Lecture 4. Introduction to Viral Replication - Influenza Virus Replication 3

Question 1

What does NS1 do?

Answer 1

NS1 has multiple proposed/identified post-transcriptional effects
Inhibits nuclear export of cellular mRNA
Increases translation of viral mRNAs
Interferon (IFN) antagonist

Question 2

How does NS1 inhibit nuclear export of cellular mRNA?

Answer 2

NS1 prevents 3’ end processing of cellular mRNAs by binding the 30 kDa component of the cellular polyadenylation/cleavage complex & preventing its binding to cellular poly(A) signal - therefore no cleavage of pre-mRNA
Binding poly(A) polymerase-binding protein and preventing its functional interaction with poly(A) polymerase - therefore no polyadenylation
Inhibition of cellular polyadenylation and therefore nuclear export

Question 3

How does NS1 increase the translation of viral mRNAs?

Answer 3

NS1 binds to the 5’ end of viral mRNAs in the cytoplasm and recruits translation initiation factors, which in turn recruit ribosomes

Question 4

What cells can’t viruses without NS1 grow in?

Answer 4

Cells with an intact IFN system
Apathogenic in normal mice

Question 5

How well do NS1 (-) viruses grown in IFN deficient cell lines?

Answer 5

Reasonably well (~10 fold lower yield than WT)

Question 6

When do NS1 (-) viruses remain pathogenic?

Answer 6

In STAT 1 -/- mice (mice that lack an immune system)

Question 7

How does NS1 prevent IFN transcription, synthesis and activation?

Answer 7

NS1 binds RIG-I and blocks sensing of viral RNA, thereby preventing induction of IFN transcription
Inhibition of cellular mRNA export blocks synthesis of IFN itself, and IFN-induced proteins
NS1 binds to, and blocks activation of the IFN-inducible dsRNA dependent kinase PKR

Question 8

What is the role of NS2?

Answer 8

Nuclear export protein, overrides movement of RNP into the nucleus and returns them to the cytoplasm

Question 9

How can RNPs re-enter the cytoplasm?

Answer 9

M1 protein and NS2 bind to RNP
Interact with and inhibit CRM1 protein

Question 10

What is an example of a drug that inhibits CRM1?

Answer 10

LMB

Question 11

How is an influenza virus particle assembled?

Answer 11

Envelope proteins have to be at cell membrane (HA, NA, M2)
Envelope proteins start to invaginate cell whilst 8 segments of the viral genome are packaged
New particle buds off

Question 12

What does NA remove from HA and other cell surface proteins in the ER and why?

Answer 12

Removes sialic acid
Prevents aggregation of new virions with each other and with the surface of the infected cell

Question 13

What are zanamivir and oseltamivir?

Answer 13

Analogues of sialic acid
Bind to and inhibit neurominidase
Only work if you give to people so it’s in the system before they are infected

Question 14

What are the two ways to ensure 1 of each of the 8 segments within the influenza virus?

Answer 14

Random and specific

Question 15

How does random packaging ensure 1 of each of the 8 segments end up in the virus?

Answer 15

Package >8 and/or live with inefficiency
Particle: pfu ratio is around 50 (for every 50 particles produced, only 1 is infectious)
9 segment viruses can be made/found
Requires minimal packaging sequence common to all segments.

Question 16

How does specific packaging ensure 1 of each of the 8 segments end up in the virus?

Answer 16

Unique cis-acting signals in each segment (specific signals are hard to find)
Each segment contains 1 or more unique sequences that act as segment-specific packaging signals
Overlap coding sequence

Question 17

What evidence is there to suggest selective packaging of each segment into the virion?

Answer 17

All influenza viruses have the same specific arrangement of RNPs, implying a specific process

Question 18

What should you print for notes in the exam?

Answer 18

The replication cycle with labels you dingus