Alexey A. Matskevich

RNase H-mediated retrovirus destruction in vivo triggered by oligodeoxynucleotides

The HIV-1 RNase H can be prematurely activated by oligodeoxynucleotides targeting the highly conserved polypurine tract required for second strand DNA synthesis. This inhibits retroviral replication in cell-free HIV particles and newly infected cells. Here we extend these studies to an in vivo model of retroviral replication. Mice that are chronically infected with the spleen focus-forming virus and treated with oligodeoxynucleotides that target the polypurine tract, exhibit either transient or long-term reductions in plasma virus titer, depending on the therapeutic regimen. Treatment prior to, during or shortly after infection can delay disease progression, increase survival rates and prevent viral infection. This strategy destroys viral RNA template in virus particles in serum as well as early retroviral replication intermediates in infected cells. As it targets events common to the replication cycle of all retroviruses, this approach may be broadly applicable to retroviruses of medical and agricultural importance.

Stimuli-dependent cleavage of Dicer during apoptosis.

miRNAs (microRNAs) play important roles in diverse physiological processes, including stress response, apoptosis and carcinogenesis. Even though the role of individual miRNAs has been demonstrated, expression of proteins involved in miRNA production in response to acute stress or harmful agents has not been extensively investigated. Here, we have studied the role of Dicer, one of the central proteins of the miRNA processing machinery during apoptosis, and show that down-regulation of Dicer results in accelerated apoptosis of HeLa cells, triggered by TNFalpha (tumour necrosis factor alpha). We have also investigated the integrity of Dicer, and provide evidence that Dicer is a target for caspases during apoptosis. The cleavage of Dicer is stimulidependent and more pronounced when apoptosis is induced by PKC (protein kinase C) inhibitors, and can also be observed in HIV-1-infected cells at late stages of infection. Thus the apoptotic machinery may regulate the miRNA pathway by affecting individual proteins, such as Dicer.

Silencing of HIV by hairpin‐loop‐structured DNA oligonucleotide

We describe inhibition of HIV replication by a partially double‐stranded 54mer oligodeoxynucleotide, ODN, which consists of an antisense strand targeting the highly conserved polypurine tract, PPT, of HIV, and a second strand, compatible with triple‐helix formation. Upon treatment of HIV‐infected cells with ODN early after infection no viral nucleic acids, syncytia or p24 viral antigen expression was observed. The ODN‐mediated effect was highly sequence‐specific. The ODN against HIV‐IIIB was effective preferentially against its homologous PPT and less against the PPT of HIV‐BaL differing in two of 24 nucleotides and vice versa. It may be interesting mechanistically as an antiviral drug.

Oligonucleotide-mediated retroviral Rnase H activation leads to reduced Hiv-1 titer in patient-derived plasma

Background:The retroviral RNase H is essential for viral replication. This component has not yet been extensively studied for antiviral therapy. It can be activated by an oligodeoxynucleotide (ODN) resulting in self-destruction of the virions. Objective:To examine antiviral potential of ODN in clinical samples using plasma of HIV-1-infected patients. Design:Plasma of 19 HIV-1-infected patients from Zurich and 10 HIV-1 isolates from Africa and drug-resistant strains were processed for ex-vivo treatment. Methods:Cell-free virions were treated with ODN in the plasma and HIV RNA was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, infectivity of the treated virions was tested on primary human peripheral blood mononuclear cells. Results:Cell-free virions in plasma contained significantly less intact HIV RNA upon treatment with ODN (P = 0.0004), and their infectivity was decreased 52-fold (P = 0.0004). In 39% of the Zurich samples, infectivity was reduced more than 10-fold, in 33% more than 100-fold, and in 28% more than 1000-fold. Also, the isolates from Africa exhibited a 63-fold reduction in infectivity (P = 0.0069) with 80% of the isolates responding more than 10-fold, 40% more than 100-fold, and 10% more than 1000-fold. Conclusion:Significant reduction of plasma HIV RNA levels and infectivity of treated virions was achieved on the basis of induced self-destruction of HIV observed with clinical samples. Reduction of viral load ex vivo was designed as model for potential effects in vivo. Premature activation rather than inhibition of a viral enzyme could be a model strategy for future antiretroviral control.

Vero cells as a model to study the effects of adenoviral gene delivery vectors on the RNAi system in context of viral infection.

Technology based on RNA interference (RNAi) is a promising source for new antiviral therapies. Although the application of RNAi has been studied extensively, significant problems with using RNAi remain. Very few studies have specifically assessed model systems for testing the effects of viruses or gene delivery vectors on the RNAi system. Since viruses have developed efficient strategies to circumvent the interferon (IFN) response, an IFN-deficient model system should be considered. Here we show that in Vero cells, which lack IFN-α and IFN-β genes, knockdown of Dicer, a key RNAi component, led to accelerated death of cells infected with other evolutionary distinct viruses: influenza A virus, vesicular stomatitis virus and poliovirus. We also demonstrate that transduction of Vero cells with adenoviral vector with subsequent infection with influenza A virus also resulted in increased mortality of infected cells. These effects were much weaker in IFN-producing A549 and Hela cell lines. Thus, the Vero cell line could serve as an interesting model for studying the effects of gene delivery vectors on the RNAi system in the context of virus-related disorders.

Silencing of viral RNAs by small double-stranded siDNA

Background We are developing an alternative approach to siRNA, which may be designated as siDNA, small interfering DNA, by using hairpin-loop-structured DNA oligodeoxynucleotides (ODN), targeted to viral or cellular mRNAs. ODNs activate the viral RNase H in retroviral particles and cellular RNases H inside the cell. Also Ago2 may play a role. Other inhibitory mechanisms such as translational arrest may contribute.

Silencing of HIV RNA by a Hairpin-loop DNA

We describe an RNA silencing, which inhibits HIV replication by a hairpin-loop DNA. A partially double-stranded 54mer DNA oligonucleotide (ODN) was targeted to the polypurine tract, PPT of HIV. It inhibits virus replication. We demonstrate that it prevents steps before DNA provirus formation. The effect of the ODN on HIV replication in cell culture is highly sequence-specific and sensitive to changes in length and single mismatches on either strand of the DNA. An ODN against HIV-IIIB was ineffective against HIV-Ba-L and vice versa, whereby their PPTs differ by two of 24 nucleotides. Thus, the structure and sequence of both strands of the ODN are important in cellular assays. In vitro the ODN leads to an RNA-DNA hybrid formation at the PPT, a structure which is cleaved by the RT/ RNase H in permeabilized virus particles. The hybrid at the PPT is preferentially recognized by the RT/RNase H for initiation of the second-strand DNA synthesis. This recognition is its normal biological function and shown here with the ODN. A cell extract containing cellular RNase H activities or RISC proteins, is unable to induce such a cleavage. In summary, the ODN mimicks a real step in viral replication, whereby the viral RNA is cleaved prematurely before DNA transcription is completed. The mechanism is reminiscent of RNA silencing by siRNA and supported by its relationship with RNaseH. The ODN may be a basis for drug design, because of low tendency for escape mutations. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005