Abstracts

Evolution of Viruses and Antiviral Defence

K. Moelling

University of Zurich, Switzerland, and Institute of Advanced Study, Berlin

Contemporary viruses can be organized in an evolutionay tree ranging from the RNA world to the DNA world, from ribozymes, via viroids, DNA-ribozymes, Influenza, retro-and para-retroviruses to DNA viruses – supporting a „virus-first“ hypothesis. Retroviruses have shaped or may have even built the human genome, where up to 50% are retrovirus-related sequences to which increasing and decreasing complexities contributed. Rudimentary reverse transcription from RNA to DNA is still ongoing today in teleomeres during embryognesis and cancer(1). Sequence analysis of the human genome witnesses our past, indicating how long HIV-like viruses, reverse transcriptase and RNAses H have been around. Endogenization of retroviruses is actively ongoing in animal models and may allow a prediction on the future of HIV in people. Evolution of HIV takes place during antiretroviral therapies. An HIV suicide approach circumvents mutagenesis and escape mutants(2,3). Co-evolution or crossing arms, also known from phage and bacteria, can be deduced from structural and functional similarities of retroviral replication and the siRNA-mediated antiviral defence machineries (4). An evolutionary relationship between siRNA and interferon can be constructed by comparing their pathways. SiRNA involving dicer as well as interferon are active antiviral defence mechanisms in mammalian cells, tested by dicer and interferon knock-down analyses (5;6). The systems differ in strength and sequence specificities.

Ref:(1) Noreen et al. Oligonucleotides (modify), (2) Matzen et al. Nat. Biotech. 25,669(2007), (3) Heinrich et al AIDS 23,213(2009), (4) Moelling et al. CSH Symp.Q.B. 71,365 (2006), (5;6) Matskevich and Moelling JGenVirol 88,2627 (2007) and BiochemJ 412,527(2008).

Silencing of HIV RNA by Activating viral RNase H

K. Moelling and J. Heinrich

University of Zurich, Gloriastr. 32, CH 8006 Zurich, Switzerland

IAC Wien

Background: We are characterizing the retroviral RT/RNase H and compared it to the PAZ/PIWI domains of the cellular Dicer complex involved in siRNA silencing. We showed similarities and demonstrated antiviral effects of Dicer in the absence of the Interferon system in virus-infected cells. Similarities between the retroviral RNase H and PIWI lead us to analyze RNA-DNA hybrid structures in comparison to double-stranded RNA as substrates.

Method: We used miRNAs as model to design a hairpin-loop DNA oligonucleotide (ODN) and investigated its effect on the virion-associated retroviral RNase H in cell-culture, animal studies and in patient-derived primary blood samples from Zurich and Africa and observed viral RNA silencing (“siDNA effects”).

Results: We are driving HIV into suicide by adding the ODN to the virus particles where it can enter, bind to the target RNA and destroy the RNA before DNA synthesis. Treatment of cell-free HIV particles, of newly HIV-infected cells – even with multi-drug-resistant HIV- or of mice, infected with the oncogenic Murine Spleen Focus Forming Virus, inhibited virus infection, replication and disease progression. It even prevented tumor formation. We are also using humanized SCID mice with ODN to test for inhibition of HIV replication. We reduced the viral infectivity by ODN-treatment of the blood of 30 primary isolates from patients by ex vivo treatment, in more than 30% by 1000-fold. Also the viral load in a mouse vagina model can be statistically significantly reduced.

Conclusion: Our approach may be applicable in humans to prevent HIV infection during sexual (as microbicide) or mother-to-child transmission or against multidrug-resistant HIV – and also other viruses (HSV etc).

Moelling et al., CSH Symp.71 (2006); Jendis et al., AIDS Res.Hum.Retroviruses (1996, 98); Moelling et al., FEBS Letters (2006); Matskevich et al., AIDS Res.Hum.Retroviruses (2006); Matzen et al., Nature Biotech (2007); Heinrich et al J AIDS (2008), Wittmer et al. Antiviral Res. 82,22 (2009)

Silencing of viral RNAs by RNases H and small double-stranded DNAs

K. Moelling, A. Matskevich, L. Elzaouk, S. Mathur, J. Heinrich, J.-S. Jung, and T. Kwok

University of Zurich, Gloriastr.32, CH 8006 Zurich, Switzerland

IUMS

We will discuss properties of Ribonucleases (RNases) H in gene silencing using short double-stranded dsDNA. Several RNases H exist, they are structurally highly conserved in many species and play a general role in removal of RNA primers required for DNA synthesis or replication. They are also related to the siRNA cleavage enzyme, Ago2, whereby PAZ and PIWI resemble RT and RNase H (1). We directly compared siRNA, antisense and dsDNA on several targets and observe differences in kinetics, cellular uptake, stabilities, sequence algorithms.

We will give examples with HIV and other viral targets.

The RNase H of HIV is an essential enzyme for retroviral replication. It is specific for cleavage of RNA in RNA-DNA hybrids and required for removal of the viral RNA genome during DNA synthesis. It is the only retroviral enzyme not yet targeted for by antiretroviral therapy. Treatment of newly infected cells with short dsDNAs inhibits HIV infection with laboratory strains as well as HIV patient isolates, including multi-drug-resistant strains (2,3). The effect is sequence-specific as tested by different HIV variants (4). HIV is a special case, because the cell-free virus particles contain the RT/RNase H. Therefore, in virus particles treated with DNA, the viral RNA is destroyed and infectivity reduced (4,5). Furthermore we established a mouse model for a retrovirus, the oncogenic Spleen Focus Forming Virus SFFV. Intravenous treatment of mice chronically infected with the SFFV, reduces the virus load in blood transiently or long-lasting, depending on the regimen of therapy. Early treatment of mice can reduce disease progression or even completely prevent infection (6). We also reduced the viral load in the blood of 30 primary patients’ isolates by ex vivo treatment, which reduced the viral infectivity in more than 30% by 1000fold (7). Also the viral load in a mouse vagina model can be statistically significantly reduced (submitted). It may be applicable to prevent HIV infection during sexual or mother to child transmission or against multidrug-resistant viruses.

Data with other viruses using siRNA, antisense and short dsDNA in a mouse model will be presented such as Influenza A (8).The role of various RNases H have been evaluated with a PPT-containing reporter coding for GFP, by knock-down of various RNases H (Kwok et al Manuscript subm).

(1) Moelling et al., CSH Symp.71 (2006); (2,3) Jendis et al., AIDS Res.Hum.Retroviruses (1996, 98); (4) Moelling et al., FEBS Letters (2006); (5) Matskevich et al., AIDS Res.Hum.Retroviruses (2006); (6) Matzen et al., Nature Biotech (2007) (7) Heinrich et al J AIDS (2008) (8)Kwok et al archives viology 154,109,2009, Kwok et al. Influenza in Biochim Biophys Acta 1709,1170(2009), Falkenhagen et al HSV in Virology J. 6,43(2009), Noreen et al on cancer in Oligonucleotides 19:2,169(2009), Hoffmann et al. cancer in Molecular Cancer Research (2009) Wittmer et al. vagina in Antiviral Res,82,22 (2009)

HIV suicide through viral RNase H

K. Moelling and J. Heinrich

University of Zurich, Gloriastr.32, CH 8006 Zurich Switzerland

Prag, Centennial Meeting of Retroviruses

We identified the RNase H of retroviruses and HIV as an essential enzyme for retroviral replication. It is specific for cleavage of RNA in RNA-DNA hybrids and required for removal of the viral RNA genome during DNA synthesis (1,2). It is the only retroviral enzyme not yet targeted for by antiretroviral therapy. RT and RNase H resemble PAZ and PIWI for siRNA silencing (3). We are driving HIV into suicide by short dsDNA, which leads to anRNA-DNA substrate for the RNase H and cleavage of the RNA. This renders the particles non-infectious including HIV from patients and multi-drug-resistant strains (4-6). In a mouse with the oncogenic Spleen Focus Forming Virus, SFFV, intravenous treatment reduced the virus load, disease progression or completely prevented infection (7). We also reduced the viral load in the blood of 30 primary patients’ isolates by ex vivo treatment in more than 30% by 1000fold (8). Also the viral load in a mouse vagina can be significantly reduced (9). HuSCID mice are under study. The approach may prevent HIV infection during sexual or mother-to-child transmission or against multidrug-resistant viruses. Also other viruses and cancer cells can be treated by activation cellular RNases H.

(1,2) Moelling et al., Nature (1971), EMBO (1988) (3) CSH Symp.71 (2006); (4,5) Jendis et al., ARHR(1996, 98); (6) Matskevich et al., ARHR(2006); (7) Matzen et al., Nature Biotechnology (2007) (8) Heinrich et al J AIDS (2008), (9) Widmer et al Antiviral Res (2009 (10) Noreen et al Oligonucleotides (2009)

Do viruses reflect evolution?            

Karin Moelling, University of Zurich, Switzerland, and Max-Planck-Institut für Molekulare Genetik, Berlin, Germany

Paris, Viruses and Microbes 2010

Virus-first hypothesis: Viruses are the most abundant entities and everywhere. This cannot have happened by infection of preexisting entities –viruses must have been around from the very beginning. Contemporary viruses can be aligned to reflect evolution and the origin of life, ranging from the RNA world to the DNA world. Replication and evolution are essential criteria for life. Two assumptions have to be made, proteins need to be subtracted form contemporary viruses, and cellular parasitism happened later. There are other exemples for free-living entities which became cellular parasites. Viruses can be organized by a gradient from RNA to DNA, or similarly from high to low genetic variability. Todays DNA world is still dependent on RNA in key reactions such as DNAN replication. Earliest replicating and evolving entities are the ribozymes. Their sequence space is outrageously high and never fully exploited. Ribozymes are reminiscent of todays plant viruses, the viroids, or Hepatitis Delta virus in humans. Size limitations of the RNA may have been overcome by accumulation of fragments, reflected by Influenza viral segmented genomes. Circular RNA as in Hanta viruses are alternatives.

Transition to the more stable DNA via hybrid intermediates may have happened even in a protein-free world by desoxribozymes and was later accelerated by catalytic protein enzymes. Proteins may have arisen before a translation machinery developed. Ribozymes helped, because ribosomes are riboyzymes. The enzyme most imprtant in generating DNA from the RNA world was the Reverse Transcriptase (which should be better designated pro– or ur-transcriptase). This is reflected by the telomerase, a rudimentary reverse transcriptase copying RNA with limited sequence information and creating DNA form RNA at the ends of chromosomes during embryogenesis and in every tumor cell today. It is also reflected by todays retro-and para-retroviruses HIV, Spuma, HBV, Caulimo). They package RNA, RNA/DNA hybrids intermediates or almost only DNA even though their life-cycles are very similar. Hepatitis B viruses with their three particle types reflect three options for compartmentalization. Icosaeders being energetically most favorable structures. Pararetroviruses stay episomal, do not integrate but may have allowed integration and incease of genome sizes. Retroviruses integrate, whereby todays assumption is, they integrate into the host genome, but they may have been the beginning of a host genome and allowed additional DNA to be integrated. Horizontal gene transfer is performed by oncogenic retroviruses today and is a widely used laboratory tool. Cellular genes are transferred as oncogenes and were the basis for cancer research. HIV with a nef-deletion reconstituted this gene from infected humans in about 15 years (Australia). Phycodanviruses are also exemples for gene transfer, they transport 22 host genes to another organism.

A proof that mainly retroviruses built our genome may come from the fact that 50% of our genome consists of retroelements. Our genome may be a graveyard of previous retrovirus infections. These range from full-sized retroviruses to solitary LTRs. Endogenization of retorviruses is actively ongoing today as exemplified by the Koala bear retrovirus which became endogenous in about 100 years. Where are the other 50% coming from? May be also from retroviruses which cannot be recognized as such any more. The oldest retroviral sequences found in rabbits are from about 25 000 years ago.

How can a parasite predate its host? It may not have been a parasite to begin with! Loss of genes may have been advantageous for viruses once the cell had formed. Exemples are bacteria, leading to mitochondria, chloroplasts or trypanosomes. Loss of independence and intracellular parasitism is also reflected by todays oncogenic retroviruses which render novel genetic information to the host cell such as growth advantages on the expense of replication competence.

Viruses may have generated the cells, they helped to develop ribosomes from ribozymes, and may have started cellular nuclei. An exemple could be the recently discovered sputnik virus in the mimivirus. The giant mimiviruses reflect a tranistion from viruses to bacteria. Formerly pox-like viruses were thought to have generated cellular nuclei.

Teh antiviral defense based on siRNA-mediated silencing uses as reverse transcriptase RNAse H related enzyme, Dicer, and the whole complex RISC, is composed like retroviruses.

There is no complete proof for a vision on the predominant role of viruses for the beginning and evolution of life – but simplicity may also be an argument. What else could have made the cell?

DNA viruses may have come from RNA viruses or may be later developments of evolution following the cell-first hypothesis.

Questions remain. Why do bacteria lack retrovirus-like phages? Could bacteria without a nucleus and high replication rates not afford such a genetic burden? Are they ahead if us in evolution? They replicate 1000fold faster than we do. Till today retroelements are shaping our genome. Viruses rarely cause diseases, only if enviromental conditions change. They guarantee evolution, as shown by genome sequence analyses which demonstrate that retroelements express non-coding RNA and regulate gene expression.

HIV suicide through viral RNase H

K. Moelling and J. Heinrich

University of Zurich, Gloriastr.32, CH 8006 Zurich, Switzerland

Cold Spring Harbor Symposium 2009

We identified the RNase H of retroviruses and HIV as an essential enzyme for retroviral replication(1,2). It is specific for cleavage of RNA in RNA-DNA hybrids and required for removal of the viral RNA genome during DNA synthesis. We validated it as a target for drug design. Out of the four known retroviral enzymes it is the only one not yet targeted for by antiretroviral therapy. Reverse Transcriptase (RT) and RNase H resemble in structure and function PAZ and PIWI, components of RISC for antiviral defence by siRNA silencing (3). We are targeting the RNase H inside cell-free virus particles by the application of a short hairpin-looped oligodeoxynucleotide (ODN) driving HIV into suicide. A short local RNA-DNA hybrid forms as substrate for the RNase H which cleaves the viral RNA. This renders the particles non-infectious including HIV from patients and multi-drug-resistant strains (4-6). In a mouse with the oncogenic retrovirus, Spleen Focus Forming Virus, SFFV, intravenous treatment with ODN reduced the virus load, delayed disease progression or completely prevented infection (7). We also reduced the HIV load in the blood of 30 primary patients’ isolates from Zurich and Africa by ex vivo treatment with ODN in more than 30% by 1000fold (8). Also the viral load in a mouse vagina can be significantly reduced in a prophylactic as well as therapeutic treatment (9). Humanized SCID mice are under study with HIV and ODN. Our approach is distinct from siRNAand antisense treatment in respect to mechanism, kinetics, and efficiency. It may prevent HIV infection during sexual or mother-to-child transmission or against multidrug-resistant viruses. Also other viruses and cancer cells can be treated by activation of cellular RNases H.

(1,2) Moelling et al., Nature (1971), EMBO (1988) (3) CSH Symp.71 (2006); (4,5) Jendis et al., ARHR(1996, 98); (6) Matskevich et al., ARHR(2006); (7) Matzen et al., Nature Biotechnology (2007) (8) Heinrich et al J AIDS (2008), (9) Widmer et al Antiviral Res (2009 (10) Noreen et al Oligonucleotides (2009)

HIV suicide and siDNA against viruses and cancer

Dr. Karin Moelling

University of Zurich, Gloriastr.32, CH 8006 Zurich, Switzerland

Korea, World Congress of Viruses and Infectious Diseases 2010

RNaseH of retroviruses and HIV is an essential enzyme for retroviral replication. It is specific for cleavage of RNA in RNA-DNA hybrids and required for removal of the viral RNA genome during DNA synthesis (1,2). It is the only retroviral enzyme not yet targeted for by antiretroviral therapy. RT and RNase H resemble PAZ and PIWI for siRNA silencing (3). We are driving HIV into suicide by short dsDNA, which leads to anRNA-DNA substrate for the RNase H and cleavage of the RNA. This renders the particles non-infectious including HIV from patients and multi-drug-resistant strains (4-6). In a mouse with the oncogenic Spleen Focus Forming Virus, SFFV, intravenous treatment reduced the virus load, disease progression or completely prevented infection (7). We also reduced the viral load in the blood of 30 primary patients’ isolates by ex vivo treatment in more than 30% by 1000fold (8). Also the viral load in a mouse vagina can be significantly reduced (9). HuSCID mice are under study. The approach may prevent HIV infection during sexual or mother-to-child transmission or against multidrug-resistant viruses. Also other viruses and cancer cells can be treated by activation cellular RNases H.

(1,2) Moelling et al., Nature (1971), EMBO (1988) (3) CSH Symp.71 (2006); (4,5) Jendis et al., ARHR (1996, 98); (6) Matskevich et al., ARHR(2006), (7) Matzen et al., Nature Biotechnology (2007) (8) Heinrich et al J AIDS (2008), (9) Widmer et al Antiviral Res (2009 (10) Noreen et al Oligonucleotides (2009)