Ben Lipkowitz
M430 Virology
Question set #3

1. Because Picornaviruses are (+) strand RNA viruses, they synthesize one 
large polyprotein straight off the viral genome. This polyprotein is 
proteolytic and selectively cleaves itself into all of the separate viral 
proteins. Two final protein products that actively cleave the Polioviral RNA
are named 2Apro and 3Cpro. 
   Poliovirus was found to use a sequence (the IRES) on its genome/mRNA 
to bring ribosomes and mRNA together to initiate translation. The IRES can
initiate translation on mRNAs that don't have a 5' capped end. The 
cap is necessary for host mRNA translation. Polio and  other picornaviruses
shut off host mRNA translation by cleaving the eIF4G portion of the eIF4F 
protein, which binds to a protein that binds to the capped end of eukaryotic
mRNA. Poliovirus mRNA is still translated because the C-terminal end of the
eIF4G protein can still pull the ribosomal 40S subunit and the IRES secondary 
structure together. All of the other host proteins except those required 
for binding to the cap structure are necessary for poliovirus mRNA 
translation. 

2. Influenza and VSV are (-) strand viruses; they must synthesize both 
mRNAs and replicate their genomes from the same piece of (-) strand RNA. 
They both do this via a mechanism that involves a buildup of nucleocapsid 
protein (NP) molecules. When the NP binds to the ssRNA that is being 
synthesized from the original genome, the viral RNA polymerase can read 
past the end of the gene. In VSV this means it can transcribe the 
intergenic region that is skipped over in mRNA transcription. When enough 
NP builds up the RNA polymerase will transcribe the entire genome.
   Measles and VSV both utilize strand slippage to edit their mRNA. 
Measles does it to change the reading frame, and VSV does it to add a 
poly(A) tail. (see pp 179 and 352 of Flint.)
   Also, Influenza and Measles contain many other similarities because 
Influenza is an orthomyxovirus and Measles is a paramyxovirus. 

3. The rhabdovirus genome is divided into individual proteins encoding 
sequences joined by short intergenic sequences, and beginning with a 
leader sequence. 
  In the mRNA transcription mode, the leader sequence is first transcribed, 
then the polymerase moves to the intergenic region. The intergenic region 
contains a sequence of 7 Uridines in a row, which causes the polymerase to 
slip and keep transcribing this section. It will terminate transcription after
adding many copies of the section to the end of the mRNA as a poly(A) 
tail, and may or may not re-initiate transcription on the next segment of 
the genome. The rate at which mRNAs are produced decreases down the length 
of the genome because it is less likely that the polymerase will 
re-initiate several times than starting over from the 3' end. 
  The genome synthesis mode occurs when there is a buildup of 
nucleocapsid protein. The protein binds to newly synthesized RNA (and is 
already bound to the original genome) which allows the RNA polymerase to 
transcribe the intergenic sequence without terminating or slipping. It can 
copy the entire genome into a (+) stranded form, which is then copied into 
(-) stranded genomes for either mRNA synthesis or packaging into viral 
particles.

4. Reovirus is known to contain dsRNA dependent RNA polymerases and 10 
segments of dsRNA. It enters cells by endocytosis, and while most other 
viruses escape the lysozome before protease activity begins, the reovirus 
remains inside. The cellular  proteases induce conformation changes and 
cause viral RNA polymerases to  synthesize new (+) strand RNA while still 
inside the viral particle! The  new RNA is extruded through pores in  the 
nucleocapsid. Each segment is transcribed by its own RNA polymerase, which 
explains why there are a maximum of 12 segments: the RNA polymerase is 
located at the fivefold axis of symmetry, of which there are 12 in a 
dodecahedron.  When new viral particles are packaged only the (+) strand 
is packed into  the nucleocapsid. The (-) strand is then transcribed by 
RNA polymerases in the capsid.  

5. a. RNA directed and DNA directed DNA polymerase activity - this is a 
strand-displacement type of mechanism, but the enzyme pauses frequently 
and may allow a recombination event to occur. This enzyme lacks the 
editing activity of most polymerases and requires Mg2+ or Mn2+ cofactors. 

DNA unwinding activity is observed - I suppose this is required to unwind 
RNA as well.

RNase exonuclease activity is used to degrade viral RNA once it has been 
transcribed into DNA. This activity allows intramolecular recombination to 
occur and the entire genome to be copied with the u5 and u3 regions in the 
correct locations. 

b. Most mammalian retroviruses use tRNApro, tRNAlys1,2 or 3 to serve as a 
primer for RNA-dependent DNA polymerization.

c. A drug which interferes with any portion of RT activity is a likely 
candidate, because RT is never (to our knowledge) used in eukaryotic 
cells. RT is necessary for viral propagation because without it the viral 
RNA cannot be copied into a provirus which is necessary for mRNA 
transcription. 
