RNA Viruses - Principles of replication and mRNA-synthesis of non-retroviral RNA viruses Flashcards
Viral RNA dependent RNA polymerase (RdRp)
1962: cell extracts of Poliovirus infected cells
- Contained activity of primer and template dependent incorporation of ribonucleotides
- This activity is insensitive for actinomycin D cellular RNA Pol II
- Activity associated with 3Dpol protein (cytoplasm)
- Comparable activity later also found in virions of (-)strand and ds RNA viruses
- RdRps act in the replication cycle (like all cellular DNA dep. RNA polymerases)
in a primer-independent way (Polio 3Dpol is an exception!)
- RdRps are mostly associated to membranes
- Initiation and termination of RNA synthesis take place at specific RNA sequences/structures
- Primary sequence similarities suggest common origin of viral and cellular RNA polymerases
- Poliovirus 3Dpol
- Loop E -> only in RdXPs
- Pattern “right hand”
Remodelling of membranes in cells by (+)-strand RNA viruses
All (+)-strand RNA viruses modify intracellular
membrane systems; generate vesicle-like structures or vesicle-networks
RNA replication always at membranes
The advantage:
- Shielding of ds RNA replicative intermediates from the innate immune system
- Resistance against RNases and proteases
- high local concentration of viral components
- Contact with ER components of the cell?
RNA Structures -> Important signals for
- Genome-replication
- Gene expression
- Translation initiation
- Genome packaging
RNA secondary structures: stem-loops and pseudo knots
stem-loop:
- base pairing only in the stem, not within the loops
Pseudoknots:
- basepairing in the stem
- basepairing between loops and external areas
Experimental determination:
- Prediction by computers
- RNase digestion (ss- and ds-specific, base-specific RNases)
- 2D-NMR
RNA signal structures in the enteroviral genome role in genome-replication and translation
- ss (+) strand RNA genome, 7.5 kb, polyA at 3’ end
5’ NTR: cloverleaf (CL), IRES (internal ribosome entry site)
CRE: intragenomic cis-active replication element
3’ NTR: Pseudoknot
3’: Poly A
Small (+)-strand RNA viruses
- small virus
- one RNA one poly protein
- processing into few mature proteins
Genome Organisation of poliovirus and cleavage of the polyprotein
- Synthesis of all proteins out of one polyprotein
- Processing by 2 viral proteases:
* 2Apro: separates structural and non-structural proteins
* 3Cpro: all other cleavages
* ?: maturation cleavage
Picornavirus proteinases
Entero-, Rhinoviruses: 2Apro: 1D-2A, 3Cpro: all other processing steps
FMD-Virus: Pro: L-1A, 2A: interrupted translation, 3Cpro: all other processing steps
Cardioviruses: 2A: interrupted translation, 3Cpro: all other processing steps
Hepatoviruses: 3Cpro: all processing steps, unknown host proteinase
Maturation cleavage in 1AB (VP0): unknown mechanism
The viral poly protein - a “Revell-Kit”?
Disassembly (processing) is prerequisite for functional assembly!
- Simultaneous disassembly of all parts
is not helpful! - Order of assembly is not arbitrary!
Processing of the viral polyprotein: - by proteases
- as strictly regulated cascade
- regulatory function for replication
Why do RNA viruses code for a single open reading frame (ORF)?
- Proteolytic cleavage of the polyprotein gives rise to equimolar amounts of mature, viral proteins
– For 1 virus particle 60 copies of the capsid proteins are required
– Excess of non-structural proteins? - Proteolytic cleavage of the polyproteins leads to intermediate and final products, which differ in their functions
- Increase of functional coding capacity
- Functionally polycistronic – genetically monocistronic
Example: 3C and 3CD are both proteases with different specificities; only after cleavage (removal of 3C) 3D becomes active as RdRp; the 3C part in 3CD is required for RNA binding activity of 3CD
RNA replication: Poliovirus
-„naked“ RNA is „infectious“ (formation of new virions after RNA transfection)
- RdRp not present in particle
- genome replication via complementary full-length (-) strand
- Poliovirus RNA genome carries at its 5 ́ end VPg (virus protein genome linked) instead of a CAP structure
Special attribute of picornaviruses:
- „virus protein genome linked“ (VPg)
- protein as primer
- covalently linked to genome
- a tyrosine residue in VPg is uridinylated and thereby linked to the genome
Asymmetric genome replication of picornaviruses
Imbalance of (+) and (-) strand RNAs in the infected cell
- ca 20-50x more (+) than (-) strand RNA
Poliovirus (-) strand-RNA synthesis
-> “Priming”-reaction at the 3’-end of the (+) strand RNA
PCbp: PolyrC binding protein 2 (host)
PAbp: PolyA binding protein (host)
1. Cloverleaf at 5 ́ end of (+) genome binds 3CDpro and PCbp; PAbp binds PolyA at 3 ́end of genome proximity of the ends; „circular“
2. VPg-peptide in 3AB (membrane protein) is cleaved off by 3CDpro
3. Binding of 3Dpol, 3CDpro and VPg to CRE
4. AAACA sequence in CRE is a template for
VPg uridylylation; VPgpUpU
5. Transfer of VPgpUpU to the 3 ́end of the genome as a complex made of VPgpUpU, 3Dpol and 3CDpro
6. Annealing of pUpU to poly A tail
7. Elongation of (-) strand
8. Replicative form (RF)
Replication in picornaviruses: the cis-active replicative element
CRE-loop-sequence is a template for uridylylation of VPg primers by 3Dpol
CRE functions independent from its position
in the genome
Poliovirus (+) strand-RNA synthesis
“Replicative form” (RF)
“Replicative intermediates” (RI)
PCbp at (+) strand cloverleaf binds membrane- bound 3AB; (+) strand cloverleaf recruites 3CDpro
(-) strand cloverleaf recruites 2C (helicase?)
RF dsRNA becomes unwound (by 2C helicase?); membrane bound 3AB is cleaved by 3CDpro and VPg is released
VPg becomes uridylylated by 3Dpol; two As at the 3 ́end of the (-) strand serve as partner for the annealing of VPg-pUpU
Elongation of VPg-pUpU-primers by 3Dpol
- total synthesis of the (+) strands
- multiple initiation events on a single template
An RNA Element at the 5′-End of the Poliovirus Genome Functions as a General Promoter for RNA Synthesis
Quality control in poliovirus replication
- Genomecircularisation:
Selective translation and amplification of RNA with correct ends - Coordination of translation with genome replication:
Only genomes which were translation matrices before (without internal stop codon) are replicated = RNA-replication in cis - Spatial coordination of RNA replication and packaging into capsids:
RNA synthesis in membrane-coated vesicles (exclusion of cellular RNAs)
-> Selection of RNA for packaging
Poliovirus RNA replication -> Basic problem: Specificity!
How does the viral polymerase find its template between all cellular RNAs?
- Specificity is guaranted by interactions of viral and cellular proteins with RNA-secondary structure elements
- Spatial proximity of synthesized viral proteins and template RNA in virus induced membrane vesicles (spherulae)
Spherulae:
- Replicating RNA resistent to RNases
- NTPs etc. must have access
- Shielding from immune system? (“innate immunity”; interferon)
Poliovirus RNA replication -> Host factors are essential
How was this discoverd?
1. Incubation of (+) strand RNA with cytoplasmic extracts of permissive cells: - Translation of the viral polyproteins
- Synthesis of (-) strand RNA
2. Incubation of (+) strand RNA with cytoplasmatic extracts of non permissive cells:
- poor translation of viral polyproteins
- no RNA replication
- addition of extracts of permissive cells starts (-) strand synthesis (rules out inhibitor!)
Message: essential host factors in permissive cells
Host factors for poliovirus
PCbp: PolyrC binding protein 2
PAbp: PolyA binding protein
Poliovirus RNA replication -> Imbalance of (+) and (-) strand RNAs in the infected cell
ca. 20-50 x more (+) than (-) strand RNA
Reason?
More effective initiation of (+) strand synthesis. How/why?
Reason?
(+) strand is needed for new virions;
(-) strand RNA is only a replication intermediate
Poliovirus RNA replication
-> Problem: Clash of replication complex and ribosome
Solution: switching between
1. Translation of RNA
2. Replication of RNA
Circularisation of the genome via protein bridge
Consequence: 5 ́and 3 ́genome end communicate
Host- and virus proteins bind specifically and in a regulated fashion to 5 ́CL, IRES and 3 ́end
e. g. host-PCbp displaced at IRES by viral 3CD
3CD binding to 5 ́and 3 ́end interferes with translation of the viral RNA
> Switch from translation to RNA replication via local concentration of 3CD
The specific RNA-interaction with proteins allows binding of either ribosomes or of replication complex
PCbp – polyrC-binding protein 2
Poliovirus genome replication -> Summary
- „Protein priming“
- Intragenomic „cis-acting replication element“ (CRE) serves as matrix for the uridylylation of VPg
- Product of minus-strand synthesis is dsRNA (RF, replicative Form)
- Multiple plus-strand synthesis occurs on one minus-strand (RI, Replicative Intermediate)
- Non-symmetric RNA-synthesis: ca. 20-50 x more (+) strand-genomes produced than (-) strands
Molecular basis for excess of (+) strand RNA in Alphaviruses
Family: Togaviridae
Genus: Alphavirus
- Sindbis Virus
- Semliki-Forest-Virus (SFV)
- Chikungunya-Virus (CHIKV)
Arbo-(Arthropode-borne) viruses
- Uncleaved intermediate of polyprotein processing is essential for (-) strand RNA synthesis
- Shutdown of (-) strand synthesis by complete cleavage of the non-structural proteins
- replicate on the outer surface of lysosomes
Replicon systems for (+)-strand RNA viruses
Replicon:
- autonomous replicating RNA molecule
- codes for all viral proteins needed for its own replication
- does not lead to formation of new virions
- gentechnically often produced by a defect/deletion in the structural proteins
Requirement
- Knowledge about genome segments required only for virion morphogenesis but not for genome replication
Strategy
- Replacing this genome segment with reporter gene(s) (enzymes, GFP, resistence genes…)
- Reporter gene expression correlates with replication efficiency of the replicon
Usage
- Quantification of genome replication efficiency
- Discrimination between signals involved in translation or replication
The picornaviral replicon
Requirements
– Capsid /Envelope proteins not essential for RNA replication; can be replaced by a reporter protein
– New RNA-synthesis is quantifyable via the activity of reporter genes
Discrimination between RNA-translation and RNA-replication
– Specific inhibitors of replication (guanidiniumchloride)
– Comparison with replication-defective replicons
Procedure:
Transcription of plasmid in vitro via T7 (phage) DdRp
Electroporation of RNA into the cells
Determination of luciferase activity
Replicon-assay
Luciferase-activity: directly proportional to the translation of “input” RNA plus translation of replicated RNA
Changes in PV-infected cells “shut-off” of the host cell
Viral proteases (2A, 3C) mediate cleavage of:
- Translation initiation factors (eIF4G, PAbp): Translational block
- Transcription factors: Transcriptional block
- Nuclear pore proteins: Reduction of host defense („Innate Immunity“)
The PV proteins 2C, 3A change the lipid metabolism:
- Proliferation and rearrangement of the ER-membranes for viral replication - Change of lipid composition
- Change of lipase-activity
- Inhibition of vesicle transport
- Inhibition of protein export (MHC-I)
- Reduction of host defense („Innate Immunity“)
Induction of apoptosis (cytopathic effect - CPE)
Inhibition of protein synthesis in poliovirus (PV) - infected cells
Protein synthesis after addition of radioactive amino acids
* In PV infected cells the cellular, cap-dependent translation is entirely shut down
* PV RNA is still effectively translated
* Mechanism: Cleavage of eIF-4G by viral proteinase
* Cap-independent translation of PV proteins via IRES
Apoptosis - Cellular defense against virus infections
Induction and inhibition ?
- active process induced by different stimuli, e.g. stress, virus infection
- programmed, morphologic changes and biochemical processes, among others cell shrinking, cytoplasm-blebbing, chromatin condensation, and intra-nucleosomal DNA degradation
Anti-apoptotic:
PV infection inhibits the synthesis of apoptotic enzymes
Pro-apoptotic:
PV proteinases cleave host proteins of the translation-(eIF4G) and DNA repair apparatus (PARP)
PARP - poly(ADP)-ribose polymerase: abundant nucleoprotein; involved in „DNA base excision repair“
Model: Programmed cell lysis at the right time facilitates virus release; early lysis interferes with viral replication
Exit from the cell
Poliovirus (PV)
* cytopathic replication: via cell-lysis
- Persistent replication: ??
Hepatitis A Virus (HAV)
* Persistent replication in cell cultures: ??
- „cell-to-cell“ spread: ?
Summary - Proteolytic cleavage of picornaviral poly protein
- Primary cleavage by 2Apro
– Separation of capsid protein segment (P1) and non-structural protein segment (P2-P3) - Secondary cleavage by 3Cpro or its precurser (3CDpro, 3ABCpro)
– Intermediate and mature viral proteins which play different roles in the viral life cycle - Maturation cleavage
– Conformational change into infectious capsid (virion)
-> Cleavage of host proteins by viral proteases
Functions of accessory proteins in the replication complex
- Restructuring of the cell and localization of RdRP
Influenza virus: RNA replication in the nucleus; nucleoprotein NP has nuclear localization signal (NLS) (cellular importins!)
HCV: Replication at membrane in the cytoplasm; NS4B induces ER-membrane pinch-off („membraneous web“) all proteins involved in replication are membrane-
bound, directly or via a protein partner
Apolipoprotein E / VLDL pathway required for virion morphogenesis
Poliovirus: 2C binds (-)-strand RNA (synthesis intermediate) and membrane; 3AB brings 3Dpol to the membrane - Positioning of RdRp to correct position in the viral genome
Sequences/secondary structures in RNA mediate specific protein binding; protein binding stabilizes secondary structures
Poliovirus:
3CD binds to cloverleaf-structure in (+)-strand-RNA; 3C-part mediated RNA-binding; 3Dpol specifically binds to cloverleaf-structure in (+)-strand-RNA; needs host factor poly rC binding protein 2 PCbp2
VSV:
P binds to N (bound to RNA); L (RdRp) binds to P accordingly mediates P binding of L to RNA - RNA helicases
- most (+) strand RNA viruses code for an RNA helicase Function: Unwinding of secondary structures and doublestrands
e.g: HCV NS3 contains helicase domain; essential for replication
- are often combined with a protease domain
or protease domains have RNA binding activity
