VIROIDS

1. Are these viruses?
   1. What are viruses? (packaged DNA or RNA but not both together)
   2. What constitute viroids? (unpackaged nucleic acid)

2. Discovery Experiments

3. Diseases (mostly plant)

   	number of nucleotides
   Potato spindle tuber	359
   Chrysanthemum stunt	354 and 356
   Citrus exocortis	371
   Cucumber pale fruit	303
   Hopstunt	297
   Avocado sunbloch	247
   Cadang-cadang (coconut palm)	246, 287, 482, and 492
   Several tomato isolates	360

4. "Life" Cycle
   1. Circular, mostly dsRNA
   2. Central 20^± bp highly conserved
   3. No AUG or CAU for translation initiation!
   4. No viroidally directed polypeptides ever detected!
   5. Situated in the nucleolus
      1. Genomic code similar to rRNA introns
      2. What's the function of the nucleolus?
   6. Infection
      1. Probably mostly from raindrop splash in plants
      2. Artificial
      3. Rubbing onto leaves
      4. cDNA infectivity
   7. Genomic replication
      1. Probably uses host's DNA-directed RNA polymerase II
      2. Rolling circle

5. Human Hepatitis Delta Agent
   1. (-?)RNA Genome = 1678 bases, 61% double helix
   2. A "Satellite RNA" of Hepatitis B
   3. Delta antigen

6. Virusoids
   1. Examples: TNV, polio DI, VSV DI (DI means defective interfering)
   2. What are they? (Like a viroid but wrapped in the helper virus's capsid.)
   3. CARNA-5 (Cucumber mosaic virus Associate RNA) contains 4 chromosomes

7. SATELLITE WARFARE

DISCOVERY OF VIROIDS

Throughout the plant kingdom were seen a number of leaf diseases for which no inclusion bodies within the cells could be seen, and yet there were hints that these were infectious diseases. Indeed, if a pulp were made of these "diseased" leaves and passed through an "ultrafilter" (the usual type of filter that bacteria cannot get through), the infection was transferable. In the next figure, the filtrate was rubbed onto the distal end and on one side of two leaves, respectively.

Furthermore, in order to show that the agent was able to reproduce, billion-fold dilutions were made and rubbed onto leaves, and more lesions arose:

Of course, up to this point, everything still points to the agent's being a virus. But one test befuddled the virologists when they did their control experiment to discover the nature of the genetic material. They ran a test not unlike that of Avery, MacCleod and McCarty - "Which enzyme would knock out the infectivity?" Normally in viral preparations, the nucleic acids are sequestered from the surrounding medium and so no enzyme is able to attack the insides of the virus particles. But look here!

RNase alone was able to destroy infectivity without breaking up any virion structure! This meant that the genetic material (RNA, in this case) was exposed to the medium. Could it be that this was an "inside-out" virus - the RNA wrapped around the outside of the capsid?!

That was easily answered by merely taking a look at the absorbance spectrum of the agent:

This is a perfect spectrum of pure nucleic acid. These things are DIFFERENT from anything we've seen before! These infectious agents consist only of RNA! (Sort of reminds us of Gierer and Schramm's experiments on TMV after they disassembled the virus and rubbed the protein and RNA components on leaves - and managed to get a small amount of infectivity from the RNA alone).

The next step along the way was to see just how discrete were these - what shall we call them? - "viroids." Were they put through sedimentation and isopycnographic centrifugations, would they show up as a blur or as discrete bands? Let's see:

It turns out that the various sorts of viroids isolated from plants in nature are each very discrete in size - numbering between 250 and 500 bases long.

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