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MM 719-722

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1. To introduce Koch's postulates.

2. To introduce the concept of the "prion" as an etiological agent of human disease.

3. To introduce the concept of the "viroid" as a disease entity.

4. To familiarize you with the current concepts of the structure, replication and pathogenicity of the prion and viroid.

Specific (terms and concepts upon which you will be tested)

Koch's Postulates

Koch's postulates state certain conditions which must be fulfilled in order to demonstrate the etiology of infectious disease. These postulates are:



Prions are infectious agents composed exclusively of a single sialoglycoprotein called PrP 27-30. They contain no nucleic acid. PrP 27-30 has a mass of 27,000 - 30,000 daltons and is composed of 145 amino acids with glycosylation at or near amino acids 181 and 197. The carboxy terminus contains a phosphatidylinositol glycolipid whose components are ethanolamine, phosphate, myo-inositol and stearic acid. This protein polymerizes into rods possessing the ultrastructural and histochemical characteristics of amyloid. Amyloid is a generic term referring to any optically homogenous, waxy, translucent glycoprotein; it is deposited intercellularly and/or intracellularly in many human diseases such as:


The prion is a product of a human gene, termed the PrP gene, found on chromosome 20. This gene contains two exons separated by a single intron. Exon I and Exon II are transcribed and the two RNAs ligated into a single mRNA. This mRNA contains an open reading frame (ORF) or protein coding region which is translated into the PrP protein. The PrP protein is a precursor of the prion protein. It is termed PrP 33-35.

The PrP 33-35 undergoes several post-translational events to become the prion protein (PrP 27-30):

In normal cells only the PrP 33-35 protein is synthesized. It is found in the neural cell membrane where it's function is to sequester Cu++ ions. In abnormal ("infected") cells, the PrP 27-30 is produced from the PrP 33-35 protein. The PrP 27-30 triggers a series of reactions that produce more PrP 27-30 proteins, i.e., PrP 27-30 induces its own synthesis. In addition to the post translational modifications, the PrP 27-30 protein differs from the PrP 33-35 protein in a single amino acid residue. Residue 178 in the PrP 27-30 contains an asparagine residue whereas the PrP 33-35 protein has an aspartate residue at this position. This causes a conformational change in the PrP 27-30 protein from an -helix to a -sheet. This conformational change in the PrP 27-30 protein has three effects:

Pathologies induced by prions

All diseases known to be of prion etiology, in animals and humans, are neurodegenerative diseases. In the human this includes: The pathological and clinical signs of these diseases suggest that they are closely related. In fact they may be variants of the same disorder. All pathological features are confined to the central nervous system. The prion protein accumulates selectively and abnormally in CNS nerve cells during the course of the disease. PrP 27-30 accumulates within the neuropil where it causes: Note that the pathology does NOT include any signs of inflammation or fever. This is evidence that the immune system does not respond to the prion protein. Since the prion protein is derived from self this is what you would expect.

These pathologies give rise to the clinical symptomology seen in these patients. These are:

1. A long incubation period (several years) which has given rise to the term "slow infection."

2. Loss of muscle coordination which leads to a difficulty in walking, indicating a functional disorder of the cerebellum.

3. Dementia characterized initially by loss of memory, diminished intellect and poor judgement.

4. Progressive insomnia characterized by a marked reduction or loss of the slow-wave and rapid-eye-movement phases. D. Transmission

Spread of the disease is via horizontal transmission, i.e., transmission from one person to another, either directly or by fomites.



Viroids are infectious agents composed exclusively of a single piece of circular single stranded RNA which has some double-stranded regions.
Structure of viroid

Because of their simplified structures both prions and viroids are sometimes called subviral particles. Viroids mainly cause plant diseases but have recently been reported to cause a human disease.

Catalytic RNAs are those that have the intrinsic ability to break and form covalent bonds; Viroids are catalytic RNA's (ribozymes) that cleave RNA to produce fragments containing a 5'-hydroxyl and a 2', 3'-cyclic phosphate.

Catalytic RNAs

This is a nonhydrolytic reaction in which the same number of phosphodiester bonds are maintained and the transesterification reaction is theoretically reversible. This reaction is considered to play an essential role in the replication of these RNAs in vivo. Such reactions are all intramolecular and hence quasi-catalytic with single turnover. These RNAs can be manipulated, however, to provide true catalytic cleavage in trans-reactions.


Circular, pathogenic RNAs are replicated by a rolling circle mechanism in vivo. There are two variations of this rolling circle mechanism:
Circular pathogenic RNAs

In the first variation (A), the circular plus strand is copied by viroid RNA-dependent RNA polymerase to form a concatameric minus strand (step 2). Site-specific cleavage (arrows) of this strand produces a monomer that is circularized by a host RNA ligase (step 3) and then copied by the RNA polymerase to produce a concatameric plus strand. Cleavage of this strand (step 5) produces monomers which, on circularization, produces the progeny circular, plus RNA, the dominant form in vivo.

In the other variation (B), the concatameric minus strand of step 1 is not cleaved but is copied directly to give a concatameric plus strand (step 3), which is cleared specifically to monomers for ligation to the circular progeny. Those RNAs that self-cleave only in the plus strand in vitro are considered to follow this route.

Human pathologies induced by viroids

The only human disease known to be caused by a viroid is hepatitis D. This disease was previously ascribed to a defective virus called the delta agent. However, it now is known that the delta agent is a viroid enclosed in a hepatitis B virus capsid. For hepatitis D to occur there must be simultaneous infection of a cell with both the hepatitis B virus and the hepatitis D viroid. There is extensive sequence complementarity between the hepatitis D viroid RNA and human liver cell 7S RNA, a small cytoplasmic RNA that is a component of the signal recognition particle, the structure involved in the translocation of secretory and membrane-associated particles. The hepatitis D viroid causes liver cell death via sequestering this 7S RNA and/or cleaving it.


The hepatitis D viroid can only enter a human liver cell if it is enclosed in a capsid that contains a binding protein. It obtains this from the hepatitis B virus. The delta agent then enters the blood stream and can be transmitted via blood or serum transfusions.


Prusiner, S.B., 1991. Molecular Biology of Prion Diseases. Science 252:1515-1522.

Medori, R., et al., 1992. Fatal Familial Insomnia, A Prion Disease with a Mutation at Codon 178 of the Prion Protein Gene. The New England Journal of Medicine 326:444-449.

Taylor, J.M., 1992. The Structure and Replication of Hepatitis Delta Virus. Annual Reviews of Microbiology 46:253-276.

Touchette, N., 1993. ß-sheet structure is key issue in prion disease. The Journal of NIH Research 5:57-59.

Barinaga, M., 1993. Ribozymes: killing the messenger. Science 263:1512-1514.

Prusiner, S.B., 1995. The Prion Diseases. Scientific American 272: 48-57.


1. Koch's postulates are a means of relating a given set of clinical symptoms to infection with a particular etiological agent.

2. Prions are infectious agents composed solely of glycoprotein. They are products of a human gene which accumulate in tissue as amyloid.

3. Amyloid deposition in tissue is a pathological manifestation of many diseases, of both prion and non-prion etiology. These diseases include Alzheimer's disease, *Creutzfeldt-Jakob disease, Down's syndrome (mongolism), *fatal familial insomnia, *Gerstmann-Straussler syndrome, *kuru and leprosy. (* indicates prion diseases).

4. The accumulation of amyloid induces these pathologies in the host: astrocyte gliosis, depletion of neuronal dendritic spines, spongiform encephalopathy.

5. In prion disease there is a long incubation period before one sees loss of muscle coordination, dementia and/or progressive insomnia.

6. Prions induce no immune reactions within the human.

7. Viroids are infectious agents composed solely of circular single-stranded RNA which folds over on itself to form some double stranded regions. These are catalytic RNAs (ribozymes).

8. The only human disease known to be caused by a viroid is hepatitis D; in this case the viroid is enclosed in a hepatitis B virus capsule.

9. The hepatitis D viroid manifests its disease potential by sequestering and/or destroying human liver 7S RNA.

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