tobacco mosaic virus

Question 1

what supergroup/superfamily is TMV part of?

Answer 1

alphaviruses (+ssRNA viruses)

Question 2

what are the symptoms of TMV infection?

Answer 2

either mosaic leaves (discoloured leaves, representative of systemic infection) or necrotic lesions (representative of a infected tobacco plant with the N gene which is resistant to systemic infection)

Question 3

how does TMV get into plant tissue?

Answer 3

mechanical transmission (damage to leaf cells)

Question 4

what does it mean that TMV is “extremely stable”?

Answer 4

it remains infectious even in dead plant debris in soil, in cigarettes, etc.

Question 5

what are some of the key virology discoveries that TMV research has helped with?

Answer 5

• (1898) TMV was 1t filterable virus (contagious living fluid)
• (1935) crystallization of TMV
• (1938) first EM images of TMV
• (1954) helical structure of TMV discovered
• (1955) discovery of a) TMV self-assembly and b) genetic material is RNA
• (1956) discovered that RNA is encased in helical structure of TMV

Question 6

what was the experiment that concluded that RNA is the genetic material in TMV?

Answer 6

the famous “reconstitution experiment” conducted by fraenkel-conrat in 1950s. found that:

• CP virion alone was not infectious
• RNA alone was infectious
• CP + RNA was infectious (mixed CP from one strain and RNA from another, resulting in a virion with same CP as RNA strain)

Question 7

how was TMV quantified?

Answer 7

using a local lesion assay on N gene tobacco leaves (similar to a plaque assay)

• abrasives (to produced lesions) were spread over a leaf, then viral stock was spread over the leaf and waited for symptoms to develop
• number of local lesions were counted and concentration of original viral stock was calculated

Question 8

how is the TMV capsid assembled and genetic material packaged into it?

Answer 8

• using double disc structural units
• hairpin structure at 3’ end of RNA is origin of assembly, and wraps around the double discs as they assemble, and the 5’ end threads through the interior of the helix

Question 9

what is coat-protein mediated protection?

Answer 9

adding TMV coat-protein genes to the tobacco plant genes (1st demonstration of transgenic resistance). in theory, excess coat-protein expressed from tobacco plant genes in the tobacco cell will interact with the CP of TMV, ‘blocking’ TMVs disassembly, all leading to TMV resistance

Question 10

what is the genome structure of TMV?

Answer 10

• +ssRNA genome (6.4 kb) therefore genomic RNA acts as 1st mRNA
• contains a 5’ cap
• 3’ end contains a tRNA structure for histidine (instead of poly A tail)
• contains 4 ORF (ORF1: MTR & HEL, ORF2: RdRP, ORF3: MP, ORF4: CP)
• ORF3 and ORF4 can only be translated as sub-genomic RNAs
• has a leaky stop codon between ORF1 and ORF2 (results in a 126 kDa protein and a 183 kDa protein)

Question 11

what happens if there is a translational readthrough between ORF1 and ORF2 of TMV genome?

Answer 11

ORF1 (MTR/HEL) produces a 126 kDa protein, whereas when there is a translational readthrough due to the leaky stop codon, a 183 kDa protein is made (MTR/HEL/RdRP)

Question 12

what are the 3 stages of TMV movement within an infected plant?

Answer 12

1. intracellular movement (upon entry, viral replication complexes form on the ER, which move to other locations of the infected cell producing multiple VRCs in the same cell)
2. intercellular/cell-cell movement (VCRs dock at plasmodesma, get through using movement proteins, and complete replication process in neighboring cells)
3. long-distance movement (virions gain entry to vascular tissue of plant to rapidly reach distal parts of the plant)

Question 13

what is the plasmodesmata (PD)?

Answer 13

the intercellular bridge (almost like channels) connecting plant cells

Question 14

what is a gene sequence that all plant viruses encode?

Answer 14

they all encode at least one movement protein