Lecture 6

Question 1

What are the three supergroups of +ssRNA viruses?

Answer 1

Alphavirus
Picornavirus
Flavivirus

Question 2

How are the +ssRNA viruses divided into groups?

Answer 2

Based on the phylogenetic relationship of RdRP (RNA dependent RNA polymerase

Question 3

What are the symptoms for the Systemic infection of TMV?

Answer 3

1) Colour changes among plants

2) Entire plant is infected

Question 4

What are the symptoms for the N gene infection of TMV?

Answer 4

There are necrotic local lesions on the plants, but it depends on the tobacco’s genotype, because the disease can cause different phenotypes

Question 5

What is special about TMV?

Answer 5

It is easily transmissible, and extremely stable

1) The virus is stable in dead plant debris in soil
2) Remains infectious in cigar/cigarettes
3) Virus is extremely infectious and sustains infectious in harsh conditions

Question 6

What are the key discoveries from research on TMV?

Answer 6

1898: TMV as the 1st filterable virus
1935: Wendell Stanely crystallization of TMV
1938: Ruska obtained the first EM graphs of TMV particles
1954: Helical structure of TMV particle
1955: Fraenkel-Conrat TMV self-assembly and RNA being the genetic material
1956: Franklin describing RNA helical structure within TMV particle

Question 7

What did the reconstitution experiment determine?

Answer 7

RNA and not capsid protein is the genetic material

Question 8

How was the reconstitution experiment done?

Answer 8

Common strains and HR strains were put into a hybrid virus as inoculum, and the progeny virus was made, which showed that RNA is the genetic material

Question 9

How was the local lesion assay to quantify TMV performed?

Answer 9

Francis Holmes (1929)
Spread abrasives on surface of leaf
Gently rub leaf surface with a finger and a dilution of a viral stock
Wait for infection and symptoms to develop
Count the number of local lesions
Calculate the timer of the original viral stocks
This is reminiscent of plaque assay for bacteriophages and animal viruses

Question 10

How are capsids assembled?

Answer 10

Structural unit: double disk
16.3 copies of CP per helical turn
2,130 CP subunits per virion
Built up as double discs

Question 11

How are genomes packaged?

Answer 11

5’ genome segment threads through the interior of the elongating helix
One of the first models on virion assembly
1) Hairpin structure that contains OAS
2) First double disk recognizes hairpin structure, and the RNA threads through and disk rotates

Question 12

How were coat protein mediated resistance in crop plants shown?

Answer 12

Abel (1986)
First demonstration of a transgenic resistant against a plant virus via genetic engineering:
Transgenic plants expressing TMV CP exhibited delayed onset of disease

Question 13

What is the theory for coat protein mediated resistance?

Answer 13

“Coat protein mediated protection”

Excess CP from the trans gene blocks virus disassembly, leading to resistance against the virus

Question 14

What is the genome structure for TMV?

Answer 14

+ssRNA genome, 6.4 kb (not base pairs since it is single stranded)
Genomic RNA is as the 1st mRNA to translate replication-related enzymes
5’ end: cap structure
3’ UTR: 3 pseudo-knots
3’ 3nd: tRNA structure for histidine

Question 15

What are the expression strategies for TMV?

Answer 15

MTR, Hel, RDRP, Sg promoter
There is a leaky stop codon: stop codon, but in rare cases, transcription continues even though should have stoped
Sg promoter helps with allowing 3’ ORF to translate
3’ proximal ORFS (MP and CP) can only be translated on sgRNAs
Supression of stop codon in ORF1 leads to continued translation of ORF2 (translational read through)

Question 16

What are the conserved motifs in the alpha viruses?

Answer 16

126 kDa protein (MTR, HEL)

183 kDA protein (MTR, HEL plus RdRP)

Question 17

How does a virus move within plants?

Answer 17

3 days: Initial viral infection
4 days: Floam tissue becomes infected
5 days: Virus moves across plant
25 days: Entire plant is infected with TMV

Question 18

What are the three stages of TMV movement within an infected plant?

Answer 18

Stage 1: Intracellular Movement: Upon entry, viral replication complexes form on ER, which move to other locations of the infected cell, producing multiple VRCs in the same cell
Stage 2: Intercellular (aka cell-to-cell movement): VRC’s dock at plasmodesma, traverses it with the help of movement protein (MP), and complete the replication process in neighbouring cells. Infected cell infects neighbouring cells
Stage 3: Long distance movement: Virus enters float and infects entire plant: Virions gain entry into the sieve elements of the phloem, move rapidly in it to reach distal parts of the plants

Question 19

What does the intercellular bridge do?

Answer 19

The intercellular bridge connects plant cells
Viruses highjack and travels through the micro channel from cell to cell, until they reach the float and then can infect entire plant
The virus will move up the channel in the ATPase dependent manner, and the viruses will squeeze through the channel
Size exclusion limit
Larger molecules must be transported through PD via active process
Viruses and viroids move between cells via PD carried out by MP

Question 20

How was the discovery of movement proteins made?

Answer 20

Temperature sensitive TMV mutant, Ls1 and Complementation by transgenic MP
All plant viruses encode at least one MP

Question 21

What are the properties of MP of TMV?

Answer 21

Binding RNA to form very thin (1.5 - 2 nm) and long RNPs (in vitro)
N terminal region of MP increases SEL of PD
Interacts with both the ER and actin filaments
Interaction with microtubules (C-terminal 66 amino acids)
Later shown to be involved MP degradation and not movement

Question 22

What was the old theory of TMV cell-to-cell movement?

Answer 22

MP complexes with TMV RNA to form a thin thread, moves on actin filament, interacts with p38, increases SELF, squeeze through central cavity of PD
Phosphorylation by cellular kinase releases MP, frees viral RNA

Question 23

How was TMV MP shown to be associated with peripheral ER in plant cells?

Answer 23

A gfp-tagged infectious clone of TMV was inoculated into tobacco leaves, MP: GFP fusion expressed, fluorescence microscopy detects GFP signals 
Movement protein (MP) shown to target ER network

Question 24

What is the new research to refute the old theory of TMV cell to cell movement?

Answer 24

Tracking of TMV movement with time lapse fluorescence microscopy initially infected cells vs movement between cells suggest that TMV moves as a viral replication complex (VRC) and not as MP-RNA thin threads as original proposed
14 hpi: rapid movement of VRC
16 hpi: VRC is stationary
18 hpi: No longer moves
20 hpi: where virus crosses micro channel and infects neighbouring cells. VRC moves into adjacent cells

Question 25

What is the composition and structure of VRC?

Answer 25

CPE: X-bodies (viroplasm) 
Helicase domains of 126 kD protein self-interact, forming hexameric ring-like structures 
MTR domain anchors at ER
Components of VRC:
Replicase proteins (126 & 180 kDa)
Movement Protein (MP) 
Viral RNA 
Ribosomes 
ER membrane Both 126 kD and MP are part of the VRC, which moves on actin filaments

Question 26

How are TMV as vector for VIGS and protein expression in plants?

Answer 26

VIGS: Virus-Induced Gene Silencing
RNA-based system for gene regulation and defence
Both PDY and PDS protect chlorophyll
TMV was engineered to produce mRNA for PDS or PSY, triggering RNA silencing
Infected leaves exhibit photo bleaching

Question 27

What is the next TMV battery project?

Answer 27

Dr. Ghodsii and Dr. Culver
Growing modified TMV in tobacco, purification, deposition on electrode surface in columns, coated with nickel to produce nano-rods
Increased surface area, energy and conductivity, leading to up to 10-fold increase in capacity.