TWiV 162: Transcription

transcriptionHosts: Vincent Racaniello, Rich Condit, and Alan Dove

Vincent, Rich, and Alan continue Virology 101 with a discussion of transcription, the process of making mRNA from a DNA template.

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Weekly Science Picks

Rich – 3D vaccinia virion
AlanHappy Holidays from NOAA (YouTube)
Vincent – 17 year old wins $100,000 science prize

Listener Pick of the Week

SarahCritical Thinking (pdf),  diagnosing respiratory infections by gene signature (Cell Host Micro), and One Health Initiative (CDC page)

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TWiV 106: Making viral DNA II

the 5prime end problemHosts: Vincent Racaniello, Dickson Despommier, and Rich Condit

On episode #106 of the podcast This Week in Virology, Vincent, Dickson, and Rich continue Virology 101 with a second installment of their discussion of how viruses with DNA genomes replicate their genetic information.

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  • Figures for this episode (pdf)
  • Letters read on TWiV 106
  • Video of this episode – download .mov or .wmv or view below

Weekly Science Picks

Rich – Google Health
Dickson – The Neandertal genome
Vincent – Lab techniques videos (thanks, Erik!)

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TWiV 100: TWiV catches a big fish

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and David Baltimore

Vincent, Alan, and Rich celebrate the 100th episode of the podcast This Week in Virology by talking about viruses with Nobel Laureate David Baltimore.

Click the arrow above to play, or right-click to download TWiV #100 (68 MB .mp3, 94 minutes)

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Alan – TimeTree
Rich –
The neurons that shaped civilization
Vincent – Ahead of the Curve: David Baltimore’s Life in Science by Shane Crotty

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A new target for hepatitis C virus

When infection with hepatitis C virus goes from acute to chronic, severe liver disease may occur which requires organ transplantation. Nearly 200 million people are chronically infected with HCV, necessitating approaches to preventing and treating infections. No HCV vaccine is available, and current antiviral therapy consists of administration of interferon plus ribavirin, a combination that is effective about half the time and is associated with undesirable side effects. New antiviral compounds that target a viral protease and RNA polymerase are currently in clinical trials may eventually reach the market. But our experience with HIV-1 has shown that combinations of three drugs are the most effective for derailing the emergences of drug resistant viruses. The third target for HCV could be NS5A, a viral protein without a known function.

To identify new inhibitors of HCV, a chemical library of one million compounds was screened for the ability to inhibit viral replication in cell culture. The active compound were then subjected to a second screen to eliminate inhibitors of known viral enzymes: the viral protease, RNA polymerase, and helicase. One of the remaining inhibitors was further refined chemically until a very potent derivative was obtained. This molecule, called BMS-790052, has a 50% inhibitory concentration in the picomolar range, and inhibits all the viral genotypes tested. It is the most powerful inhibitor of HCV discovered.

The compound was tested for safety and bioavailability in various animal species. After oral administration, the compound was found in plasma and liver, despite a molecular mass of over 700 daltons. Six different levels of the compound were tested in HCV infected individuals. No adverse effects were reported, and the highest amount administered reduced viral levels in the blood 2,000 fold after one day. These results are promising, but larger trials will now be needed to further confirm the safety and efficacy of the drug.

What is the target of BMS-790052? Two lines of evidence suggest that the compound inhibits the viral protein NS5A. The drug appears to bind NS5A, and viruses resistant to the drug have amino acid changes in this protein. Although NS5A is known to be required for viral replication, its precise function is not known. Because NS5A does not have an easily assayable enzymatic function, it has not previously been a target of drug discovery. The identification of a compound that inhibits NS5A function is an important step forward in HCV drug development. The general approach used to discover BMS-790052 should be useful in identifying inhibitors of other viral proteins that do not have well defined and measurable activities.

I discussed this paper on Futures in Biotech episode #60. If you would like to listen only to the conversation about BMS-790052, download this mp3 file, or listen to the discussion below.

[audio:http://www.virology.ws/fib60.mp3 | titles=FIB 60]

Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O’Boyle DR 2nd, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, & Hamann LG (2010). Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature, 465 (7294), 96-100 PMID: 20410884

Virology lecture #6: RNA-directed RNA synthesis


Download: .wmv (324 MB) | .mp4 (76 MB)

Visit the virology W3310 home page for a complete list of course resources.

TWiV 60: Making viral RNA

TWiV_AA_200Hosts: Vincent Racaniello and Dickson Despommier

Vincent and Dick continue Virology 101 with a discussion of how RNA viruses produce mRNA and replicate their genomes.

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Weekly Science Picks
Dick The Double Helix by James D. Watson
Vincent
Worms and Germs Blog

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Below is a video of TWiV 60, which highlights the diagrams I referred to during the podcast.

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186 MB .mov video file

584 MB .wmv video file

Influenza viral RNA synthesis

Once the (-) strand influenza viral RNAs enter the nucleus, they serve as templates for the synthesis of mRNAs. These molecules are then transported back to the cytoplasm, where they direct the synthesis of viral proteins. However, the mRNAs are not complete copies of the viral (-) strand RNAs – they are missing sequences from both the 5′- and 3′-ends. Therefore, to produce more viral (-) strand RNAs that are needed to assemble new virions, a full length (+) strand is produced, which in turn is copied to a full-length (-) strand RNA. The (-) strand RNAs are then used to assemble new virions. This process is best understood by referring to the figure, which distinguishes the processes of mRNA synthesis and replication. The (-) strand RNAs are those which are present in virions. The same processes occur for all eight segments of influenza viral RNA.

influenza-rna-synthesis

The enzyme that reproduces influenza RNA is known as an RNA-dependent RNA polymerase. This enzyme, which consists of the viral proteins PA, PB1, and PB2, is present in every virus particle. If this enzyme were absent from virions, they would never initiate infection, because the (-) strand viral RNAs cannot be translated into protein, and the cell has no enzymes which can copy such long RNA molecules. Of course, additional molecules of the viral RNA polymerase are made in infected cells, but the enzyme that is brought in with the virion is crucial for initiating the infectious cycle.

The influenza viral RNA polymerase is a primer-dependent enzyme. The enzyme cannot copy the (-) strand RNA template without a small piece of RNA that aligns on the template RNA and provides a starting point for RNA synthesis. The primers for influenza viral mRNA synthesis are produced from the cell’s own collection of mRNA molecules. The influenza viral RNA polymerase actually cleaves cell mRNAs near their 5′-ends, generating the primers it requires for RNA synthesis. This process has been called cap snatching, because the primers are made from the 5′-ends of cell mRNAs, which have an unusual chemical structure called a ‘cap’ (box labeled ‘c’ in illustration). Cap-snatching is also a feature of mRNA synthesis of other viruses.

Because RNA-dependent RNA polymerases are not found in mammalian cells, they are an excellent target for inhibition by antiviral compounds. At least one new antiviral drug that targets this enzyme, T-705, is currently in development.