Viral Genetics - DeLuca Flashcards
Composition of a virion:
Nucleic acid (genome), surrounded by a protein coat (capsid), and sometimes coated by a lipid membrane.
Describe the Hershey & Chase experiment:
- Phage radiolabeled sulfur and radiolabeled phosphorus (protein & nucleic acid).
- Infected E. coli and blended the cells. Radio-sulfur primarily in the supernatant, and radio-nucleic acid found in the cell pellet.
- Future experiments showed progeny virions only had radio labeled phosphorus detected.
Information NOT contained in virus genomes:
- Translational machinery (ribosomal RNA/proteins).
- Genes which encode energy metabolism or membrane biosynthesis
- Telomeres/centromeres
All viruses with an RNA genome must encode?
Either RNA dependent RNA polymerase (RdRp) to synthesize RNA, or reverse transcriptase to synthesize DNA from RNA.
What 2 functions do viral genomes serve during multiplication?
- Template for mRNA to be translated into viral proteins.
- Template to synthesize new genomes to be packaged into new virions.
(-) sense RNA viruses must:
Copy their genome into a (+) strand in order to be translated into protein. Must package their own RdRp.
If you had a tube of ssRNA, how could you test to know if it is (+) or (-) strand?
You could inject the genome into a cell. If it is (+) sense, it is infectious and will replicate. If it is (-) sense, it is not associated with its RdRp so it cannot replicate.
Different requirements for small and large DNA virus replication?
Small DNA viruses have a greater dependence on host replication machinery whereas larger DNA viruses can encode their own machinery.
Strategy of dsDNA viruses?
Replication: Host-cell OR virus derived DNA-dependent DNA polymerase.
mRNA synthesis: Host-cell RNA pol II
Strategy of ssDNA viruses
Either (-) or (+) strand must be converted to dsDNA.
Replication: Cellular DNA pol.
mRNA: Cellular RNA pol II
Strategy of dsRNA viruses
Replication: Virus encodes a RdRp.
mRNA: Viral RdRp
- Virions are packaged with virus encoded RdRp.
Strategy of (+)ssRNA viruses
Replication: Viral RdRp
mRNA: Genome serves as mRNA
*(+) ssRNA genomes are already infectious. RdRp is not packaged in new virions.
Strategy of Flaviviruses
(+)ssRNA viruses whose genome acts as an mRNA which is translated into a single a polyprotein. Viral/cellular proteases cleaves out individual gene products from one messenger.
Strategy of Coronaviruses
Large (+)ssRNA viruses whose genome encodes nested reading frames.
Strategy of (-)ssRNA viruses
Replication: Virus encoded RdRp
mRNA: virus encoded RdRp
Strategy of (+)ssRNA viruses with DNA intermediate (Retroviruses)
Replication and mRNA synthesis: Virus encoded reverse transcriptase copies viral RNA into (-)ssDNA. dsDNA is then made and integrated into host genome. Cellular RNA pol. II transcribes viral genes.
Strategy of gapped DNA viruses
Replication: Utilize host-cell repair machinery to become dsDNA viruses.
Importance of plaque assays
Localized regions of CPE in cell culture caused by 1 infectious particle.
Allows for isolation of clonal stocks.
Complementation in virology
Phenomenon in which two nearby mutants (which cannot grow), will grow if put into the same cell. Requires trans acting genes (acts on other genomes).
Describe the use of complementing cell-lines.
Cell lines which produce a viral gene product which acts in TRANS to propagate a mutant virus. Used to produce vectors, vaccine strains, and to study viral protein function.
Recombination between viruses:
If you infect a cell with two mutant viruses, you will get: A recombinant virus that resembles WT, complementation, and a double mutant.
The frequency of recombination is proportional to…
The distance between the mutations.
Describe a marker rescue experiment:
A mutant virus has a mutation in a gene encoding protein A. Individual genes of the wt virus are cloned into plasmids.
Step 1: The cloned wt plasmids are transfected into cells along with the mutant virus.
Step 2: Progeny are analyzed for ability to grow like wt virus or expression of protein A.
Recombination between fragments of a wt virus and a mutant genome can be used to map mutations and functions on the genome. This is called:
Marker Rescue
Mutations in specific genes can be introduced into wt virus genomes to create a mutant by recombination. This is called:
Marker Transfer
Describe cloning and manipulation in Bacterial Artificial Chromosomes (BACs)
E. coli F-plasmids have replication functions.
By marker transfer, you can take the replication function from E. coli F-plasmids and insert it into a viral genome.
You then insert this construct back into E. coli and replicate the entire viral genome. You can then introduce a mutated gene by homologous recombination and isolate the plasmid, and transfect mammalian cells to make recombinant viruses.
Why are (+)ssRNA viruses easy to reverse engineer? Why doesn’t this work in coronaviruses?
Their genomes are already infectious. This won’t work for coronaviruses because they have nested genomes.
