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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

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retroviruses

PoliovirusSindbis  virus Influenza  virus

VSV

reovirus

hepaNNs  B  virus

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• 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

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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

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TranslaNonal  machinery:  Ribosomes

RNA=pink,  yellowProtein=blue

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TranslaNonal  machinery:  tRNAs

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Transla'onal  machinery

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

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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

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5’-­‐end  dependent  ini'a'on

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

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Ribosome  shun'ng

-­‐80  kcal/mole

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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

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Internal  ini'a'on

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IRES  =  internal  ribosome  entry  site17

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eIF4G  footprint

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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

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all  eIFs

all  eIFs  except  eIF4E

eIF2,  eIF3

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• 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  

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AminoacylPepNdylExit

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Methionine-­‐independent  ini'a'on

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

• RNA  mimics  tRNAi

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RNA  genome  of  cricket  paralysis  virus

Binds  40S  ribosome  independent  of  iniNaNon  proteins

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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)

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Polyprotein  synthesis

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Leaky  scanning

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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

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Suppression  of  termina'on

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Suppression  of  termina'on

• 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

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Ribosomal  frameshiOing

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Model  for  -­‐1  frameshiOing

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

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eIF2α  kinases

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PKR

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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

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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

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Vaccinia  virus  encoded  pseudosubstrates  mimic  eIF2α

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Targets  of  viral  inhibitors  of  eIF2α  phosphoryla'on

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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

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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

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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

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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

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Two  ways  that  miRNAs  regulate  transla'on

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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

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Virus-­‐encoded  miRNAs

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

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