Genetic economy: one primary transcript- multiple translation products

Question 1

NS2-3 cleavage in noncp pestiviruses is temporally regulated by DNAJC14 (Jiv)

Answer 1

Early phase: Efficient RNA replication
Late phase: Low level RNA replication/ Virion morphogenesis
* Jiv is a cellular cofactor activating the BVDV NS2 autoprotease, thereby promoting NS2-3 cleavage
* NS3 is an essential component of the viral replicase
* NS3 cannot be substituted by NS2-3 in the viral replicase
* In contrast to HCV, uncleaved NS2-3 is essential for virion morphogenesis
-> Pestiviruses use regulated polyprotein processing to enlarge their functional protein repertoire

Question 2

Noncp pestiviruses depend for their replication in cell culture on DNAJC14 expression

Answer 2

The functional importance of NS2-activation by DNAJC14 is
-> established for noncp BVDV-1 (A) and indicated for CSFV (C)
-> but unproven for BVDV-2 (B), BDV (D) and novel pestiviruses which represent tentative pestivirus species (E-J)
-> Noncp pestiviruses (with the exception of APPV) depend for their replication in cell
culture on cellular DNAJC14 expression

Question 3

Reinitiation of Translation

Answer 3

*Reinitiation is another strategy for producing two proteins from a single mRNA *10% of eukaryotic mRNAs contain uORFs (for instance, fibroblast growth factor 5)

Reinitiation depends on:
- sequence context of the uORF AUG
- RNA secondary structure between uORF and downstream ORF
- distance between the two ORFs
- viral factors

Question 4

Start codon readthrough (leaky scanning)

Answer 4

Scanning -> Start of translation at the 1. AUG

Leaky scanning -> poor initiation context (Kozak-rule) - first AUG is often ignored -> Start of translation at the 2. AUG
-> Transport of the initiation complexes from the cap to AUG not linear along the mRNA

Question 5

Multiple ways to maximize the coding potential of a mRNA: Sendai-Virus P/C gene

Answer 5

Sendai-Virus P/C gene:
- 1 gene
- multiple mRNA variants/proteins

Mechanisms:
- RNA editing
- Leaky scanning
- Ribosome shunting

Efficiency of initiation:
ACG 81 < AUG 104 < AUG 114

Shunting: AUG 183 / AUG 201

RNA editing: introduction of 0-2 G residues
0 x G = P
1 x G = V (N-term. part identical until frameshift side)
2 x G = W

Question 6

Ribosomal frameshifting - when ribosomes move into a different reading frame

Answer 6

RNA signals: „slippery sequence“ followed by a pseudoknot or a stem-loop
A „slippery sequence“ forces the ribosome into the -1 reading frame for Coronavirus and most retroviruses (Yeast Ty1: +1 reading frame) upstream of the stop codon

Retroviruses:
- Ribosome pausing because of downstream pseudoknot/stem-loop structure
- Slippage of two tRNAs into -1 frame before/after peptidyl transfer
- each tRNA pairs with the mRNA in the first two nucleotides of each codon
- 3’ base of the 3’ codon is available for binding of next incoming tRNA
- free codon in mRNA resulting in „-1 reading-frame“ of the mRNA

Efficiency (2 and 20%) depends on the sequence context of the „slippery site“
-> Regulation

Question 7

Ribosomal “frame shifting” - Rous Sarkoma Virus (RSV)

Answer 7

gag ORF: Stop: 95% of the ribosomes
pol ORF: Shift: 5% of the ribosomes

RNA secondary structures downstream of “frame shift” sites promote ribosomal frameshifting
-> RNA pseudoknots: tertiary RNA structures with base-paaring in „loop“-region

Question 8

“RNA editing”

Answer 8

• mRNA sequence does not match the coding template sequence in the viral genome
• Alteration of the mRNA sequence by
  
  • Insertion of additional nucleotides not specified by the template during synthesis
  • posttranscriptional alteration of single bases in situ
• Potential of RNA editing
  
  • shift in the reading-frame
  • C-terminal extension (stop-codon is changed to regular codon for an amino acid)
  • change in the amino acid sequence

Question 9

Posttranscriptional RNA “Editing” of Hepatitis Delta Virus (HDV)

Answer 9

dsRNA adenosine-deaminase:
- (+)-Strang RNA: I instead of A
- I basepairs with C
- in the genome: C instead of U

mRNA:
- G instead of A and UGG (Trp) instead of UAG (stop)

protein:
- L-HDAg (packaging) instead of S-HDAg (replication)

Question 10

Posttranscriptional RNA “Editing” of Hepatitis Delta Virus (HDV)

Answer 10

• adenosine desamination of A leads to I
• during replication I basepairs with C (not A with U); mRNA contains Trp-codon not the stop codon
• S-HDAg (Replication) replaced by L-HDAg (Assembly)

Question 11

Suppression of termination - Mo-MLV (Moloney murine leukemia virus)

Answer 11

• Amber stop codon (UAG) > Gln (CAG) - Efficiency of suppression 4 – 10%
• Regulation of the relative ratio of
  Gag (structural protein) vs Pol (enzyme)

Question 12

Regulation of translation: Changing the host cell

Answer 12

• „Host-cell shutoff“
• Interferon-induced cellular defense strategies
• Viral defense mechanisms

Question 13

Inactivation of the cellular eIF4F-complex by viral factors

Answer 13

Function of eIF4F (eIF4E + eIF4G + eIF4A)
- Cap-recognition
- Binding to 40S subunit
- Fusing of RNA secondary structure

eIF4A helicase
eIF4E cap-binding
eIF4G linking 5’ cap poly(A), eIF4E-binding protein

Question 14

Proteolytic cleavage of eIF4G

Answer 14

Cap-dependent initiation of translation

IRES-dependent initiation of translation
- eIF4G- cleavage by viral proteases
- cleaved eIF4G: no eIF4E-binding, but support of IRES dependent translation

Question 15

Proteolytic cleavage of PABP by Poliovirus proteinases

Answer 15

PABP interacts among others with eIF4B, eIF4G, PAIP
- important for circularization/ translation of mRNA

Question 16

Circularisation and translation of Poliovirus RNA

Answer 16

Early infection (All picornavirus RNAs (and HAV RNAs during entire infectious cycle): Strongly competitive environment, few free ribosomes and initiation factors -> Competition for translation factors

Late infection (Enteroviruses, rhinoviruses: Cleavage of eIF4G/PABP liberating ribosomes and initiation factors for IRES-driven translation) -> Release of translation factors

Question 17

Inhibition of host protein synthesis by jamming of eIF4G-PABP interaction in rotavirus infection

Answer 17

• circularization of mRNA via interaction of eIF4G and PABP stimulates translation

Rotavirus
* Viral mRNA has cap, but no poly(A)
* Viral NSP3 binds to eIF4G and so blocks the interaction eIF4G-PABP
* Viral NSP3 binds to the 3 ́end of the viral mRNA and replaces PABP in stimulation of translation for viral mRNA

Question 18

Interferon (IFN)-effect

Answer 18

• Virus-infected cells secrete IFNalpha and IFNß (Type1 IFN)
• IFN generates antiviral state in non-infected cells (ca. 300 IFN regulated genes), e.g. via RNAse L
• INF induces the synthesis of protein kinase R (PKR)
• viral dsRNA activates PKR; it then blocks translation initiation
• INF induces the synthesis of Mx protein; which inhibits viral replication e.g. of influenza viruses and other (-)-strand RNA viruses

Question 19

Inhibition of translation initiation as a cellular defense mechanism

Answer 19

• Interferon induces the synthesis of
  PKR and (RNAse L)
• Viral dsRNA activates PKR
• Active PKR phosphorylates among others eIF2α initiation factor -> Inhibition of initiation of translation

Question 20

Effect of eIF2alpha phosphorylation upon the translation initiation

Answer 20

• eIF2GTP brings initiator tRNAi to the Initiation complex
  -> eIF2GTP recycling
  -> activated PKR
  or
  ->- eIF2-P-GDP binds eIF2B (irreversible)
• no recycling of eIF2*GTP
  -> inhibition of translation initiation

Question 21

Viral strategies for inhibition of eIF2alpha-phosphorylation

Answer 21

“Cloaking” of dsRNA via viral RNA-binding proteins
- HSV-1 Us11
- Vaccinia E3L
- Reovirus omega3

Activation of cellular PKR-inhibitor I-P58
- Influenza virus

Inhibition of eIF2alpha phosphorylation
- HCMV

eIF2alpha dephosphorylation
- HSV-1 34.5

Inhibition of PKR activation by dsRNA
-> PKR-binding RNAs
- Adeno VA RNA
- EBV EBER RNA
- HIV TAR?

-> PKR-binding proteins
- HHV8 vIRF-2
- Vaccinia K3L
- EBV SM