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Ambisense RNA Genomes

by Vincent Racaniello, PhD
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    00:00 Finally the last negative strand of virus genome we will look at it is actually not just negative, it's called ambisense. It has both plus and minus polarities, but I will tell you why we call it negative sense in a moment. These are the Arenaviruses and the example of course is Lassa virus. And I particularly like Lassa virus, because this is the virus that made me want to become a virologist. A book describing the discovery of this virus published in the 1960s called 'Fever', I read just out of college and that made me want to be virologist. The genome of this virus is shown at the top of the slide. About half of the genome is plus stranded, that’s shown in green, and the other half is negative stranded, it's shown in yellow. When this genome first enters the cell, even though part of it is plus stranded and could be translated, it's not. This genome is replicated to form an mRNA from the three prime half called the NP mRNA. So that’s the obligatory first step, and that is carried out by an RNA polymerase in the virus particle, right, because the cell can't do this, and because we have to make this mRNA as the first step, the RNA polymerase has to be in the particle, that's why we defined this as a negative strand RNA virus, because those viruses have to have the polymerase in the particle. That NP mRNAs is translated to form a protein, and that protein participates in the replication of the genome from this first ambisense product to the one in the middle which is called the anti-genome. You can't really call these plus and minus because they are mixture, they are ambisense, so we call it genome and anti-genome, and from that anti-genome we make an mRNA called the GP mRNA, which is then translated and that accesses the five prime half of the genome. So these are ambisense genomes because they have both plus and minus strand characteristics.

    01:56 Now the virology field was revolutionized about 30 years ago by making DNA copies of viral genomes by the recombinant DNA technology. And this allows you to introduce any change into a virus at will, it makes studying these viruses in the laboratory, making vaccines, making therapeutic products very easy. We call this an infectious DNA clone. Whether the virus has a DNA or a RNA genome, its genome can be made into DNA and cloned into a bacteria plasmid, which can be grown in bacteria produced in high quantities and then we can introduce all sorts of changes into it. We can make deletion, insertion, substitution, nonsense or missense mutations to study the virus. We can introduce foreign proteins to use the virus as a vector; for example one of the Ebola virus vaccine that's currently being shown to be very successful in West Africa is actually a rabies-like virus with a Ebola protein coding region inserted into it. That was made possible by this technology. So you can design any virus that you'd like, within reason of course. You can reconstruct the genome from the sequence, which I will tell you about in a moment. It's called synthetic virology and because of the power of this technology a lot of people are worried about it, and so there have been a lot of discussions over whether this is safe or not. Now in this example, we are reconstructing influenza virus from a DNA copy of the RNA virus genome, and this we're showing for simply one of the eight segments, you have to do this eight times to get an influenza virus. You start with the yellow molecule labeled minus RNA, that's the virus RNA genome. You make a double-stranded DNA copy of it, you put it in a plasmid and that plasmid when introduced into cells, will produce both negative stranded viral RNA, as well as plus stranded mRNA to make protein. So you put eight of these plasmids into cells, one coding for each of the viral segments, and out comes infectious virus. We call this transfection. We introduce a DNA into cells and out comes virus. Let me tell you an interesting use of this technology. Back in 1918 there was a huge and very serious outbreak of influenza virus; it is often called Spanish flu, although that's not where it originated. Now this outbreak killed millions of people globally, it coincided with World War I, so troop movements probably had a lot to do with it spread, but we didn't have this virus isolated from this outbreak, we didn’t isolate influenza virus until 1933. So in the 1990s, investigators determined the sequence of this virus because it had never been isolated. They determined the sequence from material that had been obtained from people who died and were frozen in Alaska.

    05:02 They opened the grave, they took biopsies of the lungs, these people were known to have died from influenza virus, and then they extracted enough material to determine the genome sequence.

    05:12 They also got more sequence from pathology slides that had been prepared from Army recruits who had died of the infection, their lungs had been sectioned, and preserved and stored, and we could recover a little bit of sequence from that as well. So the sequence of all eight segments was determined, it was built into DNA, and the virus was recovered. So we reconstructed the 1918 virus when we'd never had it before. We reconstructed it from the sequence, so this can be done with just about any virus, and people have been studying this virus and we've learned a lot about it. We’ve learned many important things and of course all of these studies are done under high containment, because if this virus got out it could likely be very dangerous.


    About the Lecture

    The lecture Ambisense RNA Genomes by Vincent Racaniello, PhD is from the course Viruses.


    Included Quiz Questions

    1. Production of infectious virus after transformation of cells by viral DNA
    2. Introduction of viral proteins into cells
    3. A way to make clonal virus stocks by plaque assay
    4. A way to make mutations by treating viruses with ultraviolet light
    5. A way to create vaccines for viruses
    1. Minus strand will be used to make mRNA by enzyme Polymerase programmed by viral genome
    2. Plus strand will be used to make viral proteins
    3. Minus strand will be used to make viral proteins
    4. Plus strand will be used to mRNA by DNA polymerase programmed by cell genome
    5. Plus strand will be used to make mRNA by RNA polymerase programmed by viral genome
    1. ...they have both plus and minus strand characteristics
    2. ...they have more plus and little minus strand genome
    3. ...their have little minus and more plus strand genome
    4. ...their minus strand is used to make NP mRNA
    5. ...their minus strand cannot be calved my endonucleases
    1. ...transfection
    2. ...transformation
    3. ...transportation
    4. ...transmigration
    5. ...induction
    1. Synthetic Virology
    2. Viral therapy
    3. Structural virology
    4. Histo Virology
    5. Molecular virology

    Author of lecture Ambisense RNA Genomes

     Vincent Racaniello, PhD

    Vincent Racaniello, PhD


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    well explained. differences well spelt out. now I get viral genomes
    By Masoh V. on 18. December 2017 for Ambisense RNA Genomes

    First time I get to understand the various confusing genomes. wonderful