ADENOVIRUS

1. Anatomy and symptoms
   1. Variations - 31 serotypes
   2. In vivo disease is self-limiting (human croup, sore throats, respiratory diseases, infectious canine hepatitis, in vitro neoplasias, diseases of hamsters, monkeys, mice, chickens, etc.)
   3. At least 14 different capsomers make up the dodecahedral virion
   4. dsDNA
      1. Always linear: 2.4 x 10⁷ dal; 50-60% G+C
      2. Terminal palindromes 0.1 kbp
      3. Evidence of a small degree of eukarytic character; has a 55 kdal protein
      

   5. The structure of the virion
      Icosahedral: 1.75x10⁸ dal total; 240 hexons + 12 pentons in capsid; 10-30 nm spikes. Three high-arginine proteins in core to neutralize the DNA.

2. Vegetative Replicative Cycle
   1. "Latent" period is about 24 hours
      0. Starting with adsorption and then viropexis of some sort
      1. Early: m-RNA's yielding early proteins including a T-antigen
         1. Host transcriptase-2 is used
         2. Host DNA synthesis is slowed after awhile
      2. Late: Begins at about 8 hours
         1. DNA is made along with other late functions
            Bi-directional DNA synthesis starting at the termini
            
         2. "VA"-RNA's: small, plentiful and of unknown function.
         3. RNA processing was first discovered with AV-2
      3. Ramifications of the restriction map of the DNA
         1. A new way of elucidating the genetic map
         2. Use of restriction fragments to ascertain identities of genes
      4. Ramifications of the map of early and late mRNA's
      5. Translation occurs in the cytoplasm
      6. Virion assembly occurs in the nucleus

   2. Maturation
   3. Escape from the cell

3. in vitro characteristics
   1. Oncogenic characteristics
      1. AAV: AV's own nemesis! (A parvovirus that is a 'satellite virus')
         1. 20-25 nm dia; 6x10⁶ dal (=18kbp=6000 aa); 32 capsomers; size of ribosome
         2. "+" and "-" strands distributed among virions; 30% T; dsDNA infection with AV
         3. Identical terminal redundancies that are highly conserved
   2. "Grazing"
      1. Characteristics of hemagglutination
      2. Hyaluronidase
      3. "Grazing" types

4. Adeno-Associated Virus (AAV)
   1. Steps Leading to AAV's Discovery
   2. Anatomy
      1. Structure of the virion - a dependent parvovirus
      2. Size and nature of the genome
         1. ssDNA; 1.5x10⁶ dal
         2. complementary strands
         3. terminal palindromes
         4. replication of the genome
   3. Occurance
   4. Life Cycle
5. African Green Monkey Connection

AAV: The Virus with Seemingly Only ONE Gene!

Tissue cultures are grown in many different ways. Shown here are the top and side views of a 'tissue culture flask.' The cells settle and form a monolayer on the bottom surface of the flask (and are depicted here like shingles in the top view.

As it was seen via EM that many AV suspensions were crowded with smaller viruses, a mix of these was added to a tissue culture of HeLa cells (human cervical cancer cells from "Helen Lane"). After awhile, the cells began leaking out huge numbers of both types of virus particles. In order to initially characterize the genetic material in these two viruses, some ₁₄C-uridine and ₃H-thymidine were added.

As can be seen in this NaBr isopycnographic analysis (density gradient centrifugation), both viral bands turned out to contain DNA because both are labelled with tritium (₃H).

When the DNA was isolated from the smaller particle and subjected to CsCl isopycnography, the virologists were surprised to find a doublet of DNA bands. Each of these bands, which showed no hyperchromic effect, and had base ratios where A did not equal T, and G not equal C, were judged to be ssDNA. Furthermore, as shown by the diffusion width of the bands, the ssDNA must have a molecular weight around 1.5 million daltons - very small for DNA!

However because when the bases were summed from both strands, A=T and G=C, it was thought that these might well be complements of one another, and so an annealing test was run, and they were indeed found to be complements.

Thus it appeared that among the population of extracellular virions, half had one of the two strands ("Watson") and the others contained "Crick."

Work now turned to the molecular biology of these two viruses. Starting with their capsids, they wanted to see if there was any similarity between the AV and its much smaller parvoviral associate (parvo is Latin for small). So the first step was to do some immunochemistry on the two viruses, and this required the two viruses be separated using "sedimentation centrifugation." Shown here is a now antiquated way of making gradients (look here for a faster way).

The parvovirus (PV) and AV were thus purified, above, and now specific antibodies were to be made in some mice, which were injected with these preparations. A couple of weeks later and after several "booster" shots, serum was collected:

A quick check of the antibody content of these sera was made using the simple Ouchterlony test. A few millimeters of suspensions of the purified viruses were added to test tubes, a small about of glycerol or sucrose was dissolved into the viral suspensions to make them dense. These "heavy" solutions were then overlayered with the "lighter" antisera. If the antibodies were active against the viral antigens, a visually apparent flocculence of precipitin would form at the interfaces. In the following figure, you see that precipitin is formed in tubes 1 and 4. Because none was formed in tubes 2 and 3, it seems that the antibodies against PV cannot react with those of AV, and vice versa. Thus no "CRM" - cross reaction! This is great! Our antibody reagents are very specific and ready for further utilization.

While we have the two viruses purified from each other, we set out to cultivate them in tissue cultures of HeLa cells in plates as shown next. One of the first things we notice is that when both were added to a plate, we have gotten clumps of cells that microscopically have all the earmarks of being tumorous. One of the common traits of tumor cells is that they lose contact inhibition and continue to grow even when they touch each other. They continue to grow and pile up on one another.

This phenomenon of in vitro oncogenesis (tumor formation) will warrant further and very important study!

But moving on with the investigation of these parvoviruses, we inspect our yields in the above plates. We do some Ouchterlony's on the supernates of each of the plates:

We notice that NO parvovirus was produced, that plenty of AV was made, and that when both were used in the inoculum, both viruses were produced. This last pair of tubes shows us that PV must require the presence of AV to grow. Furthermore, because the yield of AV is decreased when both are present (cf tube 4), it seems as if PV is detrimentally parasitizing AV. Could PV be a virus infection of a viral infection!? If so, then it should be a very specific parasite. Let's give it a name: adeno-associated virus: "AAV."

One of the things we know about this AAV is that it has a very small genome: 1.5 million daltons (ssDNA). This would have the maximum coding capacity for a peptide of about 1,500 amino acids long. As the capsomer is 1,200 amino acids long, it appears very much like this virus has one ONE GENE!

So let's confirm this. Our plan is to isolate and make some of its dsDNA, and then transcribe it in vitro to mRNA(s), and then translate those mRNAs a la Nirenberg to proteins, and hopefully come up with AAV capsomer! To help us know our results, we shall add some ₁₄C-alanine to the system. Any protein(s) made should incorporate this commonly used amino acid and should thus become radioactive and easily picked out of the mix of Nirenberg components such as enzymes, tRNA's, ribosomes, etc., etc.

So this is what we expect: The Nirenberg Translation System's contents, above, will be put atop a sucrose sedimentation gradient and spun. The in vitro synthesized capsomer ought to spin out as a single discrete radioactive band. Later antiserum work ought to show us that this band is indeed capsomer! Neat! A great confirmation experiment IF it works!

We did all those wonderful things, and now what did we get? (Take a peek at that link!)

Later the world's sequencing labs set to work on this and found three starts in different reading frames on "Watson" and two starts in different reading frames on "Crick."

And when the stops were identified, the whole genetic map of this AAV looks like this:

Indeed the whole genome is needed to make the capsomer, but nested within are also four other out-of-reading-frame "open" sequences that code for other functions. The occurance of dissimilar proteins supports the out-of-reading-frame phenomenon seen here. Thus AAV not only requires AV to perform most of its functions, but AAV also can do a little for itself! How amazing it is that so much "wisdom" can be encoded within such a small genome using antiparallel and overlapping genes!" (Sol Spiegelman).

GO BACK TO AAV Outline

The Discovery of AV's Grazing

In early days of virology, one of the questions being asked was about the nature of the attachment sites on the host cells. It was soon found that when adenovirus suspensions were added to defibrinated human blood, the blood cells clumped together - hemagglutination:

The clumping had a number of characteristics of the agglutination that occurs between antibodies and their target cells as seen by Burnet (Univ of St. Andrews, Scotland). There was an optimal concentration when there were just enough "linkers" (Ab's or AV's) added to the blood cells to form links between the cells:

However, unlike a Burnet Ab system, the hemagglutination with AV was transient. After a few hours, the blood cells went back into suspension.

The mystery for this was soon revealed when they first noted that it was to the surface layer of hyaluronic acid that the AV's attached to the cells.

But, while the AV could not penetrate the cells (not the AV's host), the viral attachment molecules seemed to incorporate hyaluronidase activity, and proceeded to breakdown the cells' surficial hyaluronic acid - not unlike cattle graze a meadow. Thus, after awhile, the cells are devoid of hyaluronic acid, the viruses fall off and the bridges disappear and the cells wander off back into suspension.

However, our friend Mary Lou threw in the proverbial monkey wrench: she dumped some of her AV into our previously treated and now dispersed blood cells:

It sooned was revealed that there are different sorts of hyaluronic acid, and that there were different strains of AV. Each strain could graze its own type of hyaluronic acid.

GO BACK TO AV Outline

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