Uses Of Reverse Genetics

Question 1

What are FORWARD genetics?

Answer 1

observe phenotype
look for change in sequence
link gene to phenotype

Question 2

What are REVERSE genetics?

Answer 2

change a gene
cause a phenotype
link gene to phenotype

Question 3

What are drawbacks of forward genetics?

Answer 3

• need obvious phenotype

- sometimes multiple mutations responsible, which one is critical?

Question 4

What is an example of reverse genetics?

Answer 4

• mutate/knock out genes

- e.g. without UBX in Drosophila, duplication of the thorax

Question 5

What do the 4 proteins of Bunyavirus do?

Answer 5

M - Gn/Gc - spikes
S - NP - binding protein
L - makes RdRp
NSs - not part of structure

Question 6

What phenotypes were observed when NSs expression was blocked in Bunyavirus?

Answer 6

Growth in cell culture reduced

Pathogenicity reduced

infection caused much IFN production

NSs is meant to block the host innate immune response

Question 7

How are reverse genetics used to study function of viral protein motifs?

Answer 7

scan sequence & look for motifs
late motifs - recruit host cell factors for viral release
delete late domain, look for phenotype

e.g. PPPY (Nedd4, ESCRT complex) motif involved in VSV release

Question 8

How are reverse genetics used to study the function of sequence signals?

Answer 8

make deletions within non-translated regions

look for phenotype

e.g. HAZV - mutageneis showed terminal sequences were most important, PE1 PE2

Question 9

What are the basic principles of reverse genetics?

Answer 9

sequence viral genome 
copy into cDNA
clone into plasmid
engineer desired mutations
use engineered plasmid to re-generate viral genome

Question 10

How are DNA viruses engineered?

Answer 10

Purify virus DNA genome

modify using standard tools of recombinant DNA technology

Question 11

What is the issue with engineering large DNA viruses?

Answer 11

difficulties in handling large DNA molecules

lab techniques cannot be used

Question 12

How can large DNA viruses be mutated using recombination?

Answer 12

compile DNA genome on a BAC
engineer a fragment with desired mutation
transfect BAC
site-specific recombination

Question 13

Why is engineering RNA viruses harder than engineering DNA viruses?

Answer 13

generate cDNA version first
modify the cDNA to correspond to desired RNA changes

use DdRp - cDNA to express the viral RNA in cells

Question 14

How to generate (-) sense RNA viruses from cDNA?

Answer 14

need to supply proteins that allows for mRNA synthesis

• Nucelocapsid (RNP)
• Polymerase (2/3 proteins)

wrap up viral genome in N protein
binds polymerase & transcribes

Question 15

What is the basic structure of influenza virus?

Answer 15

8 segments
negative strand
each segment in form of RNP

assembly requires PB1, PB2, PA and NO

Question 16

Why is the influenza vaccine base on strains ‘10 months ahead’?

Answer 16

make prediction in march
look at strains infecting people in southern hemisphere winters

3 chosen strains

GISRS

Question 17

How is the 3-strain influenza vaccination made?

Answer 17

PR8, WT co-infect
segments can mix
265 new genotypes
long process to screen for the reference strain - ‘seed’

Question 18

What are the drawbacks of the influenza vaccine?

Answer 18

reassortment is slow - 3 motnhs
dependence on 170mil egg production
some H5N1 strains grow poor in eggs

Question 19

WHy is it better to generate influenza vaccine by reverse genetics?

Answer 19

8 cDNAs encode exact desired RNE genome of the 'seed'
no eggs
takes 1-2 weeks
grown in culture
rapid response to pandemics