BackgroundCurrent therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis.ResultsCurrent HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies.ConclusionsABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0159-3) contains supplementary material, which is available to authorized users.
These data demonstrate the great potential of adenovirus dodecahedron in combination with WW domains as a protein transduction vector.
Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.Human adenoviruses (Ads) are nonenveloped viruses responsible for respiratory, ocular, and enteric infections. Their icosahedral capsid, containing the 36-kpb double-stranded DNA genome, is composed of three major proteins: the hexon, the penton (Pt) base, and the fiber. At the 12 vertices of the capsid, the protruding fiber is noncovalently attached to the Pt base, thus forming the Pt complex. It has been reported that the fiber interacts with a high affinity with a primary receptor and that the subsequent interaction of the Pt base RGD motif with cellular integrins triggers endocytosis (17). A 42-kDa protein, called the coxsackievirus and Ad receptor (CAR), is recognized by at least one serotype of each of the six subgroups of Ad except for the serotypes belonging to subgroup B (i.e., Ad3) (1, 12). Even though that Ad capsid is composed of at least 11 proteins, it has been shown that Pt alone can penetrate into human cell lines, thus making of this complex a potential vector for DNA delivery (2,5,7,8,16). Remarkably, Ad3 but not Ad5 Pt expressed in the baculovirus system led to the formation of symmetric complexes of 12 Pts called dodecahedron (Dd). We have previously shown that the non-CARbinding Ad3-Dd can be used as a gene transfer vector (2), and we show here that this virus-like particle is also able to transduce large multimeric proteins into human cells.Dd internalization. Coexpression of Ad3 Pt base and fiber proteins in the baculovirus system led to the formation of a symmetric dodecameric particle (2, 14), Pt-Dd (Fig. 1a). This complex results from the interaction between the pentameric base proteins, as attested by the Dd formation upon expression of base proteins alone (Bs-Dd; Fig. 1a). The respective roles of the fiber and the Pt base protein in Ad3 Dd entry into HeLa cells was assessed by immunofluorescence. HeLa cells grown in a 96-multiwell plate (Falcon) at 2 ϫ 10 4 cells per well were incubated in 100 l of phosphate-buffered saline (PBS) with a range of Dd concentrations. After 1 h at 37°C, cells were * Corresponding author. Mailing address: Institut de Biologie Structurale, 41 Rue Jules Horowitz, 38027 Grenoble,. E-mail: fender@ibs.fr.
ABX464 is a first-in-class, clinical-stage, small molecule for oral administration that has shown strong anti-inflammatory effects in the DSS-model for inflammatory bowel disease (IBD) and also prevents replication of the HIV virus. ABX464 which binds to cap binding complex (CBC) has demonstrated safety and efficacy in a phase 2a proof-of-concept clinical trial in patients with Ulcerative colitis. Previously, with limited technologies, it was not possible to quantify the effect of ABX464 on viral and cellular RNA biogenesis. Here, using RNA CaptureSeq and deep sequencing, we report that ABX464 enhances the splicing of HIV RNA in infected PBMCs from six healthy individuals and also the expression and splicing of a single long noncoding RNA to generate the anti-inflammatory miR-124 both ex vivo and in HIV patients. While ABX464 has no effect on pre-mRNA splicing of cellular genes, depletion of CBC complex by RNAi leads to accumulation of intron retention transcripts. These results imply that ABX464 did not inhibit the function of CBC in splicing but rather strengthens it under pathological condition like inflammation and HIV infection. The specific dual ability of ABX464 to generate both anti-inflammatory miR-124 and spliced viral RNA may have applicability for the treatment of both inflammatory diseases and HIV infection.
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