A humanized mouse model for HIV-2 infection and efficacy testing of a single-pill triple-drug combination anti-retroviral therapy.

While HIV-2 is a causative agent for AIDS in addition to the better studied HIV-1, there is currently no suitable animal model for experimental studies for HIV-2 infection and evaluating promising drugs in vivo. Here we evaluated humanized mice for their susceptibility to HIV-2 infection and tested a single-pill three drug formulation of anti-retrovirals (NRTIs abacavir and lamivudine, integrase inhibitor dolutegravir) (trade name, TriumeqR). Our results showed that hu-mice are susceptible to HIV-2 infection showing persistent viremia and CD4 T cell loss, key hallmarks of AIDS pathogenesis. Oral drug treatment led to full viral suppression and protection from CD4 T cell depletion. Cessation of therapy resulted in viral rebound and CD4 T cell loss. These proof-of-concept studies establish the utility of hu-mice for evaluating HIV-2 pathogenesis in more detail in the future, testing novel therapies and providing pre-clinical efficacy data of a three drug combination to treat HIV-2 infections.

Heat shock protein 70 inhibits HIV-1 Vif-mediated ubiquitination and degradation of APOBEC3G.

The cytidine deaminase APOBEC3G, which is incorporated into nascent virus particles, possesses potent antiviral activity and restricts Vif-deficient HIV-1 replication at the reverse transcription step through deamination-dependent and -independent effects. HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inducing APOBEC3G polyubiquitination and its subsequent proteasomal degradation. In this study, we show that overexpression of heat shock protein 70 (HSP70) blocked the degradation of APOBEC3G in the ubiquitin-proteasome pathway by HIV-1 Vif, rendering the viral particles non-infectious. In addition, siRNA targeted knock-down of HSP70 expression enhanced the Vif-mediated degradation of APOBEC3G. A co-immunoprecipitation study revealed that overexpression of HSP70 inhibited APOBEC3G binding to HIV-1 Vif. Thus, we provide evidence for a host protein-mediated suppression of HIV-1 replication in an APOBEC3G-dependent manner.

HIV-1 RT-dependent DNAzyme expression inhibits HIV-1 replication without the emergence of escape viruses.

DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tR(M)DtR(L), or one of two truncated tRNAs (Lys-3), tR(M)DΔARMtR(L) or tR(M)D3'-endtR(L). Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations.

Oral pre-exposure prophylaxis by anti-retrovirals raltegravir and maraviroc protects against HIV-1 vaginal transmission in a humanized mouse model.

Sexual HIV-1 transmission by vaginal route is the most predominant mode of viral transmission, resulting in millions of new infections every year. In the absence of an effective vaccine, there is an urgent need to develop other alternative methods of pre-exposure prophylaxis (PrEP). Many novel drugs that are currently approved for clinical use also show great potential to prevent viral sexual transmission when administered systemically. A small animal model that permits rapid preclinical evaluation of potential candidates for their systemic PrEP efficacy will greatly enhance progress in this area of investigation. We have previously shown that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and displays CD4 T cell loss typical to that seen in the human. Thus far systemic PrEP studies have been primarily limited to RT inhibitors exemplified by tenofovir and emtricitabine. In these proof-of-concept studies we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically administered chemo-prophylactics. Our results showed that oral administration of either of these drugs fully protects against vaginal HIV-1 challenge in the RAG-hu mouse model. Based on these results both these drugs show great promise for further development as orally administered PrEPs.

RNA interference targeted to the conserved dimerization initiation site (DIS) of HIV-1 restricts virus escape mutation.

Short hairpin RNAs (shRNA) targeting viral or cellular genes can effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication. This inhibition, however, may induce mutations in the targeted gene, leading to rapid escape from the shRNA-induced inhibition. We generated a lymphoid cell line that stably expressed a 19-bp shRNA targeting a well-conserved dimerization initiation site (DIS) of HIV-1, which strongly inhibited viral replication, thereby delaying virus escape. Furthermore, treatment of HIV-1 infection with DIS- and vif-shRNA combination therapy resulted in superior anti-viral responses compared to vif-shRNA monotherapy. Continuous challenge with HIV-1, however, generated virus mutants that could overcome the RNA interference restriction. Such anti-genes may be promising tools for HIV-1 gene therapy for HIV/acquired immunodeficiency syndrome.

Inhibition of HIV-1 replication by long-term treatment with a chimeric RNA containing shRNA and TAR decoy RNA.

Combinatorial therapies for the treatment of HIV-1 infection are effective for reducing patient viral loads and slowing the progression to AIDS. Our strategy was based on an anti-HIV-1 shRNA vector system in which HIV-1 vif-shRNA was fused to a decoy TAR RNA (mini-TAR RNA) to generate vif-shRNA-decoy TAR RNA under the control of the human U6 Pol III promoter. Upon expression in human cells, the RNA molecule was cleaved into its component parts, which inhibited HIV-1 replication in a synergistic manner. This chimeric RNA expressed a dual RNA moiety and greatly enhanced the inhibition of HIV-1 replication under the production of resistant virus by short interference RNA (siRNA) in long-term culture assays. We suggest that this technique provides a practical basis for the application of siRNA-based gene therapy in the treatment of HIV/AIDS.

Micro RNA like inhibition of HIV-1 replication.

In this study, we investigated an RNA (R-Psi-sgRNA) that suppresses HIV-1 replication. This RNA is expressed by a plasmid vector (pR-Psi-sgRNA-ter) that was constructed accidentally. To examine if this effect is caused by RNA interference, R-Psi-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important RNase III enzyme for RNA interference. The RNA was cleaved into fragments of approximately 20 nucleotides. We then performed an HIV-1 p24 assay with the RNA fragments to evaluate their effect on HIV-1 replication. HIV-1 replication was suppressed.

Suppression of hepatitis C virus replication by baculovirus vector-mediated short-hairpin RNA expression.

Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. Here, we report on the inhibition, by baculovirus-based vector-derived shRNAs, of core-protein expression in full-length hepatitis C virus (HCV) replicon cells. shRNAs were designed to target the highly conserved core region of the HCV genome. In particular, the core-shRNA452 containing nucleotides 452-472, as the target in the HCV core gene, dramatically inhibited the expression of the HCV core protein in replicon cells. Furthermore, HCV core-protein expression was inhibited more strongly by the vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped baculovirus vector than by the wild-type baculovirus vector.

Non-sequence specific RNA inhibition of HIV-1 replication.

In this study, we investigated an RNA (R-Psi-sgRNA) that suppresses HIV-1 replication. This RNA is expressed by a plasmid vector (pR-Psi-sgRNA-ter) that was constructed accidentally. To examine if this effect is caused by RNA interference, R-Psi-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important RNase III enzyme for RNA interference. The RNA was cleaved into fragments of approximately 20 nucleotides. We then performed an HIV-1 p24 assay with the RNA fragments to evaluate their effect on HIV-1 replication. HIV-1 replication was suppressed. We are now analyzing the sequences of the RNA fragments.

Suppression of HIV-1 replication by a combination of endonucleolytic ribozymes (RNase P and tRNnase ZL).

We examined the combinatorial action of RNase P and tRNase ZL-mediated specific inhibition of HIV-1 in cultured cells. We designed two short extra guide sequences (sEGS) that specifically recognize the tat and vifregions of HIV-1 mRNA and mediate the subsequent cleavage of hybridized mRNA by the RNase P and tRNase ZL components. We constructed an RNase P and tRNase ZL-associated vif and tat sEGS expression vector; which used the RNA-polymerase III dependent U6 promoter, as an expression cassette for EGS. Together, the RNase P and tRNase ZL-associated sEGS molecules allow more efficient suppression of HIV-1 mRNA production when separately applied. The possibilities offered by the vector to encode sEGS will provide a powerful tool for gene therapy.

Long-term transgene expression and inhibition of HIV-1 replication by a Cre/loxP-EBNA-1/oriP HIV-1-dependent ribozyme vector: Applications for HIV-1 gene therapy.

The cleavage of target mRNA by ribozymes is being exploited as a means of gene silencing in nucleic-acid-based therapies. We previously established an HIV-1-dependent ribozyme-expression vector system, based on Cre-loxP technology with an LTR-gag-p17 promoter as a molecular switch for use in acute HIV-1 infection. The simultaneous expression of the Cre protein and loxP homologous recombination induced a high level of HIV-1-replication inhibition, but ribozyme expression was transient. In the current study, we overcame this limitation by inserting EBNA-1 and oriP genes from the Epstein-Barr virus (EBV) into the vector. When this plasmid was introduced into HeLa CD4(+) cells, we observed long-term expression of both the EGFP reporter gene and the ribozyme. Moreover, HIV-1 replication was inhibited in the long-term in transfected cells. These data suggest that the HIV-1-dependent ribozyme-expression vector containing EBNA-1/oriP sequences would be a useful tool in HIV-1 gene therapy applications.

Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL.

The tRNA 3'-processing endoribonuclease (tRNase Z or 3' tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3' trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.

Suppression of human immunodeficiency virus type 1 (HIV-1) replication by an HIV-1-dependent double locked vector with the Cre/loxP system.

We previously demonstrated the function of an HIV-1-dependent ribozyme expression vector, with which the site-specific excision of loxP sequences can be achieved by using the Cre-loxP system (ON/OFF) as a molecular switch in an acute HIV-1 infection. However, this expression system also revealed the lower, non-specific expression of the anti-H1V-1 ribozyme in the absence of tat. To circumvent this problem, we used the more efficient HIV-1-dependent Cre recombinase gene expression vector, encoding the LTR-gag-p17 (extending from the 5'-LTR to the middle of the gag gene (pLTR-gag-p17-Cre)). Comparatively, the pLTR-gag-p17-Cre induces a higher Cre-protein expression level in an HIV-1 infection-dependent manner than the minimal pLTR-Cre. Furthermore, we constructed the ploxP-Rz-U5 and pLTR-gag-p17-Cre plasmids and also combined them into a single vector, pLTR-gag-p17-Cre/loxP-Rz-U5, for a comparison of their anti-HIV-1 activities. The resultant simultaneous expression of the Cre protein and the homologous recombination of the two loxP sequences induced a high level of HIV-1 replication inhibition (95%). Significantly, a high steady-state of ribozyme expression was observed in the RT-PCR analysis. These data imply that targeting the HIV-1 genes with the pLTR-gag-p17-Cre/loxP-Rz-U5 vector, which mediates HIV-1-dependent ribozyme expression, would be a useful tool for HIV-1 gene therapy applications.

Effective inhibition of HIV-1 replication in cultured cells by external guide sequences and ribonuclease P.

We examined the suppressive effect of HIV-1 RNA gene cleavage on HIV-1 expression, using the catalytic RNA subunit RNase P and the 3'-half tRNA(Try) [external guide sequence (EGS)] in cultured cells. HIV-1 expression was inhibited by the tRNA(met)-EGS-U5 and U6-EGS-U5 from the tRNA(met) and U6 promoters, respectively. There was no difference in the inhibitory effects on HIV-1 expression between the tRNA(met) and U6 promoters.

A new modified DNA enzyme that targets influenza virus A mRNA inhibits viral infection in cultured cells.

DNA enzymes are RNA-cleaving single-stranded DNA molecules. We designed DNA enzymes targeting the PB2 mRNA translation initiation (AUG) region of the influenza A virus (A/PR/8/34). The modified DNA enzymes have one or two N3'-P5' phosphoramidate bonds at both the 3'- and 5'-termini of the oligonucleotides, which significantly enhanced their nuclease resistance. These modified DNA enzymes had the same cleavage activity as the unmodified DNA enzymes, determined by kinetic analyses, and reduced influenza A virus replication by more than 99%, determined by plaque formation. These DNA enzymes are highly specific; their protective effect was not observed in influenza B virus (B/Ibaraki)-infected Madin-Darby canine kidney cells.

Development of HIV-1 dependent gene expression vector with the Cre/loxP (ON) system.

We used the HIV-1 5'LTR and the Cre/loxP system to develop an anti-gene expression system. The LTR promoter of HIV-1 has a specific activity that includes the intermediary region of gag, as shown in a previous report. We constructed the U5-region of HIV-1 as the target of the ribozyme expression vector (pCre/loxP-Rz), with the Cre/loxP system under the control of this LTR promoter. The function of this vector is to induce the HIV-1 dependent ribozyme-mediated inhibition in a dose-responsive manner. Furthermore, ribozyme mRNA expression was detected in the presence of pNL4-3 in HeLa-CD4+ cells. These studies are expected to yield novel applications of antiviral strategies for HIV-1.

Inhibition of HIV-1 replication by an HIV-1 dependent ribozyme expression vector with the Cre/loxP (ON/OFF) system.

Antiviral strategies to inhibit HIV-1 replication have included the generation of gene products that provide the intracellular inhibition of an essential viral protein or RNA. When used in conjunction with the HIV-1 long terminal repeat (LTR), an inducible promoter dependent on the virus-encoded trans-activator (tat), relatively high background activity is still observed in the absence of tat (Caruso & Klatzmann, 1992; Dinges et al., 1995). In order to circumvent this problem, we used the Cre/loxP (ON/OFF) recombination system as a tool for our investigation. In the present study, we constructed a loxP-cassette vector with the ribozyme (Rz) expression portion under the control of the tRNAi(Met) promoter between two loxP sequences (plox-Rz-U5). We also constructed an HIV-1 LTR promoter-driven Cre recombinase gene (pLTR-Cre). These vectors were triple-transfected into HeLa CD4 cells with the HIV-1 pseudotype viral expression vector. Basal activity was not detectable before HIV-1 infection. The LTR-dependent Cre protein product in HIV-1 infected HeLa CD4 cells expressed the ribozyme by inducing loxP homologous recombination, which strongly inhibited the HIV-1 gene expression. These results demonstrate the potential of an anti-ribozyme with the Cre/loxP system for controlling HIV-1 infection via gene therapy.