reverse genetics
viral genomes cloned as dsDNA clones to manipulate it and see what phenotype it produces
infectious clone
a full length copy of the viral genome cloned into a plasmid or a vector
forward genetics
identification of rare viruses with a certain phenotype and identifying the mutation/gene responsible
whats needed for a + strand RNA in reverse genetics
just the viral genome is required to initiate an infection
what is used to develope a precise 3’ end (3)
restriction digestion sites
HDV ribozymes
Poly A signals
why does a precise 3’ end matter
to recognize the genome for replication or transcription
how to generate a precise 5’ end in reverse genetics for RNA
T7 promotor for transcription
how does DNA launched reverse transcription work
host polymerase 2 transcribes the plasmid DNA right into the viral genome RNA
how does RNA launched reverse transcription work
using a T7 transcription promotor in vitro to generate the viral RNA genome from the DNA template
what are the 4 plasmids are needed for full - strand RNA
N, P, L, RNA
elements (plasmids) of RdRp in non segmented - RNA genomes
P
L
reverse genetics for unimolecular - strand RNA
- RNA is made into cDNA with a T7 promotor added upstream
- the cDNA/T7 plasmid is added to permissive cells with three other plasmids (N, L, and P)
- in the cell, T7 polymerase transcribed viral RNA and the N, L, and P bind to it and help create infectious particles
transcription of mRNA requires that the genome is wrapped in what
N (nucleotides)
what must a cell express for reverse genetic system of a unimolecular - strand RNA
T7 RNA polymerase
what are the 3 plasmids for RdRp in segmented genomes
PA
PB1
PB2
is viral RNA ever naked? what is it?
no it is always in an RNP which contains NP and RdRp
3 subunits of RdRp
PB1
PB2
PA
explain the 12 plasmid system for influenza
there are 8 plasmids expressing vRNA and 4 helper plasmids containing the viral proteins that are needed
tell me about the 8 plasmid system for influenza
each plasmid is bidirectional and contains -vRNA and proteins needed
how is each palmist bidirectional is 8 plasmid influenza
each plasmid contains RNA polymerase 1 and 2. 1 creates the -vRNA and 2 creates the mRNA for the proteins needed
what does T7 polymerase transcribe from cDNA in - unimolecular RNA viruses
- viral RNA
what does T7 polymerase transcribe from the cDNA in reoviruses
it will transcribe a + RNA so it can get a complimentary strand created to make dsRNA
whats a BAC
larger plasmid vector that can carry very large DNA inserts and replicate stably inside E coli
very large DNA genomes are cloned into what
BAC
