replication neg RNA virus

Question 1

which are the only RNA viruses replicating in the nucleus? What is the advantage?

Answer 1

Borna Virus and Influenza virus
advantage: using the splicing apparatus

Question 2

what are the fundamental characteristics of RNA minus strand replication?

Answer 2

1.- strand is not infectious
2.viral enzymes for formation of + strand needed
3.active RdRp in virus particle
4.replication via complete + strand intermediate

Question 3

what are the characteristics of the genome of Orthomyxo-,, Paramyxo, and Rhabdoviridae?

Answer 3

-serves as template for mRNA synthesis
-always in a complex with proteins
-without proteins not infectious
-viral RdRp packed into virion

Question 4

what are the characteristics of the Rhabdovirus (Vesicular Stomatits Virus VSV)?

Answer 4

Genome: not segmented and replicated in cytoplasm
5 Proteins: G (Glycoprotein), L and P (RNA polymerase), M (Matrix), N (Nucleocapsid protein –> transcription according to the attenuation model
3’end: Leader-N-P-M-G-L-5’end

viral RdRP (L): transcribes 5 monocistronic mRNAs
capping and polyadenylation

Question 5

describe the cell cycle of VSV

Answer 5

1.binding and fusion: release of helical nucleocapsid
2.synthesis of 5 mRNA and leader RNA
3.Translation of viral mRNA
4.+ Strand synthesis
5.- strand synthesis
6.processing and localization of G-Protein
7.Packaging and release with M-Protein as driving force

Question 6

what is the Attenuation model? In which virus does it play a role?

Answer 6

in VSV virus:
start of the mRNA synthesis at the 3’ end of the N gene, not the 3’ end of the genome (inefficient reinitiation after termination of transcription) –> attenuation: decreasing amounts of transcript from 3’ to 5’ (N>P>M>G>L) –> severely reducced amounts of L (RdRP)

Poly A via stuttering of viral polymerase at intergenic region

Question 7

VSV transcription: what is the mechanism behind the attenuation of transcription? What model?

Answer 7

1.Initiation of mRNA synthesis by binding of L (RdRP) in complex with three P at the 3’ end of N gene
2-Termination of mRNA synthesis at intergenic region (ig)
3.inefficient reinitiation after termination of transcription at the 3’ end of the P gene
–> start stop model

Question 8

How is the Poly A tail synthesized in VSV?

Answer 8

-Synthesis of 7A residues in IG region by VSV polymerase complementary to 7Us
-stuttering of polymerase: those 7 Us are transcribed multiple times
-Termination of mRNA synthesis at ig region after 200 A
-Inefficient reinitiation –> capping

Question 9

VSV: how does the switch between mRNA and genome synthesis work?

Answer 9

switching from mRNA synthesis (early) to replication (late) via changes in the composition of the replicase complex:
L+P = transcription
L+N+P = replication

intracellualr accumulation of amount of N decisive for switch to replication:
N=low= mRNA synthesis
N=high= replication

Question 10

what does the crystal structure of a nucleocapsid-like Nucleoprotein-RNA complex of respiratory syncytial virus look like?

Answer 10

10 N-proteins in complex with RNA Ring –> serves as template for viral RNA synthesis

Question 11

How does the rabies virus switch from transcription to replication?

Answer 11

switch controlled by intracellular concentration of M-protein

low amount of M: Transcription
high amount of M: replication

Question 12

Why are animal viruses relevant research objects?

Answer 12

-often very expensive outbreaks
-importamt experimental models
-transmission from animals to humans: zoonosis

Question 13

why does rabies virus package - strand RNA into its virion?

Answer 13

no selective packaging but strand imbalance: more - than +
very strong RNA promotor at 3’ end of + strand –> overproportional - strand synthesis

Question 14

how is the genome organized and structured in influenza virus?

Answer 14

-segmented genome: 8 segments –> 1-2 proteins (each associated with polymerase complex and NP)
-splicing –> nucleus
-reassortment
-viral RdRp (3 subunits: PB1, PB2, PA)
-cap snatching

Question 15

what is cap snatching? How does it work?

Answer 15

5’ ends of cellular pre-,RNA in nucleus is cleaved off and serves as primer for viral mRNA synthesis

mechanism:
hydrolysis of a random capped nuclear mRA via nuclease activity of viral polymerase complex.

Question 16

why is the influenza genome so variabel? why is it so problematic?

Answer 16

antigenic shift/reassortment:
exchange of surface proteins via reassortment (segments switched) leads to massiv change in antigenicity of the virus –> also possible between fowl, swine, humans: cross infections between swine and human possible but only rarely between human and birds

PROBLEM: no acquired protective immunity via previous infections or vaccinations

Question 17

describe the replication cycle of influenza a virus

Answer 17

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 including cap snatching
5.mRNA splicing of NS2/M2
6.synthesis of PA/PB1/PB2 (nuclear import)
7.export and translation of viral mRNAs
8.processing and localization of HA, NA

Question 18

what role does the ribonucleotiprotein complex (RNP) play in influenza virus?

Answer 18

genomic RNA never naked –> always associated to RNP

polymerase complex mediates circularisation of genome segments

Question 19

how does the mRNA synthesis of influenza A virus work?

Answer 19

permanent synthesis of capped and polyadenylated mRNA in nucleus:

3 RNA species: vRNA (-strand RNA), viral mRNA (cap, Poly A) and cRNA (+ strand RNA)

viral pol snatches cap –> cap-primer dependent initiation of transcribtion and elongation by viral polymerase
replication is mediated by NP

cRNA synthesis in cis
vRNA synthesis in trans

mRNA splicing

Question 20

what controls/changes the activity of the replication complex in influenza virus? what is the consequence?

Answer 20

binding of specific sequences in the viral RNAs to pol subunits

–>functionally different replication complexes catalyse transcription or replication

Question 21

what are the functions of the 5’ end cap in influenza virus?

Answer 21

-RNA stabilisation
-essential for translation initiation
-essential for binding mRNA to eIF4A which mediates ribosome binding to cap

Question 22

how is the replication of influenza virus regulated? Why is there an inbalance between vRNA and cRNA?

Answer 22

RNA promotor: corksrew structure of communicating ends –> differently efficient RNA promotors in vRNA (weak promotor for sythesis of cRNA) and cRNA (strong promotor for synthesis of vRNA) –> vRNA:cRNA=10:1

–> because cRNA is only a replication intermediate, vRNA is packed into the virion

Question 23

how are RNA segemnts of Influenza virus packed into new virions?

Answer 23

segment specific packaging signals at the 5’ and 3’ ends (NTR)

8 helical rods (genome) with different lenghts in the virion

Question 24

why is there genetic diversity of negative strand RNA viruses?

Answer 24

missing accuracy of RNA polymerase (RdRp has no proof readign activity)

segmentation allows reassortment

Question 25

How does cap snatching work in Hantavirus?

Answer 25

snatched from mature mRNA in cytoplasm (viral N-protein binds to cap of cellular mRNAs –> cellular mRNA is degraded in P-bodies but N-protein protects cap)

Question 26

compare - and + strand RNA genomes

Answer 26

-strand vs + strand:
1. genome packed into RNP complex vs not packed into RNP
2.stable against RNases vs instable
3.naked RNA non infectious vs naked RNA infectious
4.serves as matrix for +strand RNA synthesis vs serves as matrix for protein and- strand RNA synthesis
5.nucleoproteins are cooperative to maintain ss form vs RNA replication in cytoplasm, codes for helicase