BIOLOGY 416
NAME:_____________________
MOLECULAR VIROLOGY
EXAM #1
WEDNESDAY FEB. 16, 2005
(A) Answer each of the following questions in the space provided.
(B) Be sure to write legibly.
A. Answer the following short answer questions. (46 POINTS TOTAL)
1. Describe the major mechanistic differences between DNA and RNA recombination (6
points).
DNA recombination – breaking and reformation of covalent bonds within the
nucleic acid strands
RNA recombination – does not involve breaking of strands; instead the RNA
polymerase switches templates by ‘jumping’. This occurs during replication of the
RNA molecule.
2. Describe in detail a quantitative assay that can be used to measure the amount of virus
in a sample. Provide enough information for the reader to perform such an assay (8
points).
Do a plaque assay:
Titrate 10x serial dilutions of virus on a susceptible monolayer of cells;
following addition of known amount of virus (usually 250 microliters), cells
overlaid with agar and incubated. Several days later plaques are visualized by
staining live cells and observing 'holes' of dead cells, count plaques.
Calculate pfu per mL.
Pfu/mL. = # of plaques x dilution plated x 1mL/0.250mL
3. List three features that are found in most eukaryotic genes (6 points).
Promoter, introns, exons, start sites and termination sites.
_______Initials
4. Virus X produces a replication protein in both brain and kidney cells, but at different
levels. Describe how you would examine the replication protein of this virus in these
cells. What reagents would you need (8 points)?
Do a Western blot on cytoplasmic extracts of both brain and kidney cells that
have been infected with the virus. You will need to run an SDS-PAGE gel of
the cells extracts which will then be transferred to a nitrocellulose membrane.
The membrane will then be blocked with nonspecific protein such as milk and
then probed with a primary antibody that is specific for the replication
protein. A secondary antibody with some kind of label
(chemiluminescent or radioactive) will be used to detect the primary
antibody. You will need film which can be exposed to the nitrocellulose blot so
that you can visualize the protein bands.
5. The genome of a (+) strand RNA virus is said to be infectious. Explain? (4 points).
The genome of a (+) strand RNA virus directly acts as an mRNA. When
introduced into cells it will use the cells translation machinery to produce
viral proteins which in turn will replicate the genome and result in the
production of virus particles. Thus, the RNA genome is itself infectious.
6. For each of the polymerase enzymes listed below give the template specificity and the
product (ie DNA-dependent DNA polymerase) (8 points).
Reverse trancriptase: RNA-dependent DNA polymerase
Eukaryotic RNA polymerase II: DNA-dependent RNA polymerase
Plus strand RNA virus polymerase: RNA-dependent RNA-polymerase
Minus strand RNA virus polymerase: RNA-dependent RNA-polymerase
BIO 416 Exam 1
Page 2
_______Initials
7. Explain the function of a cis-acting packaging sequence (6 points).
The capsid/coat protein of a virus recognizes the packaging sequence and then
encapsidates / packages that genome. It is cis-acting because the packaging
sequence helps package only the genome on which it is found.
B. Answer the following TRUE (T) OR FALSE (F). ( 3 points each, 30 POINTS
TOTAL)
___F__
8. Primary cells are usually used in the study of viruses because of their ability
to be propagated indefinitely and provide a homogeneous population of
cells to infect.
___F__
9. Recombination in RNA viruses via template switching occurs prior to RNA
replication.
___F__ 10. All mutations produce an observable phenotype
___T___ 11. Restriction endonucleases are present in many bacterial species as a
mechanism to control the entry of foreign DNA.
___F__ 12. In a persistent infection, infectious virus is never released from the cells.
___F___ 13. Penetration of viruses or their genomes into the host cell is an energyindependent event.
_ _F____ 14. The genome of a minus-strand RNA virus is sufficient to initiate an
infection when introduced by itself into a susceptible cell.
___F___ 15 Complementation between two distinct parental viruses results in progeny
virions containing new genotypes.
___T___ 16. Reassortment of influenza genome segments can occur when two
genetically different viruses infect the same cell.
BIO 416 Exam 1
Page 3
_______Initials
__T____ 17. The high error frequency of DNA viral genomes can be attributed to the
lack of proof-reading by their DNA polymerases.
C.
Answer the following problem solving question (24 points)
18. As the senior virologist at Special Pathogens Branch of the CDC, you are given a
very small sample of a novel virus that produced encephalitis in humans in the
southwestern United States. Your job is to characterize the virus and assign it into
an appropriate virus family. Describe how you would carry this out and pay
attention to detail. Make sure that you describe how you would (a) physically
analyze the virus and (b) genetically analyze the virus (what is the genome, and
what is the replication strategy).
a) 1. Amplify virus in cell culture or animal host.
2. Harvest and purify it.
3. Examine i) particle, ii) proteins, and iii) nucleic acid.
i) particle: determine if it has envelope (detergent treatment) and look at
it by EM or X-ray crystallography.
ii) Analyze protein components by gel
iii) Determine RNA/DNA, ss/ds and linear or closed circular using specific
nucleases. Also determine how many fragments per genome by analysis
on gel by Southern or Northern blotting.
b) Prepare cDNA.
1. Sequence: Determine gene order and nature of proteins.
2. Probe function by mutagenesis.
These steps will help you determine the family to which this virus
belongs.
BIO 416 Exam 1
Page 4