Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays.

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to nonspecific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with nonspecific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can nonspecifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

Heterocyclic compounds that inhibit Rev-RRE function and human immunodeficiency virus type 1 replication.

A cell-based screening assay was performed to identify compounds that inhibited the postintegration stage of the human immunodeficiency virus (HIV) life cycle. This assay utilized a cell line that contains the HIV gag and pol genes expressed in a Rev-dependent fashion. The cell line produces about 10 to 15 ng of p24 per milliliter of medium over a 24-h period in the form of viruslike particles. Any compound that inhibits a postintegration step in the HIV life cycle scores in this assay by decreasing particle production. Forty thousand compounds were screened, and 192 compounds were selected from the original screen because they showed more than 50% inhibition at a 10 muM concentration. The cumulative evidence presented in this study strongly suggests that 2 of the 192 compounds work as inhibitors of HIV Rev function. This was determined by a variety of cell-based assays, although the compounds do not interfere with Rev-RRE (Rev response element) binding in vitro. Both compounds inhibit replication of the lab isolate NL4-3 as well as an HIV primary isolate from Brazil (93BR021) and thus are promising leads as therapeutic candidates that target HIV replication through inhibition of Rev function.

Silencing of HIV-1 gene expression by siRNAs in transduced cells.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.

Small molecule modulators of HIV Rev/Rev response element interaction identified by random screening.

A high throughput scintillation proximity assay with biotinylated human immunodeficiency virus (HIV) Rev protein and tritiated Rev response element RNA was used to screen over 500,000 small molecules. Several chemical classes of inhibitors and two chemical classes of enhancers of binding were identified, with the molecular weight range being 400-600. The most common structural motif of inhibitor was an acidic moiety at the end of a linear aromatic system. Most of these modulators had EC(50) values in the 1-10 microM potency range, with several below 1 microM. Several classes displayed structure-activity relationships suggesting specific molecular interactions between small molecule and macromolecule. Several molecules were confirmed as inhibitors in a gel shift assay and by surface plasmon resonance analysis. Furthermore, one inhibitor was shown to bind the Rev protein with a binding constant equal to its IC(50) value, consistent with the mechanism of inhibition being binding Rev. Thus, small molecules can modulate this macromolecular protein-RNA interaction in vitro. However, no compound demonstrated HIV antiviral activity in a relevant cell-based assay.

Synthesis of biomimetic analogs of neomycin B: potential inhibitors of the HIV RNA Rev response element.

The aminoglycosidic antibiotic, neomycin B, is an inhibitor of the binding of Rev to RRE. This paper reports on the synthesis of analogs of neomycin B as potential anti-HIV compounds designed to function as inhibitors of Rev/RRE binding.

[Effect of 5-azacytidine on expression of DNA sequences homologous to ENV gene of murine mammary tumor virus in normal human lymphocytes]. / Vliianie 5-azatsitidina na ékspressiu posledovatel'nostei DNK, gomologichnykh geny ENV virusa raka molochnoi zhelezy myshei, v limfotsitakh zdorovykh donorov.

Expression of DNA sequences, related to MMTV env gene, in peripheral blood lymphocytes, which was strictly specific for human mammary carcinoma, has been previously reported. These sequences (homologous to env gene site coding for MMTV gp52 envelope antigen) expressed in T cells can play the key role in virus infection transmission and propagation. In order to elucidate the possible routes of env MMTV-homologous sequences expression, we tried to induced it in donot T lymphocytes by various methods: hormone and virus treatment (related genome "saving" at the expense of the added virus envelope), T cell culturing with conA, interferon-2, and 5-azacytidine. RT-PCR with primers specific for the gp52-coding area of MMTV env gene showed expression of env-homologous sequences in donor T cells cultured in medium with 5-azacytidine. Indirect immunofluorescence with monospecific serum to MMTV gp52 detected gp52 analogous genes only in cultures with 5-azacytidine but not other agents. We therefore suggested that MMTV env-homologous sequences in donors are situated in the methylated promoter zone. Expression of these sequences in T cells, specific for human mammary carcinoma, can be due to demethylation of the promoter and induction of env-homologous sequences to the level of translation of gp52 analogous antigens or by initial location of some of the expressed sequences in the demethylated zone of the genome.

ADAR1 is a novel multi targeted anti-HIV-1 cellular protein.

We examined the antiviral activity of ADAR1 against HIV-1. Our results indicated that ADAR1 in a transfection system inhibited production of viral proteins and infectious HIV-1 in various cell lines including 293T, HeLa, Jurkat T and primary CD4+ T cells, and was active against a number of X4 and R5 HIV-1 of different clades. Further analysis showed that ADAR1 inhibited viral protein synthesis without any effect on viral RNA synthesis. Mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the rev RNA, in the region of RNA encoding for Rev Response Element (RRE) binding domain and in env RNA. These mutations inhibited the binding of rev to the RRE and inhibited transport of primary transcripts like gag, pol and env from nucleus to cytoplasm resulting in inhibition of viral protein synthesis without any effect on viral RNA synthesis. Furthermore, ADAR1 induced mutations in the env gene inhibited viral infectivity.

HIV-1 viruses detected during episodic blips following interleukin-7 administration are similar to the viruses present before and after interleukin-7 therapy.

BACKGROUND: administration of recombinant human interleukin (IL)-7 leads to CD4 and CD8 T-cell expansions in HIV-infected individuals, demonstrating promising capacity for immune reconstitution. However, a proportion of patients treated with recombinant human IL-7 experience transient increases in plasma HIV-RNA ('blips'), possibly reflecting 'purging' of a quiescent reservoir that provides a barrier to viral eradication. OBJECTIVE: to identify the sources of HIV detected during transient viremic episodes following IL-7 administration, viral quasispecies were analyzed in a total of 281 primary sequences derived from seven patients who experienced the episodic blips following IL-7 therapy. METHOD: the C2-V3 regions of the HIV-1 env gene were sequenced from HIV-1 RNA in plasma and HIV DNA from peripheral blood mononuclear cells (PBMCs) obtained at baseline (day 0 of recombinant human IL-7 therapy), during the episode of viral blips (day 4), and at a time when levels of plasma HIV-RNA had returned to less than 50 copies/ml (day 28). RESULTS: the HIV sequences detected during transient viremia following IL-7 administration were closely related to those of the plasma viruses present before and after cytokine administration. All virus quasispecies detected during blips were also present in proviral sequences in PBMCs. CONCLUSION: the low level viremia induced by IL-7 likely reflects predominantly transient induction or release of virus from a preexisting pool rather than activation of silent quasispecies.

HIV type 1 subtype diversity and drug resistance among HIV type 1-infected Kenyan patients initiating antiretroviral therapy.

The treatment of HIV-1 infection with antiretroviral drugs has greatly improved the survival of those who are infected. However, HIV-1 diversity and drug resistance are major challenges in patient management, especially in resource-poor countries. To evaluate HIV-1 genetic diversity and drug resistance-associated mutations among drug-naive patients in Kenya prior to antiretroviral therapy (ART), a genetic analysis of HIV-1 pol-RT and env-gp41 was performed on samples collected from 53 (18 males and 35 females) consenting patients between April and June 2005. The average age, baseline CD4(+) T cell counts, and viral loads were 38 (range, 24-62) years, 475 (range, 203-799) cells/mm(3), and 4.7 (range, 3.4-5.9) log(10) copies/ml, respectively. Phylogenetic analysis revealed that 40 samples (75.5%) were concordant subtypes for the two genes and 13 (24.5%) were discordant, suggesting possible recombination and/or dual infections. Prevalent subtypes included A1/A1(pol-RT/env-gp41), 31 (58.5%); D/D, 9 (16.9%); A1/C, 2 (3.8%); A1/D, 4 (7.5%); G/A1, 2 (3.8%); A1/A2, 1 (1.9%); C/A1, 2 (3.8%); D/A1, 1(1.9%); and D/A2, 1 (1.9%). Major reverse transcriptase inhibitor (RTI) resistance-associated mutations were found in four patients (7.5%). Of these patients, three had nucleoside RTI resistance mutations, such as M184V, K65R, D67N, K70R, and K219Q. Nonnucleoside RTI resistance-associated mutations K103N and Y181C were detected in three patients and one patient, respectively. Multiple drug resistance mutations were observed in this drug-naive population. With increasing numbers of patients that require treatment and the rapid upscaling of ART in Kenya, HIV-1 drug resistance testing is recommended before starting treatment in order to achieve better clinical outcomes.

Reappearance of an 11-year-old sequence in an HIV-1 infected patient during treatment interruption.

HIV-1 from a patient with multi-drug resistant virus was identified as wild type during treatment interruption. The aim of the study was to describe how the viral population is affected by treatment interruptions and use phylogeny to reconstruct the evolutionary pattern. 15 samples covering 13 y and 2 treatment interruptions were analysed in both pol and env. The wild type virus found in the sample from the second treatment interruption in 2002 had not been present as a dominant population since 1994. Phylogeny showed that the 2002 sample was more closely related to wild type sequences than to other sequences sampled in 2002. This indicated that the wild type virus was caused by recruitment from the viral archives rather than reversion of previously circulating resistant strains. A few weeks after re-initiated treatment, virus showed full resistance, indicating that resistant virus was present as a subpopulation and reselected due to higher fitness in the presence of drugs. Phylogeny of env showed that CCR5 and CXCR4 viruses coexist in the patient. In conclusion, the study showed that at all times during infection, virus is archived in the cells and can be recruited when the surrounding environment changes and the archived virus is more fit.

Drug resistance and viral evolution in plasma and peripheral blood cells during structured treatment interruption (STI) and non-interrupted HAART.

BACKGROUND: Highly active antiretroviral therapy (HAART) can successfully reduce plasma and tissue levels of HIV-1 RNA and results in reductions in HIV-related morbidity and mortality, but the slow viral evolution during therapy in cellular reservoirs is a continuing problem. In addition, little remains known how viral evolutionary process may differ between cell-free and cell-associated compartments, over time, in vivo in patients receiving HAART or STI. OBJECTIVES: The main objectives of this study were to assess viral replication kinetics, drug resistance and viral evolution during HAART and STI. STUDY DESIGN: We have conducted a longitudinal study of virus culture kinetics in vitro, molecular analysis of uncultured HIV-1 variants from plasma and PBMC of 6 patients on HAART, 4 patients on STI, and 6 from treatment-naïve patients. RESULTS: Our data suggest that drug resistance mutations remained compartmentalized between plasma and PBMC. The divergent distribution of resistance mutations between plasma and PBMC coincided with divergent env gene evolution in these compartments. In contrast, the HIV strains from therapy-naive patients showed tight genetic and phylogenetic concordance between plasma and PBMC. Both STI and non-STI groups showed the presence of resistance mutations to both RT and protease inhibitors, which correlated with inadequate suppression of viremia and partially with the virus culture isolation in vitro. CONCLUSIONS: Overall, STI for HIV patients has no added advantage over regular HAART at the virologic level and in the diminution of resistance mutations that result in therapy failure. Under both forms of anti-retroviral therapies, virus could be isolated in vitro from the PBMC showing continuing low-level viral replication under suppressive therapy. Overall, these data may be useful in predicting the late emergence of drug resistance mutations via the latent integrated provirus.

Evaluation of mismatch-binding ligands as inhibitors for Rev-RRE interaction.

Drugs targeting the stem-loop IIB of Rev responsible element (RRE) of HIV-1 mRNA are potential therapeutic agents for HIV-1 infection. The stem loop is characterized by an internal loop consist of consecutive G-G and G-A mismatches, which is the single binding site for Rev protein for nuclear export of viral mRNA. We report here that ligands binding to G-G and G-A mismatches in duplex DNA also bind to the internal loop in competition with Rev peptide and lead to the dissociation of pre-formed Rev-RRE complex in a model system.

env chimeric virus technology for evaluating human immunodeficiency virus susceptibility to entry inhibitors.

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120, gp41, and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both gp120 and gp41. Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of gp120 of NL4.3/AMD3100 and gp41 of NL4.3/T20 or recombination of the gp160 genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in gp120 alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for gp41 in relation to NL4.3/T20. In conclusion, we demonstrate the use of env CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that env CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.

Rapid, transient changes at the env locus of plasma human immunodeficiency virus type 1 populations during the emergence of protease inhibitor resistance.

Plasma human immunodeficiency virus type 1 (HIV-1) populations were genetically analyzed at their most variable locus, the envelope gene, during the rapid emergence of resistance to protease inhibitor monotherapy. Plasma virus populations remained genetically constant prior to drug treatment and during the 1 to 2 weeks following initiation of therapy, while viremia fell 10- to 100-fold. Concomitant with rapid plasma viremia rebounds associated with the emergence of drug-resistant virus, marked alterations were then detected at the env locus. Plasma population changes lasted only a few weeks before the reappearance of the pretreatment envelope variants. The emergence of resistance to single protease inhibitors was therefore associated with major but transient changes at a nonselected locus. Selection for resistance to single protease inhibitors thus appears to be more complex than the continued replication of a large, random, and therefore genetically representative sampling of the pretreatment plasma population. The possibility that drug-privileged anatomical sites containing distinct envelope variants and/or a small effective HIV-1 population size account for these results is discussed.

Targeted site-specific cleavage of HIV-1 viral Rev responsive element by copper aminoglycosides.

Site-specific cleavage of the HIV-1 viral Rev responsive element by copper aminoglycosides is reported under physiological conditions. This bubble and stem-loop RNA structure is efficiently targeted at micromolar concentrations of complex. The specificity of cleavage of structured viral RNA relative to a non-cognate tRNAPhe of well-defined secondary and tertiary structure is demonstrated. Cleavage products from simpler substrates [diribonucleotide (ApA) and 2',3'-cyclic monophosphate ester (cAMP)] were analyzed by 31P NMR and demonstrate a hydrolytic mechanism in the absence of external redox agents. These results demonstrate copper aminoglycosides to be highly efficient chemical nucleases with a targeting capability for viral RNA and suggest a novel methodology to counter RNA viruses.

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.

Inhibition of the HIV-1 rev-RRE complex formation by unfused aromatic cations.

RNA viruses cause a wide range of human diseases. Development of new agents to target such viruses is an active area of research. Towards this goal, a series of diphenylfuran cations as potential inhibitors of the Rev-RRE complex have been designed and synthesized. Analysis of the interaction of the diphenylfurans with RRE and TAR RNA model systems by gel shift assays indicates that they exhibit both sequence and structure-dependent binding modes. Our results show a strong interaction between the diphenylfuran ring system and RRE bases, while the TAR interactions are much weaker with the compounds that are the best inhibitors of Rev-RRE.

Modulation of the Rev-RRE interaction by aromatic heterocyclic compounds.

The HIV-1 Rev protein regulates the nucleocytoplasmic distribution of viral precursor RNAs that encode HIV-1 structural proteins. Rev-mediated viral RNA expression requires a sequence-specific interaction between Rev and a viral RNA sequence, the Rev responsive element (RRE). Because the Rev-RRE interaction is essential for HIV-1 replication, anti-viral agents that selectively block this interaction may be effective anti-HIV-1 therapeutics. Here, we show that certain aromatic heterocyclic compounds, in particular, a tetracationic diphenylfuran, AK.A, can block binding of Rev to its high-affinity viral RNA binding site. AK.A abolishes Rev-RRE interactions at concentrations as low as 0.1 microM. Inhibition appears to be selective and results from competitive binding of the drug to a discrete region within the Rev binding site. Interestingly, the molecular basis for the AK.A-RNA interaction, as well as the mode of RNA binding differs from previously described aminoglycoside Rev inhibitors. Analysis of a variety of aromatic heterocyclic compounds and their derivatives reveals stereo-specific features required for the inhibition. Our results further demonstrate the feasibility of identifying and designing small molecules that selectively block viral RNA-protein interactions.

Eilatin Ru(II) complexes display anti-HIV activity and enantiomeric diversity in the binding of RNA.

Eilatin-containing octahedral ruthenium complexes inhibit HIV-1 replication in CD4+ HeLa cells and in human peripheral blood monocytes with IC(50) values of approximately 1 microM. Similar metal complexes that lack eilatin display 15-100-fold lower anti-HIV activities. [Ru(bpy)(2)"pre-eilatin"](2+), a complex that contains a nonplanar analogue of eilatin, shows significantly lower nucleic acid binding and lower anti-HIV activity than eilatin complexes. This result indicates that the extended planar surface presented by eilatin is important for both activities. Rev peptide and ethidium bromide displacement assays are used to probe the nucleic acid affinity and specificity of Lambda- and Delta-[Ru(bpy)(2)eilatin](2+). Two HIV-1 RNA sites are compared and a significant binding preference for the Rev response element over the transactivation response region is found. Simple DNA duplexes show a consistent selectivity for Lambda-[Ru(bpy)(2)eilatin](2+) compared to Delta-[Ru(bpy)(2)eilatin](2+), while RNAs show more diverse enantiomeric selectivities.

Facilitation of HIV-1 isolation from patients by neuraminidase.

We previously reported that infectivities of human and other non-human lentiviruses including human immunodeficiency virus type 1 (HIV-1) are activated by desialylation of the virion surface [H. Hu et al., J Virol 70: 7,462-7,470 (1996)]. The present study was designed to determine whether neuraminidase (NA) is useful for isolation of HIV-1 from patients. Peripheral blood mononuclear cells (PBMC) or CD4+ cells isolated from the PBMC of infected individuals were cocultured with PBMC or CD4+ cells from uninfected healthy donors, and the efficiency and frequency of virus isolation in the presence of NA were compared with those by a routine conventional procedure. In a total of 41 isolation trials from 28 individuals, the presence of NA markedly increased the frequency of isolation. Furthermore, both the day when virus was first detected and the day when the virus titer was the highest in the cultures were significantly earlier in the presence of NA than in the absence of NA. The deduced amino acid sequences of the V2 and V3 regions of gp120 were identical or very similar between the isolates obtained in the presence or absence of NA, suggesting that both isolation procedures selected a similar population. NA was thus found to facilitate HIV-1 isolation and its use is recommended particularly when isolation is negative by the conventional procedures.