replication of RNA viruses Flashcards
what are the characteristics of viral RNA dependent RNA polymerase (RdRp)?
-contained activity of primer and template dependent incorporation of ribonucleotides
-act in a primer independent way
-insensitive for actinomycin D (in contrast to cellular pol)
-activity associated with 3Dpol Protein in cytoplasm
-replication always takes place at membranes
-has right hand pattern
-inition and termination of RNA synthesis take place at specific RNA sequences/structures
Which RdRp is an exception because it is primer dependent?
Polio 3Dpol
how can RNA viruses be classified?
double strand vs single srand vs ambisense
single stranded can either be + or -
- strands can either be segmented or unsegmented
unsegmented can either be circular or linear
Explain the replication cycle of poliovirus
1.binding to PV receptor capsid opening, RNA release
in cytoplasm
2.polyprotein synthesis
3.Polyprotein
4.Pv induced membrane vesicles:
- minus-strand synthesis: Priming reaction at 3’end: VPg is bound to CRE and uridylylated
-plus strand synthesis: Vpg is released and uridylylated by 3DPol, Vpg serves as primer
5.packaging and exit
what do all + strand RNA viruses do with membranes?
-modify intracellular membrane systems
-generate vesicle-like structures or vesicle networks
what is the advantage of membrane remodelling during replication of + RNA viruses?
shielding of ds DNA replicative intermediates from inate immune system
resistance against RNases and proteases
high local concentration of viral components
For what are RNA structures important signals?
-genome replication
-gene expression
-translation initiation
-genome packaging
name RNA secundary structures
stem loop: basepairing only in the stem, not within the loop
– kissing loop interaction
– multibranched loop
–interior loop
–hairpin loop
–bulge loop
Pseudoknots: basepairing in the stem and basepairing between loops and external areas
How can RNA secundary structures be determined experimentally?
-predicition by computers
-RNase digestion
-2D NMR
how is the ss +RNA genome structured?
5’ nTR with cloverleaf and IRES (internal ribosome entry side)
CRE (intragenomic cis-active replication element): lies in encoding capacity
3’NTR Pseudoknot
3’ Poly A
How does the cap independent translation in picornavirus work?
inactivation of translation of cellular mRNAs by
-viral protease that cleave essential eIF4G –> circularisation of cellular mRNA –> host cell shut off!!
-celavage of PABP (polA binding protein)
cleaved eIF4G binds to IRES of viral RNA –> circularisation –> Translation
Polyprocessing
how is the polioviral genome organised/the polyprotein cleaved?
Translation of genome into one Polyprotein
processing done by two viral proteases: 2APro (separates structural and non structural proteins) and 3CPro –> hierachy of cleavage
temporally and spatially ordered release of functional viral proteins
VPg covalently linked at 5’ end used as protein primer
RdRp not present in particle
genome replication via complementray full-length - strand
how is the picornaviral genome and proteins organised?
-VPg Protein bound at the 5’ end used as Primer
-VPG is encoded in 3B in genome
-encodes for protease, polymerase, membraneanchor and helicase/nTPase and capsid protein
How are viral polyproteins processed?
-by proteases
-as strictly regulated cascade
-regulatory function for replication
why do RNA viruses code for a single ORF?
-Proteolytic cleavage gives rise to equimolar amounts of mature viral proteins
-proteolytic cleavage of the polyproteins lead to intermediate and final products which differ in the functions
-increase of functional coding capacity
-functionally polycistronic, genetically monocistronic
how is the picornavirus replicated?
asymmetrically.
1.- strand synthesis
2.+ strand synthesis
imbalance of + and - strand in infected cell: +>-
How is the polio + strand sythesized in detail?
-CRE loop sequence is template for uridylylation of VPg primers by 3Dpol; CRE binding of VPg, 3CDpol and 3DPol (=covelent linking)
-no cap structure
-Elongation of Vpg uridinilated primers by 3Dpol (total synthesis of + strand, multiple initiation events on a single template)
what are the general steps of poliovirus replication?
1.circularization
2.inition of negative strand RNA synthesis
3.unwinding
4.initiation of positive strand RNA synthesis
How is the polio - strand synthesized?
-circularisation
-priming of - strand synthesis by specific binding of 3Dpol to pseudoknot at the 3’ end of plus RNA
How does CRE function in picornaviruses?
independently from its position in the genome
how does the quality control of poliovirus replication work?
1.genome circularisation
2.coordination of translation with genome replication (only genomes which have been translated before are replicated)
3.spatial coordination (RNA synthesis at membrane coated vesicles)
how does the viral polymerase of poliovirus find its template between all cellular RNAs?
-interactions of viral and cellular proteins with RNA secondary structure elements
-spatial proximity of synthesized proteins and template RNA in virus induced membrane vesicles (spherula)
why are there more + strands than - strands during the poliovirus RNA replication?
initiation of + strand synthesis is more effective because:
1.+ is eventually packed,
2.- is only an intermediate
how is the problem of clashing of replication complex and ribosome in poliovirus solved?
switching between translation and replication:
-functional circularisation of genome via protein bridge: host and virus proteins bind specifically and in regulated fashion to 5’CL, IRES and 3’end
-switch from translation to replication via local concentration via 3CD –> specific RNA interaction with proteins allows binding of either ribosomes or replication complex (3CD binding interferes with translation of viral RNA)
what is the molecular basis for excess of + strand during replication of RNA viruses?
uncleaved intermediate of polyprotein processing is essential for - strand RNA synthesis
shutdown of - strand Synthesis by complete cleavage of the non-structural proteins
what is the alpha virus cycle?
-uptake in coated vesicles
-release of virus into cytoplasm via endosome
-replication on the outer surface of lysosomes
-secretory pathway via ER/Golgi
-packing
-release
what is the genome structure like of the alpha virus and what is the advantage?
has an RNA genome and 1 subgenomic mRNA for encoding the structural proteins.
has cap structure, no VPg
advantage; much more structural proteins can be produced
what is the replicon system in regard to + strand RNA viruses? where is it used?
used in: quantification of genome replication effeciecy and discrimination between signals involved in translation or replication
replicon: autonomous replicating of RNA molecule, codes for all vieal proteins needed for its own replication, does not lead to formation of new virions
strategy: know which genome segments are required for virion morphogenesis but not for genome replication –> replacing this genome segment with reporter genes –> reporter gene expression correlates with replication efficiency of the replicon
Explain the picornaviral replicon
-capsid and envelope proteins are not essential for RNA replication –> they can be replaced by a reporter protein –> new RNA sythesis is quantifyable via activity of reporter genes
how can be discriminated between RNA translation and RNA replication in a picornaviral replicon?
use specific inhibitors of replication or compare with replication-defective replicons
explain the shut off of the host cell after polio infection. Which method can be used to show this phenomenon?
viral proteases 2A and 3C mediate cleavage of:
1.translational inition factors eIF4G and PAbp –> translational block
2.transcription factors –> transcriptional block
3.nuclear pore proteins –> reduction of host defense
2A and 3C also change the lipid metabolism
cytopathic effec t by induction of apoptosis
show by measuring the rate of protein Synthesis over time by using radioactive amino acids –> uninfected cells are constantly rising whereas infected cells fluctuate
at what timepoint does apoptosis take place in Polio infection?
Programmed cell lysis at the right time facilitates virus release, early lysis interferes with viral replication
