RNA Viruses - Principles of replication and mRNA-synthesis of non-retroviral RNA viruses

Question 1

Viral RNA dependent RNA polymerase (RdRp)

Answer 1

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”

Question 2

Remodelling of membranes in cells by (+)-strand RNA viruses

Answer 2

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?

Question 3

RNA Structures -> Important signals for

Answer 3

• Genome-replication
• Gene expression
• Translation initiation
• Genome packaging

Question 4

RNA secondary structures: stem-loops and pseudo knots

Answer 4

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

Question 5

RNA signal structures in the enteroviral genome role in genome-replication and translation

Answer 5

• 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

Question 6

Small (+)-strand RNA viruses

Answer 6

• small virus
• one RNA one poly protein
• processing into few mature proteins

Question 7

Genome Organisation of poliovirus and cleavage of the polyprotein

Answer 7

1. Synthesis of all proteins out of one polyprotein
2. Processing by 2 viral proteases:
   * 2Apro: separates structural and non-structural proteins
   * 3Cpro: all other cleavages
   * ?: maturation cleavage

Question 8

Picornavirus proteinases

Answer 8

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

Question 9

The viral poly protein - a “Revell-Kit”?

Answer 9

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

Question 10

Why do RNA viruses code for a single open reading frame (ORF)?

Answer 10

• 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

Question 11

RNA replication: Poliovirus

Answer 11

-„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

Question 12

Asymmetric genome replication of picornaviruses

Answer 12

Imbalance of (+) and (-) strand RNAs in the infected cell
- ca 20-50x more (+) than (-) strand RNA

Question 13

Poliovirus (-) strand-RNA synthesis
-> “Priming”-reaction at the 3’-end of the (+) strand RNA

Answer 13

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)

Question 14

Replication in picornaviruses: the cis-active replicative element

Answer 14

CRE-loop-sequence is a template for uridylylation of VPg primers by 3Dpol

CRE functions independent from its position
in the genome

Question 15

Poliovirus (+) strand-RNA synthesis

Answer 15

“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

Question 16

Quality control in poliovirus replication

Answer 16

1. Genomecircularisation:
   Selective translation and amplification of RNA with correct ends
2. Coordination of translation with genome replication:
   Only genomes which were translation matrices before (without internal stop codon) are replicated = RNA-replication in cis
3. Spatial coordination of RNA replication and packaging into capsids:
   RNA synthesis in membrane-coated vesicles (exclusion of cellular RNAs)
   -> Selection of RNA for packaging

Question 17

Poliovirus RNA replication -> Basic problem: Specificity!

Answer 17

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)

Question 18

Poliovirus RNA replication -> Host factors are essential

Answer 18

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

Question 19

Poliovirus RNA replication -> Imbalance of (+) and (-) strand RNAs in the infected cell

Answer 19

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

Question 20

Poliovirus RNA replication
-> Problem: Clash of replication complex and ribosome

Answer 20

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

Question 21

Poliovirus genome replication -> Summary

Answer 21

• „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

Question 22

Molecular basis for excess of (+) strand RNA in Alphaviruses

Answer 22

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

Question 23

Replicon systems for (+)-strand RNA viruses

Answer 23

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

Question 24

The picornaviral replicon

Answer 24

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

Question 25

Replicon-assay

Answer 25

Luciferase-activity: directly proportional to the translation of “input” RNA plus translation of replicated RNA

Question 26

Changes in PV-infected cells “shut-off” of the host cell

Answer 26

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)

Question 27

Inhibition of protein synthesis in poliovirus (PV) - infected cells

Answer 27

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

Question 28

Apoptosis - Cellular defense against virus infections

Answer 28

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

Question 29

Exit from the cell

Answer 29

Poliovirus (PV)
* cytopathic replication: via cell-lysis
- Persistent replication: ??

Hepatitis A Virus (HAV)
* Persistent replication in cell cultures: ??
- „cell-to-cell“ spread: ?

Question 30

Summary - Proteolytic cleavage of picornaviral poly protein

Answer 30

• 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

Question 31

Functions of accessory proteins in the replication complex

Answer 31

1. 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
2. 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
3. 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