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Transla'onLecture 10

Virology W3310/4310Spring 2012

The difficulty lies, not in the new ideas, but in escaping old ones...

-‐-‐JOHN MAYNARD KEYNES

1

retroviruses

PoliovirusSindbis virus Influenza virus

VSV

reovirus

hepaNNs B virus

2

• Found on most eukaryo/c mRNAs except organelle mRNAs and certain viral mRNAs

• 5'-‐7-‐methylguanosine (m7G) joined to second nucleo/de of mRNA by 5'-‐5' phosphodiester linkage

• Directs pre-‐mRNAs to processing and transport pathways, regulates mRNA turnover, required for efficient transla/on by 5'-‐end dependent mechanism

N N

N

O

HN

H2N

CH3

O

CH2 O P O PO

O-

O

O-

OHO P O CH2

O

O-

O

O O(CH3)

POO-

O CH2 O

O O(CH3)

P OOO-

5'5'

(m7)

3'

base 1

base 22'3'

5'

OH2'

3' 2'

guanosine

5’-‐cap structure

3

5’-‐untranslated region

•3 – >1,000 nt in length, typically 50 – 70 nt•OWen contains RNA secondary structures; must be unwound to allow passage of ribosome

•Length and secondary structure influence translaNon efficiency

3’-‐untranslated region

•Can regulate translaNon iniNaNon, translaNon efficiency, mRNA stability

•poly(A) tail, necessary for efficient translaNon

4

TranslaNonal machinery: Ribosomes

RNA=pink, yellowProtein=blue

5

TranslaNonal machinery: tRNAs

6

Transla'onal machinery

• Ribosomes• tRNAs• ini/a/on proteins (eIF)• elonga/on proteins (eEF)• termina/on proteins (eRF)

7

Two mechanisms of ini'a'on

• 5’-‐dependent iniNaNon (most mRNAs): iniNaNon complex binds to 5’-‐cap structure, and scans in a 3’-‐direcNon to the iniNaNon codon

• Internal ribosome entry: iniNaNon complex binds at, or just upstream of, the iniNaNon codon

8

5’-‐end dependent ini'a'on

9

Other mechanisms for decoding have been discovered in virus-‐infected cells

13

Ribosome shun'ng

-‐80 kcal/mole

14

direct transloca'on

5' scanning

Ribosome shun'ng–35s mRNAs of plant pararetroviruses

–late adenovirus mRNAs

–P/C mRNA of Sendai virus

ShunNng is predicted to decrease dependence of mRNAs for eIF4F during iniNaNon by reducing the need for mRNA unwinding

15

Internal ini'a'on

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IRES = internal ribosome entry site17

eIF4G footprint

19

Hepa''s C virus IRES

• 40S subunit binds IRES without iniNaNon proteins

• eIF3 binds IRES, needed for subunit joining

• HCV coding sequence is part of the IRES

20

all eIFs

all eIFs except eIF4E

eIF2, eIF3

21

• Viral IRESes– Picornaviruses– Flaviviruses (hepaNNs

C virus)– PesNviruses (bovine

viral diarrhea virus, classical swine fever virus)

– Retroviruses (SIV, MMLV, HTLV, FLV)

– Insect picorna-‐like viruses (cricket paralysis virus, PlauNa stali intesNne virus)

• Cellular IRESes– BiP– c-‐Myc– Antennapaedia– Ornithine decarboxylase– Fibroblast growth factor– Vascular endothelial growth factor

– protein kinase p58PITSLRE

22

AminoacylPepNdylExit

23

Methionine-‐independent ini'a'on

• Can assemble 80S ribosomes without any eIFs or Met-‐tRNAi

• RNA mimics tRNAi

24

RNA genome of cricket paralysis virus

Binds 40S ribosome independent of iniNaNon proteins

25

Maximizing the coding capacity of the viral genome

• Polyprotein (Picornaviridae, Flaviviridae, Togaviridae, Arenaviridae, Bunyaviridae, Retroviridae)

• Internal iniNaNon (IRES) (Picornaviridae, Flaviviridae)

• Leaky scanning (Retroviridae, Paramyxoviridae)

• Re-‐iniNaNon of translaNon (Orthomyxoviridae, Herpesviridae)

• Suppression of terminaNon (Retroviridae, Togaviridae)

• Ribosomal frameshiWing (Retroviridae)

27

Polyprotein synthesis

28

Leaky scanning

29

Suppression of termina'on

eRF1 and eRF3 recognize all 3 stop codons (UAA, UAG, UGA

But occasionally stop codons may be recognized by a charged tRNA

30


31


• Suppressor tRNAs: recognize terminaNon codons and insert specific amino acid, e.g. suppressor tRNA that inserts selenocysteine for UGA

• Normal tRNAs may misread terminaNon codons

32

Ribosomal frameshiOing

33

Model for -‐1 frameshiOing

34

Regula'on of transla'on in virus-‐infected cells

35

eIF2α kinases

37

PKR

38

PKR and cellular an'viral response

• PKR induced and acNvated by virus infecNon

• Leads to inhibiNon of host translaNon, apoptosis

• Different viral mechanisms have evolved to inacNvate the PKR pathway

40

Adenovirus VA RNA I prevents ac'va'on of PKR

dsRNA

active

VA RNA I

eIF2α subunitphosphorylation

P P

inactive

PKR

inactive

no phosphorylation of eIF2α subunit

active protein synthesis

protein synthesis inhibited

41

Vaccinia virus encoded pseudosubstrates mimic eIF2α

42

Targets of viral inhibitors of eIF2α phosphoryla'on

43

Regula'on of eIF4F ac'vity

• eIF4F = eIF4G + eIF4E + eIF4A

• Cleavage of eIF4GI, II in cells infected with picornaviruses

• Cleavage by proteinases 2Apro, Lpro

• Viral IRES does not require intact eIF4G

45

microRNA (miRNA)

• Small non-‐coding RNAs encoded within the viral or cellular genome

• Transcribed as 60-‐70 nt pre-‐miRNA by either pol II or pol III

• Processed by the cell to ~21 nt

48

miRNA

• >1000 human miRNAs, regulate ~60% of protein-‐coding genes

• Control gene expression post-‐transcripNonally via either repression of translaNon or mRNA degradaNon

• Mainly target 3’-‐UTR of RNA

49

miRNA

• miRNA funcNon in a complex, the miRNPs, composed of Ago, Dicer, and TRBP

• Complementarity to 3’-‐UTR determines whether the mRNA is degraded or translaNon is repressed

50

Two ways that miRNAs regulate transla'on

51

Viruses and cellular miRNAs

• miR-‐122 is a liver-‐specific miRNA required for HCV replicaNon

• miRNAs target viral or cellular genes needed for viral replicaNon

52

Virus-‐encoded miRNAs

• Viral miRs block apoptosis, facilitate immune escape, prevent cell cycle arrest, promote latency

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