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TWiV 66: Reverse transcription

17 January 2010 by Vincent Racaniello

Hosts: Vincent Racaniello and Dickson Despommier

Vincent and Dickson continue virology 101 with a discussion of information flow from RNA to DNA, a process known as reverse transcription, which occurs in cells infected with retroviruses, hepatitis B virus, cauliflower mosaic virus, foamy viruses, and even in uninfected cells.

This episode is sponsored by Data Robotics Inc. To receive $50 off a Drobo or $100 off a Drobo S, visit drobostore.com and use the promotion code VINCENT.

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Click the arrow above to play, or right-click to download TWiV #66 (50 MB .mp3, 68 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email.

Links for this episode:

  • Discovery of RNA tumor viruses
  • Reverse transcriptase found by Temin and Baltimore (pdfs)
  • Figures for this episode
  • Video of this episode – download .mp4 or .wmv or view below

Weekly Science Picks
Vincent Joint UN Programme on HIV/AIDS

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

Filed Under: This Week in Virology Tagged With: AIDS, caulimovirus, foamy virus, HBV, hepatitis b virus, HIV, retroelement, retrovirus, reverse transcriptase, reverse transcription, rna tumor virus, TWiV, viral, virology, virus

TWiV 64: Ten virology stories of 2009

3 January 2010 by Vincent Racaniello

3D_InfluenzaHosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich discuss ten compelling virology stories of 2009.

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Click the arrow above to play, or right-click to download TWiV #64 (68 MB .mp3, 94 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email.

Ten virology stories of 2009:

  1. Pandemic influenza: Swine-origin H1N1 virus (TWiV 36)
  2. XMRV, prostate cancer, and chronic fatigue syndrome (TWiV 50, 55)
  3. AIDS vaccine ‘success’ (TWiV 51)
  4. Colony collapse disorder (TWiV 46, 49)
  5. AIDS-like disease in wild chimps (TWiV 45)
  6. Diverse viral community in Antarctic lake (TWiV 58)
  7. Polyomavirus seroepidemiology in humans (TWiV 26)
  8. Poxvirus threatens UK red squirrels (TWiV 63)
  9. Polio spreads from Nigeria (TWiV 29)
  10. How mosquitoes survive Dengue virus infection (TWiV 21)

Picture book on viruses for kids (Thanks Soraia!)

Weekly Science Picks
Rich Surely You’re Joking, Mr. Feynman! by Richard P. Feynman, Ralph Leighton, Edward Hutchings, and Albert R. Hibbs
Alan Spaceweather.com
Vincent The Art and Politics of Science by Harold Varmus

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

Filed Under: This Week in Virology Tagged With: AIDS, antarctic virus, CFS, colony collapse, Dengue, H1N1, HIV, honeybee, influenza, mosquito, polio, poliomyelitis, polyomavirus, poxvirus, prostate cancer, squirrel, swine flu, TWiV, viral, virology, virus, xmrv

Viruses and journalism: Poliovirus, HIV, and sperm

18 December 2009 by Vincent Racaniello

polio_hivIn the summer of 1989, two papers about viruses were published in high-profile journals. One described the engineering of a recombinant poliovirus bearing on its surface an antigen from HIV-1. The second paper claimed that transgenic mice could be made by adding DNA to sperm before using them to fertilize eggs. Both reports played a role in a television interview I did with Earl Ubell of CBS News.

I was reading the Cell paper describing a new way to make transgenic mice when I received a call from CBS News. They wanted me to comment on a report that had just been released by the journal Nature, describing the production of a recombinant poliovirus bearing an antigen from HIV-1. When rabbits were inoculated with the recombinant poliovirus (the authors called it a chimeric virus), antibodies were made which could block infection with HIV-1. CBS wanted to talk because I had developed the technology to genetically engineer polioviruses.

Within an hour, science correspondent Earl Ubell arrived in my lab with a camera crew. We chatted while they set up their equipment and began to record one of my postdoctoral fellows, Gerardo Kaplan, moving about the laboratory. Ubell asked me what I thought about the polio-HIV story. I told him that it was interesting but would probably not be useful for preventing AIDS because only a small piece of HIV-1 protein could be inserted into the poliovirus capsid. Then we started talking about the sperm – transgenic mouse story, which he had heard about. I was more positive about that story; I noted that the findings could revolutionize biology.

In a few moments a microphone was clipped to my shirt and the camera began recording our conversation. Ubell began asking me questions about the polio-HIV experiment. We discussed how the virus was engineered, why it was possible to insert foreign protein into poliovirus, and the utility of this discovery. I was not positive about the future of this technology for vaccines, but I did point out its potential for research. His last question was about the sperm – transgenic mouse paper: what did I think about that? I responded, “I think that if this report is correct, then it’s going to be fabulous”. Then he packed up and left.

Several weeks later one of my students sent me a copy of the final program that had been shown on television. I was surprised to find that just one of my comments was used, and it had nothing to do with the polio-HIV story. It was the answer I had given in response to Mr. Ubell’s question about the sperm – transgenic mouse experiment, which of course was not the topic of the TV piece. I don’t know who made the juxtaposition, but I found it duplicitous. As a result of this experience, for the next 20 years I limited my contact with journalists.

Here is Mr. Ubell’s June 1989 report for CBS News. To his credit, he did get the science right, and he pronounced my name correctly. Unfortunately, while poliovirus antigen chimeras have been useful in the research lab, they have not been used as vaccines. And making transgenic mice by putting DNA into sperm turned out to be wrong.

Lavitrano, M. (1989). Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice Cell, 57 (5), 717-723 DOI: 10.1016/0092-8674(89)90787-3

Filed Under: Commentary, Information Tagged With: AIDS, HIV, journalism, poliovirus, sperm, transgenic mice, viral, virology, virus

TWiV 62: Persistence of West Nile virus

13 December 2009 by Vincent Racaniello

The_Persistence_of_MemoryHosts: Vincent Racaniello, Dickson Despommier, and Alan Dove

On episode #62 of the podcast This Week in Virology, Vincent, Dickson, and Alan discuss STEP HIV-1 vaccine failure caused by the adenovirus vector, presence of West Nile virus in kidneys for years after initial infection, adaptation of the influenza viral RNA polymerase for replication in human cells, and the significance of the D225G change in the influenza HA protein.

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Click the arrow above to play, or right-click to download TWiV #62 (47 MB .mp3, 66 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email.

Links for this episode:

  • HIV vaccine failure probably caused by adenovirus vector used
  • Persistence of West Nile virus in kidneys for years (JID and ProMedMail) (thanks, Lenn!)
  • Adaptive strategies of influenza RNA polymerase for replication in humans
  • New CDC estimates of 2009 H1N1 infection in US
  • Receptor binding specificity of 2009 H1N1 virus
  • Distribution of sialic acids in human respiratory tract

Weekly Science Picks
Dick Smallpox – The Death of a Disease by DA Henderson
Alan Olympus Bioscapes Digital Imaging Competition
Vincent Microbe Magazine

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

Filed Under: This Week in Virology Tagged With: adenovirus, AIDS, d225G, H1N1, HIV, influenza, kidney, persistence, RNA polymerase, sialic acid, step, swine flu, viral, viral receptor, virology, virus, West Nile virus

AZT inhibits XMRV

8 December 2009 by Vincent Racaniello

aztXenotropic murine leukemia virus related virus (XMRV) has been implicated in prostate cancer and chronic fatigue syndrome (CFS). Because XMRV is a retrovirus, it has been suggested that it might be susceptible to some of the many drugs available for treatment of AIDS. Of ten licensed compounds evaluated for activity against XMRV, just one, AZT (azidothymidine), was found to inhibit viral replication.

Compounds used to treat HIV-1 infection fall into distinct classes: protease inhibitors (Ritonavir, Saquinavir, or Indinavir), nucleoside reverse transcriptase inhibitors (NRTI, AZT, 3TC, Tenofovir, D4T), non-nucleoside reverse transcriptase inhibitors (NNRTI, Efavirenz, Nevirapine), integrase inhibitors (118-D-24), and fusion inhibitors (Maraviroc). None of the HIV-1 protease inhibitors, NNRTI, or integrase inhibitors blocked XMRV replication.  Of the NRTIs, only AZT significantly inhibited viral replication. Fusion inhibitors were not examined in this study.

AZT was the first drug licensed to treat AIDS. It is phosphorylated to the active form by cellular enzymes. Phosphorylated AZT is an inhibitor of viral reverse transcriptase because it acts as a chain terminator when incorporated into DNA:

azt_mechanism

Because AZT has a N3 (azido) group on the ribose instead of a hydrogen, the next base cannot be added to the DNA chain and synthesis stops.

The relative selectivity of this drug depends on the fact that reverse transcription takes place in the cytoplasm, where the drug appears first and in the highest concentration. But the presence of AZT monophosphate causes depletion of the intracellular pool of ribosylthymine 5′-triphosphate (TTP). Therefore AZT has substantial side effects which include muscle wasting, nausea, and severe headaches. AZT treatment can also damage bone marrow, which requires multiple transfusions of red blood cells. The drug was used extensively because there was no alternative until other antivirals were developed.

AZT can be taken orally but it is degraded rapidly by liver enzymes. Patients must take the drug two or three times a day to maintain an effective antiviral concentration. The drug is modestly effective in infected adults, leading to a transient increase in CD4+ T-cell counts.

Much effort has been devoted to discovering alternatives to AZT, and several nucleoside analogs that have therapeutic value, such as 3TC, are available. However 3TC does not inhibit XMRV replication.

It is not known if treatment with AZT will effect either prostate cancer or CFS. If prostate cancer is triggered when XMRV inserts into chromosomal DNA, then the drug will not likely block progression of the disease because the drug does not eliminate infected cells. Whether reduction of viral loads by AZT treatment has a positive therapeutic outcome remains to be determined. Because AZT is approved for use in humans, such studies can proceed immediately, without the need for extensive toxicity studies in animals.

Sakuma R, Sakuma T, Ohmine S, Silverman RH, & Ikeda Y (2009). Xenotropic murine leukemia virus-related virus is susceptible to AZT. Virology PMID: 19959199

Filed Under: Basic virology, Information Tagged With: AIDS, antiretroviral, azt, CFS, chronic fatigue syndrome, fusion, HIV-1, integrase, nnrti, nrti, prostate cancer, viral, virology, virus, xmrv

Raltegravir inhibits murine leukemia virus: implications for chronic fatigue syndrome?

20 November 2009 by Vincent Racaniello

RaltegravirThe finding that a retrovirus, XMRV, is associated with chronic fatigue syndrome has lead to the suggestion that the disease might be treated with some of the antiviral drugs used to treat AIDS. The integrase inhibitor Raltegravir has been found to block the replication of murine leukemia virus, which is highly related to XMRV. But the drug exacerbates autoimmune disease in mice which might rule out its use in treating CFS.

Retroviruses such as XMVR and HIV-1 have genomes composed of single-stranded RNA. This nucleic acid is converted to a DNA copy in infected cells by the viral enzyme reverse transcriptase. The double-stranded viral DNA is then integrated into the chromosomal DNA of the host cell, a process accomplished by an viral enzyme called integrase (illustrated).

retroviral_integration

Raltegravir (pictured above left) is an inhibitor of HIV-1 integrase that was approved for use in humans in 2007. The drug blocks the integration of viral DNA into the host genome and therefore inhibits viral replication.

The mouse retrovirus murine leukemia virus (MLV) has been linked to the development of spontaneous autoimmune disease. The mechanism by which the virus induces this disease is not known, but stimulation of innate immune responses by viral DNA might be involved.

Raltegravir also inhibits integration of MLV DNA into the murine genome. When mice with autoimmune disease were treated with raltegravir, they succumbed to autoimmune disease a month earlier than untreated animals. Mice without the disease were not affected by the antiviral drug. The authors speculate that by inhibiting viral DNA integration, raltegravir increases the amount of unintegrated viral DNA, elevating innate responses and exacerbating autoimmunity.

It’s not known if raltegravir is active against XMRV, the retrovirus associated with chronic fatigue syndrome. Given the similarity between the genomes of MLV and XMRV it seems likely that the drug will inhibit the virus. If the ability of raltegravir to treat CFS is tested in clinical trials, it will be important to carefully monitor treated patients for signs of autoimmunity. CFS has an autoimmune component which could worsen with raltegrivir treatment.

An obvious question is whether raltegrivir induces autoimmunity in AIDS patients. I’m not aware of any such reports, which is probably not surprising given the fact that HIV-1 infection leads to immunosuppression.

CFS sufferers should not despair: other antiretroviral drugs, including chain terminators such as AZT, do not allow the accumulation of unintegrated viral DNA. These compounds might be useful for treating the disease.

G.B. Beck-Engeser, D. Eilat, T. Harrer, H.-M. Jack, M. Wabl (2009). Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir Proceedings of the National Academy of Sciences : 10.1073/pnas.0908074106

Filed Under: Basic virology, Information Tagged With: AIDS, CFS, chronic fatigue syndrome, HIV-1, mlv, raltegravir, viral, virology, virus, xmrv

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by Vincent Racaniello

Earth’s virology Professor
Questions? virology@virology.ws

With David Tuller and
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