Replication and RNA synthesis of (-)-strand RNA viruses Flashcards
Cellular loci of viral RNA synthesis
Cytoplasm
- Location of replication of nearly all RNA viruses
- Location of mRNA synthesis
- Replication does not take place free in the cytoplasm but at membrane structures, cytoskeleton
- High local concentration of viral components increases efficiency
- Packaging most often at membranes
- Problem: host activities located in the nucleus, like capping, must be performed by the virus
Nucleus
- The only RNA viruses replicating in the nucleus are Influenza- and Borna viruses
- Advantage: Use of the splicing apparatus
- Nuclear transport mechanisms for RNA and proteins; e.g. Influenza NP with „NLS“
Replication and RNA synthesis of (-)-strand RNA viruses
Segmented e.g. Influenza virus
non segmented (Mononegavirales, e.g. VSV)
- RNA of (-)-strand RNA viruses is not infectious
- viral enzymes for formation of (+)-strand essential
- active RdRP in virus particle
- replication via complete (+)-strand intermediate
(-)-strand-RNA viruses: Orthomyxo-, Paramyxovirus, Rhabdoviridae -> Genome (segmented or not segmented)
- serves as template for mRNA-synthesis
- always in a complex with proteins
- without those proteins not infectious
- viral RdRP always packed into virion
(-)-strand-RNA viruses
Rhabdovirus: Vesicular Stomatitis Virus (VSV)
-> The VSV is Bullet-Shaped
Genome not segmented
- Serves as template for mRNA-synthesis
- always in complex with proteins
- is replicated in the cytoplasm
Viral RdRP (L)
- Transcribes antigenome/genome
- Transcribes 5 monocistronic mRNAs
- Capping, polyadenylation
1) Binding and fusion (release of helical nucleocapsid)
2) Synthesis of 5 mRNAs (and “leader RNA”)
3) Translation of viral mRNAs
4) (+)-strand-synthesis (N, P and L)
5) (-)-strand synthesis (N, P and L)
4b) Processing and localization of G-protein
5b) Packaging and release (M-protein as driving force)
Genome Organisation and mRNA-synthesis of VSV
Attenuation model
- Start of mRNA synthesis always at 3 ́end of N gene (not 3 ́ end of genome!)
- inefficient reinitiation after termination of transcription
- Attenuation: decreasing amounts of
transcript from 3 ́ to 5 ́ (N>P>M>G>L)
leading to severely reduced amounts of L (RdRp)
- PolyA via stuttering of viral polymerase
at the 7xU sequence in the „intergenic region“
Start-stop model of mRNA-synthesis in VSV
-> Term: Attenuation of Transcription!
- Initiation of mRNA synthesis by binding of L (RdRP) in complex with 3 x P at the 3 ́end of the N gene (not at 3 ́end of the genome)
- Termination of mRNA synthesis at „intergenic region“(ig)
- Ineffcient reinitiation after termination of transcription at the 3 ́end of the P gene
- Attenuation: decreasing amounts of
mRNA transcripts from 3 ́ to 5 ́ (N>P>M>G>L)
high levels of N, far less L (RdRP)
PolyA-synthesis by VSV polymerase
- Synthesis of 7A residues complementary to 7 U residues in the IG region (sequence of IG region is a signal in cis)
- Stuttering of polymerase; those 7 Us are transcribed multiple times
- Termination of mRNA synthesis at „intergenic region“(ig) after about synthesis of 200 nucleotides (A200)
- Inefficient reinitiation at 3 ́UUGUC and „capping“ of the newly synthesized mRNA
Term: Attenuation!
VSV: Switching between mRNA and genome synthesis
- Low N concentration (early after infection) favors mRNA synthesis (RdRp is not highly processive)
- High N concentration favors replication (processive RdRp)
- Start of genome replication exactly at 3 ́ end of (-)-strand-genome
- Switching from mRNA synthesis (transcription; early) to replication (late) via changes in the composition of the replicase complex:
L + P = transcription
L + N + P = replication - Complex of N and P attaches during its synthesis to the (+)-strand and inhibits poly-adenylation and termination (allows for readthrough at IG regions)
- Intracellular accumulation of amount of N decisive for switch to replication
Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus
VSV proteins N and M
1 and 2 indicate the outer and inner leaflets of the phospholipid bilayer envelope
N-term. domain of M serves as linker to N layer (hub)
Each virion contains two nested, left-handed helices:
an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid.
Side-by-side interactions between neighboring N subunits are critical for the
nucleocapsid to form a bullet shape.
G Glycoprotein layer
Why does rabies virus package (-)-strand RNA into its virion?
- no selective packaging, but 49:1 (-):(+) strand-imbalance on synthesis level
- molecular basis: very strong RNA promotor at the 3 ́end of (+)-strand
Consequence: overproportional (-)-strand-synthesis
Proof: Rabies virus made with strong RNA promotor at the 3 ́end of (-)-strand gives a 1:1 (-):(+)-strand ratio
Consequence: both strands are packaged in equal amounts
Switch from transcription to replication controlled by intracellular concentration of M-protein
- low amount of M: Transcription
- high amount of M: Replication
Rhabdovirus: Vesicular Stomatitis Virus (VSV)
- Binding and fusion (release of the helical nucleocapsid)
- Synthesis of 5 mRNAs (and “leader RNA”)
- Translation of viral mRNAs
- Processing and localization of G-protein
- Packaging and release (M-protein as the driving force)
- (+)-strand-synthesis (N, P and L)
- (-)-strand synthesis (N, P and L)
Influenza virus
-> Spanish Flu (1918)
- wordwide pandemic
- 2 waves in spring and fall 1918
- estimated 20 million death
- Virus probably derived from birds
- Transmission to humans
- Very efficient spread human to human
- Atypical age distribution of victims
- Atypical pathology
„The 1918 flu filled hospitals,
decreased life expectancy significantly“
Influenza viruses and their virulence
- Saisonal Influenza viruses (efficient human to human transmission, 0.02 % case fatality)
- 1918 Virus/ “Spanish Flu” (efficient human to human transmission, > 1 % case fatality rate)
- H5N1 Virus from fowl (since 1997 in south-east Asia) (extremely inefficient human to human transmission, 60 % case fatality rate)
- New Flu “Schweininfluenza” Virus H1N1 (efficient human to human transmission, 0.1 % case fatality rate)
Variability of Influenza Virus: New-Mixing of RNA-Genome Segments
Problem Reassortment: “antigenic shift”
- viral surface proteins (HA/NA) are main antigens for the immunystem
- exchange of surface proteins by reassortment leads to massiv change in antigenicity of the virus
- no acquired protective immunity via previous infections or vaccinations
Viruses with high virulence
- Change of host species without sufficient adaptaion to new host
German: “hoch virulent”; falsch ist: hoch pathogen
English: both highly pathogenic or highly virulent