Introduction to Viral Replication + Influenza (2-4) Flashcards
How to do viruses synthesise proteins?
Viruses use host cell ribosome and translation machinery to synthesise their proteins from mRNA
→ but, different viruses have different genomes so they have different strategies for genome replication and mRNA synthesis
→ almost all viruses carry their own polymerases
What are the different types of virus genomes?
DNA → double-stranded - linear, circular, gapped
→ single-stranded - +ve or -ve
RNA → double stranded - segmented, non-segmented
→ single-stranded - +ve, -ve, ambisense
Why do some viruses need their own polymerases?
Our (host) polymerases are DNA-dependant
→ RNA viruses use RNA as a template so need RNA-dependant polymerases
How are viruses classified with the Baltimore system?
Class 1 → dsDNA
Class 2 → ssDNA
Class 3 → dsRNA
Class 4 → +ve ssRNA
Class 5 → -ve ssRNA
Class 6 → +ve ssRNA RT
Class 7 → gapped dsDNA
have different mechanisms to produce mRNA
What type of virus is influenza?
Class 5 orthomyxovirus
→ segmented genome
What are some if the most prevalent influenza pandemics?
1918 → Spanish flu, 25,000,000 deaths
1956 → Asian flu, 70,000 deaths
1968 → Hong Kong flu, 34,000 deaths
2009 → Swine flu (H1N1), +450 deaths
? → H5N1 again flu, +250 deaths since 2003
→ mutation might make it highly transmissible in humans
What are the proteins present on the surface of an influenza particle?
HA → haemagglutinin - present as a trimer
NA → neuraminidase - present as a tetramer
How is influenza genome material structured?
8 segments of -ve sense ssRNA
→ wrapped in nucleoprotein with a copy of viral polymerase attached - ribonucleoprotein (RNP)
→ each segment forms helix with pan handle structure
What proteins make up the influenza viral polymerase?
3 polymerase subunits
PB1 → polymerase basic protein 1
PB2 → polymerase basic protein 2
PA → polymerase acidic protein
What do the different influenza genome segments encode?
from largest - smallest
1 → PB2
2 → PB1, PB1-F2
3 → PA, PA-X
4 → HA
5 → NP
6 → NA
7 → M1, M2
8 → NS1, NS2
What is the different between human and avian influenza strain receptors?
Human strains (e.g. H1N1) → binds sialic acid linked to galactose via α(2,6) in human respiratory epithelium
Avain strains (e.g. H5N1) → binds sialic acid linked to galactose via α(2,3) in duck guy epithelium
→ sialic acid points in a different direction - leads to difference in tropism
How does influenza enter human cells?
HA trimer (on virus membrane) binds to sialic acid on human endosomal membrane - endocytosis
→ low pH changes the conformation of HA to expose the fusion peptide - so it can be inserted into the membrane
→ H+ ions enter virus via M2 - acidification causes further conformational change - α helices fold bringing membranes close together
→ fusion of membranes and release of vRNPs from M1
What occurs to influenza contents after entry into human cells?
Fusion of viral and endosome membranes allows the contents of virus to be inserted into cytoplasm - then move to nucleus
→ the nucleoproteins on vRNPs have nuclear localisation signal - allows it to interact with nuclear pore complex
→ vRNPs imported into nucleus - influenza replicates in nucleus
What are the stages in influenza virus mRNA synthesis?
- Cleavage (cap-snatching) → to use host cell translation machinery efficiently needs to make capped mRNA - steals cap from cellular mRNAs in nucleus
→ cleaves host mRNA downstream from cap - Initiation → uses cap as a primer to initiate synthesis on its own mRNA
- Elongation → mRNA synthesised, polyadenylation occurs at 17-22 bases from the 5’ end of the genome
Why does influenza mRNA synthesis need to occur before further parts of the viral life cycle?
No further parts of the life cycle can occur until viral proteins are synthesised
→ particularly NP nucleoprotein and polymerase subunits
→ mRNA synthesis is the first thing to occur once it enters nucleus
What is cap-snatching in influenza mRNA synthesis?
Stealing caps from host cell mRNA
1 → PB1 (on viral polymerase) binds 5’ end of genome
2 → PB2 binds cap of host mRNA
3 → PB1 binds 3’ end of genome and aligns this with the host mRNA at position 10-13
4 → If the alignment is correct PA cleaves the mRNA
→ therefore viral mRNA synthesis depends on cellular mRNA synthesis by RNA Pol II to supply capped primers
What are the 3 types of RNA found in influenza virus infected cells?
(+) mRNA → for protein synthesis (95%)
(-) virion RNA → genome segments that have entered
(+) cRNA anti-genome → exact copy of virion RNA to make more copies (can’t use mRNA as its modified)
→ cRNA is unstable unless wrapped in nucleoprotein, thus replication can’t occur until proteins made (i.e. mRNA)
What is the role of NP in influenza vRNA synthesis?
NP protein binds to cRNA as soon as it is made to stabilise
→ prevents polyadenalation - allows anti-termination of the poly(A) site
→ cRNA has a different sequence - doesn’t promote cap-primes initiation and no poly(A) site (no mRNA) - only vRNA is made
What is the order of RNA production for influenza?
- mRNA is synthesised, exported to cytoplasm
- Viral proteins synthesised; Pol and NP go back to nucleus
- cRNA stabilised as cRNP (binding to NP proteins)
- Many copies of vRNA can be made from each cRNP
- vRNA stabilised as vRNP - exported to cytoplasm for assembly
How are the genes on influenza gene segment 2 translated?
Leaky scanning
→ segment 2 codes for PB1 (polymerase) and PB1-F2 (virulence factor)
→ the proteins are translated from different start codons in different reading frames (different aa - different protein)
In order for a start codon AUG to be recognised it needs to be in a particular sequence context
→ if its surrounded by a strong imitation context ribosomes are highly likely to pause and start translating
How are the genes on influenza segment 3 translated?
Ribosomal frame shifting
→ segment 3 codes for PA (endonuclease, polymerase) and PA-X (host cell shut off)
→ proteins share the same common N-terminus, ribosomal frame shifting gives different C-termini
Slippery sequence (repeated A/U) causes ribosome to pause and continue translating in new reading frame
→ e.g. prefers base pair with UUG over UUU
How are the genes on influenza segment 7 translated?
mRNA splicing
→ segment 7 codes M1 (matrix), M2 (ion channel) and M3 (unknown)
→ splicing gives 3 different mRNAs, each coding for one protein
→ various splice sites produce mRNAs of different lengths
How are the genes on influenza segment 8 translated?
mRNA splicing
→ segment codes NS1 (accessory protein) and NS2 (nuclear transport)
→ splicing gives 2 different mRNAs, each coding for one protein
→ NS1 translated from unspliced mRNA, NS2 from spliced
What are the 3 functions of NS1 protein in influenza?
- Inhibits nuclear export of cellular mRNA
- Increases translation of viral mRNAs
- Interferon antagonist
(manipulated environment to make it more favourable for the virus)
