Unusual Infectious Agents

Question 1

In addition to viruses, molecular virologists also study _______ _____ which are infectious agents that are SMALLER & SIMPLER than viruses.

These include: _________, ________ & _______.

Answer 1

Subviral particles

Viroids; satellites; prions

Question 2

Viroids are _________, ________, single-stranded ______ molecules that do not encode _______ .

Some viroids are _______ (required for replication).

Viroids exhibit extensive _______ ______ ______.

They replicate ________ when introduced into host and are capable of _______ from host to host.

Answer 2

UNENCAPSIDATED; circular; RNA; protein

Ribozymes

INTERNAL BASE PAIRING

Autonomously; migrating

Question 3

What are the two families viroids are classified into?

Answer 3

1) Pospiviroidae
2) Avsunviroidae

Each have distinct structures and modes of replication.

Question 4

(T/F) Viroid species range in length from 1,700 to 4,000 nucleotides.

Answer 4

False!

Viroid species range in length from 120 to 475 nucleotides!

Viruses range in length from 1,700 to 4,000 nucleotides

Question 5

What was the first viroid to be identified?

Are all harmful?

Answer 5

The POTATO SPINDLE TUBER VIROID was the first viroid to be identified.

Some viroids are HARMLESS whereas others cause economically important CROP diseases.

Question 6

What are the other two economically important viroids aside from the POTATO SPINDLE TUBER viroid?

Answer 6

1) Coconut CADANG-CADANG viroid (lethal infection of coconut palms)

2) Apple SCAR SKIN viroid (infection causing unappealing apple appearance)

Question 7

Match the two families of viroids to their definitions:

1) Pospiviroidae
2) Avsunviroidae

A) ROD-like and BRANCHED regions, LACK a central conserved region. Replication occurs in the CHLOROPLASTS. They are functional RIBOZYMES required for its replication.

B) ROD-like secondary structure with SMALL SINGLE STRANDED regions and a central conserved region. Replication occurs in the NUCLEUS.

Answer 7

Pospiviroidae: ROD-like secondary structure with SMALL SINGLE STRANDED regions and a central conserved region. Replication occurs in the NUCLEUS.

Avsunviroidae: ROD-like and BRANCHED regions, LACK a central conserved region. Replication occurs in the CHLOROPLASTS. They are functional RIBOZYMES required for its replication.

Question 8

(T/F) Like avsunviroidae, pospiviroidae also has ribozyme activity.

Answer 8

False!

Pospiviroidae lack ribozyme activity.

Question 9

How are viroids different than viruses?

Answer 9

Viruses are parasites of the host TRANSLATION proteins.

Viroids are parasites of the host TRANSCRIPTION proteins.

Viroids depend on cellular RNA polymerases for their replication.

Question 10

Match the following steps regarding the replication of Pospiviroidae in the nucleus:

1) Step 1
2) Step 2
3) Step 3
4) Step 4

A) Concatemeric RNAs are copied back to original sequence by the RNA pol II and are imported to the NUCLEOLUS where they are CLEAVED to individual genome lengths by cellular enzymes (RNase III). The RNAs are CIRCULARIZED by host ligase enzymes

B) Viroid RNA is IMPORTED into the NUCLEUS, likely by nuclear import proteins

C) Circular RNA is then exported to adjacent cells via microchannels called PLAMODESMATA

D) Host RNA polymerase II copies the viroid RNA by ROLLING CIRLC REPLICATION which forms linear CONCATEMERIC RNAs

Answer 10

Step 1: Viroid RNA is IMPORTED into the NUCLEUS, likely by nuclear import proteins

Step 2: Host RNA polymerase II copies the viroid RNA by ROLLING CIRLC REPLICATION which forms linear CONCATEMERIC RNAs

Step 3: Concatemeric RNAs are copied back to original sequence by the RNA pol II and are imported to the NUCLEOLUS where they are CLEAVED to individual genome lengths by cellular enzymes (RNase III). The RNAs are CIRCULARIZED by host ligase enzymes

Step 4: Circular RNA is then exported to adjacent cells via microchannels called PLASMODESMATA

Question 11

What is a concatemeric RNA?

Answer 11

A concatemeric RNA is a long continuous RNA molecule that contains multiple copies of the same RNA sequence linked in series.

Question 12

Match the following steps regarding the replication of Avsunviroidae in the chloroplast:

1) Step 1
2) Step 2
3) Step 3
4) Step 4
5) Step 5
6) Step 6

A) Concatemers undergo SELF-CLEAVAGE via its ribozyme activity and genome units are CIRCULARIZED by self-ligation (with help from host ligase)

B) Viroid RNA is imported into CHLOROPLAST

C) Circular RNA is then exported to adjacent cells via microchannels called PLASMODESMATA

D) Complementary RNA concatemers are produced by chloroplast RNA polymerase via ROLLING CIRCLE REPLICATION

E) The complementary circular RNA serves as a template for SUBSEQUENT ROUNDS of concatemeric RNA synthesis (now in the original sequence)

F) Concatemers undergo SELF-CLEAVAGE via its ribozyme activity and genome are CIRCULARIZED by self-ligation

Answer 12

Step 1: Viroid RNA is imported into CHLOROPLAST

Step 2: Complementary RNA concatemers are produced by chloroplast RNA polymerase via ROLLING CIRCLE REPLICATION

Step 3: Concatemers undergo SELF-CLEAVAGE via its ribozyme activity and genome units are CIRCULARIZED by self-ligation (with help from host ligase)

Step 4: The complementary circular RNA serves as a template for SUBSEQUENT ROUNDS of concatemeric RNA synthesis (now in the original sequence)

Step 5: Concatemers undergo SELF-CLEAVAGE via its ribozyme activity and genome are CIRCULARIZED by self-ligation

Step 6: Circular RNA is then exported to adjacent cells via microchannels called PLASMODESMATA

Question 13

What are some of the similarities between the two types of viroid replication?

Answer 13

1) Both require replication by host polymerase enzymes

2) Both use the rolling circle mechanism for replication

3) Both generate concatemers

4) Both need to circularize their genome before transmission into adjacent cells

Question 14

What are some of the differences between the two types of viroid replication?

Answer 14

Pospiviroidae:
- replicates in the NUCLEUS
- lacks ribozyme activity; concatemeric RNAs cleaved by host enzymes
- concatemer is translated back into the original sequence before being cleaved + circularized

Avsunviroidae:
- replicates in the CHLOROPLAST
- has ribozyme activity; self-cleavage of concatemeric RNAs
- concatemer is cleaved and circularized and then translated back into the original sequence, where it is further cleaved and circularized!

Question 15

1) What are some common symptoms of viroid infection in plants?

2) How do viroids cause disease?

Answer 15

1) Stunting of growth, deformation of leaves & fruit, stem necrosis, and death

2) Viroids may contribute to pathogenesis by SILENCING SPECIFIC HOST GENES DUE TO VIROID-SPECIFIC siRNAs!

Question 16

How do viroids migrate?

Answer 16

1) Viroids that have completed replication are exported from the nucleus/chloroplast and move to adjacent cells via PLASMODESMATA and can migrate systemically throughout the plant via the PHLOEM (vascular tissue).

2) Specific RNA loops & structures are required for systemic transport. A variety of host proteins bind viroids; viroid transport may require INTERACTION with HOST PROTEINS.

3) Viroids can be transmitted by CONTAMINATED farm machinery, insects, contact with infected plants; may also enter POLLEN/OVULE - transmission to seed

Question 17

Briefly summarize the general path of viroid infection.

Answer 17

Import to cell organelle (nucleus/chloroplast) –> replication –> export out of organelle –> trafficking to adjacent cells –> entry into phloem –> long-distance movement to leaves & roots –> exit from phloem into new cells

Question 18

What are satellites?

Answer 18

Satellites are SUBVIRAL agents that have RNA or DNA genomes; linear & circular forms.

They may infect plants, animals or bacteria.

Satellite lack genes required for replication and therefore depend on CO-INFECTION with a HELPER VIRUS for their propagation (replicated using helper virus enzymes).

Question 19

What are the two types of satellites? Briefly describe each.

Answer 19

1) Satellite viruses: Distinct particles that contain nucleic acid genomes that encode structural proteins that encapsidate the satellite genome.

2) Satellite RNA: RNA genome is packaged by proteins encoded in the helper virus genome, May or may not encode for other proteins.

Question 20

(T/F) Satellite RNAs generally do not encode capsid proteins.

Answer 20

True!

Question 21

Answer the questions for each: Avsunviroidae, Pospiviroidae, and Satellites.

1) Requires co-infection with helper virus:

2) Encodes protein:

3) Replication done by:

Answer 21

Avsunviroidae
1) Requires co-infection with helper virus: NO

2) Encodes protein: NO

3) Replication done by: host RNA polymerase and viroid ribozyme

Pospiviroidae
1) Requires co-infection with helper virus: NO

2) Encodes protein: NO

3) Replication done by: host RNA polymerase and host RNase

Satellites
1) Requires co-infection with helper virus: YES

2) Encodes protein: YES (satellite virus)

3) Replication done by: helper virus replication proteins

Question 22

How do satellites replicate?

Answer 22

Satellite RNA genomes are replicated by a ROLLING CIRCLE MECHANISM similar to that used by viroids, except that replication is ACHIEVED by the HELPER VIURS RNA POLYMERASE and the replication takes place in the CYTOPLASM.

Question 23

(T/F) How helper virus RNA polymerase recognizes satellite RNA genomes is not clear, largely because they do not appear to have sequence or structural similarity with the genome of the helper virus.

Answer 23

True!

Question 24

Match the following steps of replication of satellite RNA:

1) Step 1
2) Step 2
3) Step 3-6

A) Depending on the satellite, the complementary concatemers may be RESOLVED INTO GENOME LENGTHS (like by ribozymes) and COPIED or it may be copied prior to cleavage.

B) Satellite RNA enters the (plant) cell and is converted TO CIRCULAR RNA (if linear)

C) Circular RNA is replicated by ROLLING CIRCLE REPLICATION by the helper virus RNA polymerase. This reaction produces CONCATEMERIC complementary products

Answer 24

Step 1: Satellite RNA enters the (plant) cell and is converted TO CIRCULAR RNA (if linear)

Step 2: Circular RNA is replicated by ROLLING CIRCLE REPLICATION by the helper virus RNA polymerase. This reaction produces CONCATEMERIC complementary products

Step 3-6: Depending on the satellite, the complementary concatemers may be RESOLVED INTO GENOME LENGTHS (like by ribozymes) and COPIED or it may be copied prior to cleavage.

Question 25

In plants, satellites may _______ or ______ disease caused by helper virus. In some cases, satellite RNAs ______ the yield of helper viruses, which may lead to _____ disease.

The symptoms associated with satellites are thought to be due to __________ of the _____ ________.

Answer 25

Attenuate; Exacerbate; Reduce; Milder

Silencing; Host Genome (similar to viroids)

Question 26

Give an example of how satellites can silence host genomes and lead to pathogenesis.

Answer 26

Some satellites (ex. cucumber mosaic virus) produce small RNAs that has complementary sequence to plant host genome which, in turn, limits the production of chlorophyll (yellow leaves appear instead of green)

Question 27

What are prions? What do they cause?

Answer 27

Prions are infectious proteins that do not consist of ANY NUCLEIC ACID.

They cause rare, fatal, neurodegenerative disorders that affect humans and other mammals called TRANSMISSIBLE SPONGIFORM ENCEPHLOPATHIES (TSE).

Question 28

What are Transmissible Spongiform Encephalopathies (TSEs) characterized by?

Answer 28

TSEs are characterized by LONG INCUBATION periods, spongiform changes in the brain associated with LOSS OF NEURONS and overall ABSENCE of host RESPONSES.

Question 29

How was variant Creutzfeld-Jacob disease (vCJD) contracted in humans?

Answer 29

Consumption of meat from animals with bovine spongiform encephalopathy (mad cow disease)

Question 30

The three types of spongiform in the brain associated with TSEs are ______, _______, and ________.

The infected brain is composed of a collection of ____ ______ that resemble a _____.

The specific symptoms that the individual experiences likely depend on:

Answer 30

Kuru; Classic CJD; Scrapie

Tiny holes; sponge

The region of the brain that is damaged

Question 31

1) What was the first TSE identified?

2) Which animals does scrapie affect?

3) What is Scrapie linked to (ex. symptoms)?

Answer 31

1) Scrapie

2) Sheep

3) It is linked to BEHAVIOURAL CHANGES, neurodegeneration, impaired motility, excessive scratching, and death usually within 4-6 weeks from symptom onset.

*scrapie is endemic in some countries

Question 32

Why was Scrapie first thought to be caused by a virus?

Why was this hypothesis rejected?

Answer 32

Sheep farmers observed that scrapie could be transmitted to healthy sheep, suggesting an INFECTIOUS AGENT was the cause of the condition. Infectivity from extracts from scrapie-affected sheep brains was shown to PASS THROUGH FILTERS with pores small enough to retain everything but viruses!

It was rejected because the scrapie infectivity was HIGHLY RESISTANT to UV radiation, formaldehyde, heat, disinfectants, autoclaving etc, and NO NUCLEIC ACID was identified.

Question 33

1) What is PrPc?

2) What is the function of wildtype PrPc?

3) What role does it play in Scrapies?

Answer 33

1) PrPc is a MEMBRANE ASSOCIATED NEURONAL GLYCOPROTEIN that is encoded by the prnp gene which is HIGHLY conserved in many mammals (humans as well).

2) The normal function of PrPc has been difficult to determine because mice lacking both copies of the prnp gene develop relatively normally & have few obvious defects (maybe for cell adhesion + communication?)

3) The infectious agent of Scrapie is due to an altered, misfolded form of PrPc.

Question 34

What is the prion hypothesis?

Answer 34

The pathogenic component of TSE is host-encoded PrPc protein with an altered, abnormal prion conformation, called PrPsc (sc for scrapie).

The prion PrPsc converts the normal PrPc into more copies of the pathogenic PrPsc form. The PrPsc can then accumulate in the central nervous system, leading to detrimental TSE symptoms (memory loss, ataxia, visual changes, dementia, death, etc).

Question 35

Briefly describe the steps of the model of prion propagation (the prion hypothesis).

Answer 35

1) Introduction of infectivity (PrPsc)
2) Heterodimer formation (PrPc + PrPsc)
3) PrPc –> PrPsc
4) Dissociation after conversion, increasing number of infectious units!

Question 36

(T/F) PrPsc is completely degraded by proteinase K in vitro, while PrPc only produces a small fragment by proteinase K in vitro.

Answer 36

False!

PrPc is completely degraded by proteinase K in vitro, while PrPsc only produces a small fragment by proteinase K in vitro.

Question 37

PrPc has ____ β sheet structure and ____ α helical content (normal protein).

PrPsc has _____ β sheet structure and ____ α helical content (pathogenic prion).

Answer 37

Low; High

High; Low