Lecture 14- Rhabdoviruses (VSV)

Question 1

VSV

Answer 1

Vesicular Stomatitis Virus

Member of the Rhabdo virus family, also rabies virus

Question 2

The Virion

Answer 2

Glycoprotein (G)
Matrix Protein (M)
Genome RNA
N protein
L and P proteins (transcriptase)

Question 3

Glycoprotein (G)

Answer 3

On outside of the virion. Glycosylated, endows virus to bind to cell receptor (it is the VAP)
G protein has fusion activity at low pH
important for receptor mediated endocytosis

Question 4

N protein (N)

Answer 4

N protein coats the RNA genome from one end to another. RNA a coil w/in internal structure of the particle. N protein reduces secondary structure and makes RNA a good template for transcriptase and replicase. Replicase and transcriptase do very well when n protein is coating, we don’t know why but very important to straighten rna and make it a good template.
One n protein for approx every 9 nucleotides of RNA.

Question 5

L protein

Answer 5

RNA pol (transcriptase)

Question 6

P protein

Answer 6

essential for the activity of RNA pol (L protein)

Question 7

Nucleocapsid

Answer 7

Ribonucleoprotein particle/RNP

RNA plus protein are in a tight complex

Question 8

Nucleocapsid

Answer 8

Ribonucleoprotein particle/RNP

RNA plus protein are in a tight complex

Question 9

Genome Organization

Answer 9

ss- RNA 
3' end
leader RNA (non coding)
N- nucleocapsid
P- phosphoprotein (binds to L)
M- Matrix
G- glycoprotein
L- RNA polymerase (requires P for activity)
There is not a start site for transcription before every gene. Only one start site for transcription and translation.  But starts and stops, can transverse full length.  Produces monocistronic RNA by stop start restart

Question 10

Transcription

Answer 10

Layout of the genome informs how much of that gene will be transcribed
Way more N mRNA than there is L
mRNAs decrease as you go along. P seems out of place, since you would only need as much P as you would L, we don’t know the deal with this but everything else makes sense.
Transcription done by virion associated transcriptase
Transcriptase and Replicase initiate at the same site on the genome
Transcriptase (replicase) initiates at nucleotide 1 (on leader peptide) at the 3’ end of the genome

Question 11

Transcription (cont)-

Production of VSV monocistronic mRNAs (transcriptase)

Answer 11

Start-stop-restart transcription (polyA200 addition)
Minus-strand template coated with N protein
Each has gene has a poly a tail and a 5’ cap (needs 5’ cap unless it’s polio)
Uncapped leader RNA produced early on in infection; does not do anything
ig is intergenic region- where rna polymerase stops
each message not actually connected
ig region is a bunch of U’s (stop part) (poly A added by slippage at U7) and then NA
Stuttering: reiterative copying of 7 U’s makes A tail
After good size poly A tail, moves forward two nucleotides and starts UUGU sequence (restart)
If transcriptase does not restart it falls off
Gradient of transcription: probability isn’t 100%. Restart probability at ig .5 to .8
If always .8, would only make about 40% as much L as N
at last ig, ig’’, stop-add poly A, no restart
L protein can synthesize 5’ cap??

Question 12

Synthesis of G protein

Answer 12

Structure: N term, signal sequence, long stretch outside the cell (virion), 20 aa “stop transfer” sequence in lipid bilayer, 29 aa tail on cytoplasmic side of plasma membrane, C terminus
29 aa tail ends up on inside of cytoplasmic side of plasma membrane, inside envelope
signal sequence determines that this protein is destined to end up in the plasma membrane (helps designate proteins)
also in lambda: N protein is an anti termination (??) factor

Question 13

Synthesis of G protein (cont)

Answer 13

Signal sequence must be recognized by SRP to get into plasma membrane
N terminal region is signal sequence to determine that this protein at the end will be in the plasma membrane, needs to be recognized by SRP, a normal host factor.
Translation occurs on the ER (Rough ER b/c it has ribosomes bound to it, translating proteins like this).
Beginning with N terminus, it is threaded through. Signal sequence is still there.
It is threaded through the lumen of the ER, eventually cleaved by signal peptidase (found in ER, normal cellular protein).
Start stop transfer sequence is going to pass through this pore in the ER, since stop transfer sequence is very hydrophobic it will stop there, and this will be the part that sticks in the membrane.
N terminus in lumen of ER, locked in membrane by stop transfer sequence, small tail sticking out of ER

Question 14

Synthesis of G protein (cont 2)

Answer 14

Protein in membrane of ER eventually ends up becoming a trimer and gets glycosylated (both happen on the way to the golgi). Ends up in the membrane of golgi. Usually, there are multiple trimers all clustered together. Membrane of golgi fuses with plasma membrane. After membrane fusion event, end up with long N terminal region sticking out of plasma membrane and 29 aa seq (C term) on the inside
Golgi functions to transport outside cell and into membrane.

Question 15

Assembly and release

Answer 15

N protein bridges inside of the envelope with nucleocapsid.
M protein has three types of interactions
1) carboxyl interacts with 29 aa tails on cytoplasmic side of g protein trimers
2) M protein also has a hydrophobic region that embeds itself in the plasma membrane (acts as a lock)
3) recruits nucleocapsid structure (RNP particle is - RNA which is coated with N protein. L and P also bound to RNA)
in some way, promotes budding
Entire particle is pushed through the membrane and released, which is how you get the bullet shape.