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
Origin of new “New Flu” virus
-> Influenza A(H1N1) A/California/D4/2009
- completely new mix of genome segments
- sensitive against Tamiflu and Relenza
- no higher virulence
Phylogenetic tree of Influenza viruses
Swine viruses and Human viruses = Closer relationship - cross infections between human and swine possible (rarely documented)
Human viruses and Avian viruses = More distantly related - almost no cross infections between human and birds
-> Genome segments can in most cases not be exchanged between avian and human Influenza viruses!
Segmented (-)-strand RNA viruses: Orthomyxoviridae
-> Structure and genome organization of influenza A virus
Segmented Genome
- 8 genome segments
- each coding 1-2 proteins
- each associated with a polymerase complex and NP
- Splicing
- RNA-replication in the nucleus
- Reassortment
Viral RdRp
- transcribes anti-genome/genome and mRNAs
- Cap-snatching: 5’ ends of cellular pre-mRNA in nucleus is cleaved off and serves as primers for viral mRNA synthesis
- 3 subunits: PB1, PB2, PA
Replicative cycle of influenza A virus
1) Binding via HA to sialic acid residues
2) Endocytosis and pH induced fusion via HA
3) Nuclear transport of nucleocapsid via NLS in NP and in PB2
4) Synthesis of 8 mRNAs in the nucleus “Cap snatching”
5a) Export and translation of viral mRNAs
6a) Synthesis of PA, PB1, PB2 (nuclear import)
5b) mRNA splicing for NS2/M2
6b) Processing and localization of HA, NA
Ribonucleoprotein complex (RNP)
- 248 bases mini RNA- segment in complex with 9 NP and Pol-complex
- normally no ring but a helical supercoil
(only due to short RNA) - genomic RNA never naked
- always associated to nucleocapsid protein (NP)
- polymerase complex (PB1, PB2 and PA) mediates circularisation of genome segments
mRNA synthesis of influenza A virus
8 (-)-strand genome segments as nucleoprotein complex
-> Synthesis of capped and polyadenylated mRNAs
Can be inhibited in vivo by alpha-amanitin as well as actinomycin D (Pol II inhibitors)!?
-> permanent synthesis of cellular pre-mRNAs in the nucleus is essential
Molecular basis: Cap snatching
3 RNA species
vRNA: viral genomic (-)-strand RNA 3 ́and 5 ́ end conserved
viral mRNA with „Cap“: 5 ́Cap and 10-13 nucleotides of a cellular mRNA and a polyA
cRNA: (+)- strand RNA (anti-genome) compl. to (-)-strand RNA genome
Capping of eukaryotic mRNAs
Pre-mRNA is modified in the nucleus
- by different phosphatases and transferases
leading e.g. to 5 ́ 7N-methylguanosyl-cap
- becomes poly-adenylated
5 ́ cap function
- RNA stabilisation
- essential for translation initiation
- essential for binding of mRNA to eIF4A which mediates ribosome binding to cap
Cap snatching in mRNA synthesis of influenza A virus -> Procedure
Hydrolysis of a random, capped nuclear mRNA via nuclease activity in the viral polymerase complex (PA)
Viral pol initiates (+)-strand synthesis by integration of a GTP compl. to
the second last nucleotide (C)
of the (-)-strand:
Cap-primer dependent initiation of transcription
Elongation by viral polymerase
mRNA synthesis and genome replication of influenza A virus
mRNA synthesis:
- PB1 gets activated by viral (-)-strand RNA
- PB2 is „cap-binding protein“
- PA catalyzes mRNA cleavage
(not PB1; new in 2009)
Genome replication:
- Mediated by NP, since PA binds to the nucleoprotein complex (not to naked RNA)
- PA essential for de novo initiation[(+) and (-)] (without cap-primer)
- PB1 catalyzes cRNA synthesis
- PB2 without function
Functionally different replication complexes catalyse transcription or replication, respectively
Binding of specific sequences in the viral RNAs to Pol subunits controls and changes the activity of the replication complex
Activation of Influenza virus polymerase complexes for transcription
1) Inactive polymerase complex
2) Binding of specific 5 ́sequence induces activation of:
- Cap-binding activity of PB2
- 3 ́(-)-strand binding activity of PB1
-> Temporal coordination!
3) Selective binding of specific 3 ́-(-)-strand sequence induces activation of:
- Endonuclease activity of PA
- Initiation of transcription
- Elongation of mRNA
4) Elongation of mRNA to the 7 U sequence in the template
5) - RNA signal (7xU) blocks elongation
- 7 U transcribed multiple times = polyA
Influenza virus
-> Regulation of replication
vRNAs: (-)-strand segments in virion
cRNAs: (+)-strand copies; replication intermediates
- RNA promotor: „corkscrew structure“ of communicating ends
RNA promotor: Bases 1-9 at 5 ́ and 3 ́ ends
as well as 11-16 at 5 ́ and 10-15 at 3 ́end
-> differently efficient RNA promotors in vRNAs and cRNAs
Promotor on vRNA weak (for synthesis of cRNA)
Promotor on cRNA strong (for synthesis of vRNA)
Consequence: ratio of 10:1 for vRNA:cRNA
By increasing the efficiency of the cRNA-promotors, equal amounts of RNA can be generated in the cell
Base paired with the other end of the same genome segment!
Structure Influenza viral riboucleoprotein (vRNP)
Each influenza viral ribonucleoprotein (vRNP) consists of one single-stranded, negative-sense genomic RNA associated with multiple nucleoprotein (NP) monomers and a single trimeric polymerase complex (composed of PB1, PB2 and PA). The 5′ and 3′ vRNA ends are complementary and base pair to form a double-stranded structure, which is bound by the polymerase complex at one end of the vRNP filament. The internal vRNA region is organized into an antiparallel double helix, the formation of which is driven by contacts between NP monomers (known as the ‘minor’ groove), and a loop can be observed at the end of the filament opposite to that bound by the polymerase complex.
Refined model of Influenza Virus Replication/Transcription
- pol complex already present in vRNP makes transcription only in cis
- pol remains stably bound to 5 ́end of vRNA - proceeds after cap snatching with elongation of mRNA
- when it comes close to the end of the vRNA
a steric hindrance supports stuttering of pol at 7U sequence to produce poly A - after termination reinitiation takes place
at 3 ́end of vRNA template (permanent contact of pol also with 3 ́end of vRNA?) - mRNA splicing and export into cytoplasm
- translation and import of pol subunits
into nucleus - cRNA synthesis at incoming vRNPs can occur by already associated pol (in cis)
- vRNA synthesis is started by newly synthesised pol complex (in trans) at 3 ́end of cRNA; 5 ́end of the newly synthesised vRNA also recruites pol; this pol complex recruits NP molecules via protein/protein interaction
- the new protein coat of the vRNA including pol complex at 5 ́end is essential for packaging
- RNA synthesis leads to replacement of pol at 5 ́end of template
Primary transcription -> vRNP
Following nuclear entry, viral ribonucleoproteins (vRNPs) that were associated with incoming viruses transcribe viral mRNAs in cis using the resident polymerase complex bound to the double-stranded genomic ends and cellular pre-mRNA caps obtained by cap- snatching from cellular RNA polymerase II (Pol II) (known as initiation). The vRNA is then threaded through (and copied by) the viral polymerase complex (known as elongation). When the 5′ end of the vRNA is reached, it is held by the polymerase to promote the generation of the poly-A tail in conjunction with the cellular SFPQ protein (known as poly-A addition).
Genome replication -> vRNP
After new viral proteins are translated by the cellular machinery, soluble polymerases mediate genome replication in trans, which is promoted by the activity of the cellular minichromosome maintenance (MCM) complex and the Tat-SF1 and UAP56 cellular proteins. The FMR1 protein stimulates the assembly of polymerase complexes and
nucleoprotein (NP) in the presence of vRNA. The specific contributions of CLE and importin-α (IMPα) are currently unknown.
Influenza virus: Packaging of the RNA-segments into the new visions
- Segment-specific packaging signals at the 5 ́and 3 ́end (NTRs) of the segments as well as ca. 80 bases of the coding sequence in the 3 ́region
- RNA-structure for specific packaging?
- Interaction of different segments needed for packaging?
- Ordered structure of the genome segments in the virion!
- 8 helical rods in the virion
- differ in length
Multiple interactions are found among the RNPs. The position of the eight RNPs is not consistent among virions, but a pattern suggests the existence of a specific mechanism for assembly of these RNPs.
Influenza virus: Packaging signals of RNA-segments
-> Applicable for gene technology, vector development
- Foreign genes can be linked up with those signals and so be co-packaged reliably into virions
Experiment:
- Instead of hemagglutinin (HA) and neuraminidase (NA) GFP and VSV-G coded on the „HA“ and „NA“ segments
That means:
- Virion contains still 8 genome segments
- Is infectious (HA and NA functional replaced by VSV-G)
- Has a segment with terminal sequences of the NA segment for variable foreign gene expression (e.g. GFP)
Comparing the heterotrimeric RdRo of Orthomyxoviruses and the monomeric RdRp of La Crosse virus (genus Bunyavirus)
All modules found in the heterotrimeric RdRp of Influenza virus can also be found in the monomeric bunyavirus polymerase
Conclusion:
Higher similarity than expected!
Common drugs for all these RdRps?
Genetic diversity of negative-strand RNA viruses
Missing accuracy of RNA-polymerases
- RdRp without proof-reading activity (missing 3 ́->5 ́exonuclease)
- 1 mistake per 103-104 bases; accordingly 1-10 mistakes in each new genome
- Formation of quasispecies
- Influenza: „Antigenic drift“
Segmentation allows reassortment
- Co-infection with two related segmented viruses allows for reassortment
- Influenza: „Antigenic shift“
Bunyaviruses (e.g. Hantaan virus, Rift Valley fever virus)
- Proliferation in insects and mammals (change of host!)
- “Hemorrhagic fever with renal syndrome” (HFRS)
Family: Bunyaviridae
Genus:
Hantavirus
Nairovirus
Orthobunyavirus
Phlebovirus
Tospovirus
Type:
Bunyamwera virus (BUNV)
Hantaan virus (HTNV)
Dugbe virus (DUGV)
Rift valley fever virus (RVFV)
Tomato spotted wilt virus (TSWV)
Main:
Crimean-Congo hemorrhagic fever virus (CCHFV)
Hantavirus induced diseases
Disease: Hemorrhagic fever with renal failure syndrome (HFRS)
Puumala-Virus: 0.1 - 0.9% mortality
South and western Germany: Puumala-Virus East and northern parts: Dobrava-Virus
Zoonotic host: Rötelmaus
Cap-snatching of Hantaviruses
- Viral N-protein binds to cap of cellular mRNAs
- After transport of mRNA into P-bodies mRNA is degraded
- During mRNA degradation the 5 ́region and cap is protected by viral N protein
- P-bodies serve as stock for N-Cap complexes
- Protein in Hantavirus preparation (viral polymerase?) shortens 5 ́cap-RNA to ca. 17 bases
- N-bound 5 ́fragment with Cap serves as primer for viral mRNA
- N mediates already during transcription translation initiation
- Role of N during packaging of 3-segment genome: Packaging in p-bodies?
Comparing (-)- and (+)-strand RNA genomes
(-)-strand RNA
- Genome packaged in viral proteins
„ribonucleoprotein(RNP)-complex“ with RdRp in the particle; „nucleocapsid RNA“
- stabilized against RNases
- only recognized and amplified from Pol as RNP-complex „naked“ RNA non-infectious
- serves as matrix for (+)-strand-RNA synthesis
- nucleoproteins are cooperative ss-RNA binding proteins and are required in RNA synthesis to maintain the ss form (no stable doublestrand made of template and newly synthesised strand)
(+)-strand RNA
- Genome (except Retroviruses) not packaged in RNP; no RdRp within the particle
- instable against RNases
- „naked“ RNA is „infectious“ (formation of new virions after RNA transfection)
- serves as matrix for protein synthesis and (-)-strand RNA synthesis
- codes in most cases for a helicase (essential for replication, strand separation?)
- RNA replication in the cytoplasm
- dsRNA can be detected in infected cells
