize a role contributed by an lncRNA, NEAT1, to HIV-1 replication. NEAT1 is essential for the integrity of the nuclear paraspeckle substructure. Based on our findings from NEAT1 knockdown, we have identified the nuclear paraspeckle body as another important subcellular organelle for HIV-1 replication.

Small molecules targeted to a non-catalytic "RVxF" binding site of protein phosphatase-1 inhibit HIV-1.

HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q(35)VCF(38), which resembles the PP1-binding "RVxF" motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the "RVxF"-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the "RVxF"-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners.

A method to study the role of DDX3 RNA helicase in HIV-1.

Viral replication requires the use of host cell proteins and enzymes. Many viruses utilize viral helicases at various stages of their life cycle; these viruses have evolved to encode directly helicase or helicase-like proteins. In contrast, the genomes of retroviruses are devoid of viral helicases. Human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle. In this chapter, we briefly summarize the approach for assaying the RNA unwinding activity of RNA helicases measuring the effect of helicase inhibitors on HIV-1 replication.