Replication of (+)-strand RNA Viruses

Question 1

Replicative cycle of poliovirus

Answer 1

• binding to PV-receptor → capsid opening → RNA release
• polyprotein synthesis
• polyprotein processing
• protein transport into vesicles →replication
• • Strand RNA can go back to cytoplasm for packaging or another cycle of protein synthesis and packaging

Question 2

Advantages of (+)-strand RNA viruses generated vesicle like structures/networks

Answer 2

Shielding of dsRNA replicative intermediates from innate immune system
Resistance against RNase and protease
High local concentration of viral components
?contact of ER components of cell →can help virus to leave cell when assembled

Question 3

RNA structures can be signals for

Answer 3

Gene expression
Genome replication
Translation initiation
Genome packaging

Question 4

RNA structures important for genome replication and translation

Answer 4

Cloverleaf and IRES (5‘ NTR)
CRE - intragenomic cis-active replication element
Pseudoknot and poly A(3‘ NTR)

IRES - internal ribosome entry site → binds ribosome for correct placement on mRNA without scanning for AUG from 5‘ end

Question 5

Cap-independent translation initiation mechanism of + strand RNA viruses as example picornavirus

Answer 5

The virus cleaves the eIF4G with protease and therefore the initiation complex can’t bind to the 5‘ cap →shut off of host cell transcription
The mRNA of the virus contains a IRES that recruits the clever initiation complex and therefore translation of the viral mRNA is possible
The PABP is cleaved as well → no circularisation

Question 6

Genome organisation of poliovirus and cleavage of polyprotein

Answer 6

Translation of the mRNA to one polyprotein: P1 (structure proteins) and P2 & P3 (replication complex/non-structural proteins)
First processing step by 2Apro → separates P1 from P23
Further processing by 3Cpro → cleaves all other proteins

The processing of the viral polyprotein is a strictly regulated cascade, has regulatory function for replication (intermediates)

Question 7

Cascade of polyprotein processing of poliovirus

Answer 7

1. co-translational cleavage by 2Apro : seperation of the capsid protrins P1
2. Secondary cleavage of P2 and P3 by 3Cpro
3. P2 cleavage into 2Apro and 2BC and P3 cleavage into 3CDpro and 3AB
4. 2BC cleavage into 2B and 2C (helicase) and 3AB cleavage into 3Aanchor and 3Bprimer and 3CDpro cleavage into 3Cpro and 3Dpol

Extra copies of proteins are used to manipulate host cell: host cell shut off and apoptosis inhibition

Question 8

RNA replication of (+)-strand RNA viruses:

Describe the model of VPg priming in picornaviruses

Answer 8

1. Cloverleave at 5‘ end of + strand RNA binds 3CDpro and PCbp (PolyrC binding protein of host) and PAbp (polyA binding protein of host) binds to PolyA at 3‘ end of genome → proximity of ends „circularisation“
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 urridyllyation; 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 PolyA
7. Elongation of (-)strand
8. Replication form (RF) dsRNA

Question 9

Steps of production of new (+)strand RNA

Answer 9

1. PCbp at (+)strand cloverleaf binds membrane bound 3AB and recruits 3CDpro; (-)strand cloverleaf recruits 2C (helicase ?)
2. RF dsRNA become unwound by 2C, membrane bound 3AB is cleaved by 3CDpro and VPg is released
3. VPg becomes uridylylated by 3Dpol; two A‘s at 3‘ end of the (-)strand serve as partner for the annealing of VPg-pUpU
4. Elongation of VPg-pUpU primers by 3Dpol
   →total synthesis of the (+)Strand
   →multiple initiation events on one template
   ⇒Replicative intermediates (RI)

Question 10

What are the three checkpoints of Quality control in poliovirus replication?

Answer 10

1. Genome circularisation: 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 plackaging into capsid: RNA synthesis in membrane-coated vesicles (exclusion of cellular RNAs)
   ⇒Selection of RNA for packaging

Question 11

How is a clashing of transcription and replication machinery prevented?

Answer 11

Switching between both:
The mRNA is circular when replicated → 3CD binding to 5‘ and 3‘ end interferes with translation of viral RNA
⇒ switch to replication via local 3CD concentration; specific protein interaction with RNA allows righter Ribosome or replication complex attachment

Question 12

How do picornerviruses shut off the host cell?

Answer 12

→viral proteases(2A, 3C) mediate the cleavage of:

 - Translational initiation factors (eIF4G, PAbp)
 - Transcription factors
 - nuclear pore proteins →reduction of innate immunity

→2C and 3A change the lipid metabolism
-Proliferation and rearrangement of ER-membranes for viral replication
→change of Lipid composition
→change of lipase-activity
→inhibition of vesicle transport
⇒induction of apoptosis (CPE) virus uses apoptosis as cell exit

Question 13

Describe how P1 is cleaved for the morphogenesis of the virion

Answer 13

P1 is cleaved form polyprotein by 2Apro, then 3CD cleaves P1 into 1AB, 1C and 1D complex. A conformational change leads to Protomer (5S) by 3CD. 5 protomers are put together to form a pentameter (14S). 12 pentameter form the procapsid (80S). the RNA is incorporated to form a Provirion, which becomes the mature vision through maturation cleavage (P0 to VP1 and VP4)

Question 14

Name the three functions of the accessory proteins in replication complex

Answer 14

1. Restructuring the cell and localisation of RdRp
2. Positioning of RdRp to correct position in the viral genome
3. RNA helicase