Magor- Topic 6: Exit of viruses

Question 1

Attachment step of poliovirus

Answer 1

That attachment step is part of the opening of the capsid, making a pore & releasing that RNA into the cytoplasm.

Question 2

Assembly of poliovirus

Answer 2

1. Virus attaches to pvr
2. Uncoating at cell membrane
3. Proteins are made as polyproteins
4. Polyproteins are cleaved into their parts & synthesized
5. Initial cleavage is autocatalyzed, but then proteases are released.
6. To amplify this, that RNA is taken over to the outside of that vacuole
7. (+) strands are replicated into (-) strands with RNA-dependent RNA polymerase
8. The (-) strand is used to make more (+) strand

Question 3

When does the assembly process of poliovirus begin?

Answer 3

Once there’s lots & lots of capsid proteins as well as lots of (+) strand RNAs around.

Question 4

In poliovirus assembly, proteins are folded in the ________, then cleaved by _________

Answer 4

polyprotein, proteases

Question 5

Intramolecular interactions are favored in the intact ________

Answer 5

polyprotein

Question 6

RNA strand inside the capsid is doing the cleavage of ______

Answer 6

VP4

That’s brilliant because VP4 is inside the capsid and seals it up and makes it really solid. That step doesn’t happen unless there’s an RNA inside that capsid.
It’s the RNA itself that’s performing the catalysis.

Question 7

Cleavage of VP0 to VP2 and VP4 requires the _______

Answer 7

RNA

Question 8

How does the virus make sure that an RNA genome gets in every virion?

Answer 8

If the RNA is not in there, the final VP4 cleavage step doesn’t happen & the capsid just falls apart.

Empty capsids just sort of disassemble, reassemble, disassemble, reassemble all the time.

Question 9

One of the polio vaccines that is in progress

Answer 9

One of the vaccines that is in progress is to make just the capsid proteins & trick it to stabilize the capsid proteins basically by recombinant DNA engineering.

They’re trying to make a vaccine in a way that doesn’t need any RNA. Get that capsid stable with just the capsid & then there will be no danger of it reverting to a live infectious virus.

Question 10

Integrase and reverse transcriptase are packaged in virion.
Why does it do this?

Answer 10

So that it doesn’t have to make those proteins before it can make copies of itself & get integrated into the genome.

Question 11

What happens once HIV is integrated into the genome?

Answer 11

once HIV is integrated into the genome, it can use the host transcriptional machinery to make copies of itself as well as it can use things like we do, like alternate splicing. That allows it to pack more in that genome.

Question 12

gag gene

Answer 12

Transcription normally starts near the gag gene.
It transcribes through the gag gene, which encodes a few different proteins involved in the capsid.

And it will make a lot of those proteins because it needs a lot more capsid proteins than it does enzyme reverse transcription & integrase.

Question 13

HIV integrates into genome then uses ______________ to make more copies.

Answer 13

host transcriptional machinery

Question 14

For every 10X that HIV makes a transcript through gag, transcribing it with RNA polymerase, for the 10th time, what happens?

Answer 14

It will go through the Pol gene & make integrase & reverse transcriptase because it needs 10 capsid proteins for one of these enzymes.

Question 15

Assembly of retroviral particles from a polyprotein precursor

Answer 15

These proteins are cleaved up, but they’re already in the order that they need to be in order to pile up & pack up and make these little units (MA=matrix, CA=capsid, NC=nucleocapsid) that will then go up to the membrane & get incorporated into that virion.

Question 16

Where do the membrane proteins gp120 and gp41 get made?

Answer 16

• synthesized together as one protein & cleaved later
• proteins that were made inside the rough ER as integral membrane proteins, ultimately end up as surface proteins. And it’s the region that has all the surface proteins that’s where the viral assembly takes place.

Question 17

Secreted or TM proteins are made on the ___________ and secreted into __________, or left in ER membrane

Answer 17

rough ER, ER lumen

Question 18

Localization of viral proteins to the cellular membrane

Answer 18

1. Rough ER buds off into Golgi, and Golgi buds off into rough vesicles that fuse with the cell membrane.
2. Assembly of enveloped viruses frequently takes place at plasma membrane

When it fuses with the cell membrane, what was on the inside of that vesicle moving towards the membrane is now on the outside of the membrane.

Question 19

DNA virus can be quite large
The larger the virus = the more _______________

Answer 19

the larger the virus= the more likely it is actually gonna bring in its own polymerase and its own RNA polymerase.

Question 20

Smaller virus like parvovirus (ssDNA virus) uses ____________ and __________________

Answer 20

host DNA polymerase and host RNA polymerase

It isn’t using any of its genome to bring in a polymerase

Question 21

Larger DNA viruses encode their own ________

Answer 21

enzymes

Question 22

________ is the biggest DNA virus (~130-300 kb)

Answer 22

Pox

It brings in its own DNA polymerase & RNA polymerase

Question 23

Why does the Pox virus bring in its own DNA polymerase & RNA polymerase?

Answer 23

They’re not as high fidelity as our polymerase and so that they can evolve.
That’s why it is advantageous for the virus to be big enough to bring in its own polymerase.

Question 24

Herpes virus will bring in its own ___________ but it uses our __________________

Answer 24

DNA polymerase; host RNA polymerase

Question 25

Structure and genome organization of Herpesvirus

Answer 25

-enveloped virus
- it has a long genome with about 200 genes. Some of those genes are involved in its lifecycle.

Question 26

DNA viruses have to do a couple of things.
They’re the same orientation, the same kind of DNA as ours but our cells are not normally synthesizing DNA. We only do that in the S phase of our cell cycle. And so the virus first has to trick the cell into entering that S cycle.
What is the reason for that?

Answer 26

The reason for that is because the DNA polymerase is not necessarily around unless it is in that S cycle & the nucleotides (dNTPS, ATPs, etc.) those aren’t freely around unless the cell is in that synthesis phase.

Question 27

Common mechanisms in exponential viral DNA replication

Answer 27

1. Synthesis is template directed -you have to have a strand of DNA to make a strand of DNA
2. Each strand is copied beginning at the origins
   (replication origins= DNA replication regions; opens a bubble in the DNA)
3. Uses a DNA dependent DNA polymerase

Question 28

Generation of dNTP for DNA synthesis is only present in ________

Answer 28

S phase

Question 29

What is the most common pattern of replication of DNA of host and viral genomes

Answer 29

Bidirectional replication

Question 30

Semidiscontinuous DNA synthesis from a bidirectional origin

Answer 30

• RNA synthesis is initiated on the strand by specialized enzyme called primase
• Synthesis of leading strand starts from replication origin on RNA primer
• Lagging strand proceeds in short pieces as template becomes open (Okazaki fragments)

Question 31

We can’t initiate DNA synthesis without something for that incoming base to attack. It needs a __________ in order to put that base in place.

Answer 31

hydroxyl (OH) group

Question 32

In the same way that the poliovirus use the tyrosine, what we do and what this virus does is

Answer 32

It synthesizes a little RNA using RNA primase

Question 33

Discontinuous synthesis leaves _________ strand unfinished.

Answer 33

lagging

Removal of the primer form the 5’ end creates a gap that cannot be filled.
Successive rounds of replication result in the loss of genetic material, unless specialized mechanisms are in place to deal with it.

Question 34

“End problem”

Answer 34

• If you make a little RNA primer & then begin your DNA synthesis, & then cleave out that RNA again, then you shorten that strand.
• We get away with that because we make big long chromosomes that are full of repetitive DNA. And so every time our cells replicate, that telomere does get a little shorter.
• Viruses could get really short very quickly if they didn’t have a way to solve this end problem. And every virus doe sit differently & one common mechanism is the rolling circle mechanism.

Question 35

Rolling circle mechanism of DNA replication

Answer 35

• Viruses’ way of solving the end problem

1. The virus circularizes its linear template
2. An enzyme nicks that ds circle, revealing an OH group
3. Replicate by continuous copying
4. Displace strand
5. Complete the lagging strand
6. Double strand DNA templates result
7. Keep making more copies by continuing around again

Question 36

How do you get one genome in each capsid?

Answer 36

1. Proteins bind to the ends of a genome unit
2. Empty capsid binds to these proteins
3. The DNA is stuffed in the capsid until it reaches those proteins at the other end
4. Cleavage of DNA

Question 37

Viral DNA synthesis occurs in specialized ___________

Answer 37

compartments

DNA templates and enzymes are sequestered into discrete sites in the nucleus.

Products of one replication cycle become templates for the next.

Question 38

Host DNA polymerases proofread during DNA synthesis.
What does this mean?

Answer 38

It make a mistake= it chews it back with exonuclease activity and then goes forward correcting mistakes.

Question 39

Viral DNA polymerases may not proofread to the same fidelity as ours. What is an advantage of this?

Answer 39

They can evolve

Question 40

Acyclovir

Answer 40

• most of us are infected with Herpes simplex I which causes cold sores. Some people suffer from much worse cold sores than others. There is no vaccine, there is definitely no cure.
• There are drugs that can be used to reduce the symptoms.
• Acyclovir can be used to treat Herpes simplex symptoms and it is an inhibitor of the polymerase, preventing the DNA amplification steps.

Question 41

Herpes simplex virus latent infection

Answer 41

In the latent infection, there are transcripts that are specifically used to keep that virus latent.

Many of those drugs then are reactivated by stress.