Translaon Lecture 10 Biology W3310/4310 Virology Spring 2014

The difficulty lies, not in the new ideas, but in escaping old ones... --JOHN MAYNARD KEYNES retroviruses

hepas B virus

reovirus Poliovirus Sindbis virus Influenza virus VSV

©Principles of Virology, ASM Press

O CH3 (m7) N HN

H N N N 2 2' 3' O O O 5' guanosine OH OH 5' CH2 O P O P O P O CH2 O- O- O- base 1 O

O

3' O 2' O(CH3)

O P O CH2 base 2 5' O O- 5’-cap structure

3' O 2' O(CH3) O P O O-

• Found on most eukaryoc mRNAs except organelle mRNAs and certain viral mRNAs • 5'-7-methylguanosine (m7G) joined to second nucleode of mRNA by 5'-5' phosphodiester linkage

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

©Principles of Virology, ASM Press 5’-untranslated region 3’-untranslated region ! •Can regulate translaon •3 – >1,000 nt in length, typically 50 – 70 nt iniaon, translaon efficiency, mRNA stability •Oen contains RNA secondary structures; must be unwound to allow passage of ribosome •poly(A) tail, necessary for efficient translaon •Length and secondary structure influence translaon efficiency

©Principles of Virology, ASM Press Translaonal machinery

• Iniaon proteins (eIF) • Elongaon proteins (eEF) • Terminaon proteins (eRF)

©Principles of Virology, ASM Press 5’-end dependent iniaon

©Principles of Virology, ASM Press ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press Other mechanisms for decoding have been discovered in virus-infected cells Ribosome shunng

-80 kcal/mole

©Principles of Virology, ASM Press –35s mRNAs of plant pararetroviruses Ribosome shunng –Late adenovirus mRNAs –P/C mRNA of Sendai virus

direct translocaon

5' scanning

Shunng is predicted to decrease dependence of mRNAs for eIF4F during iniaon by reducing the need for mRNA unwinding ©Principles of Virology, ASM Press Internal iniaon

©Principles of Virology, ASM Press IRES = internal ribosome entry site ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press eIF4G footprint ©Principles of Virology, ASM Press all eIFs

all eIFs except eIF4E

eIF2, eIF3

©Principles of Virology, ASM Press Aminoacyl Pepdyl Exit

©Principles of Virology, ASM Press Methionine-independent iniaon

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

©Principles of Virology, ASM Press RNA genome of cricket paralysis virus

Binds 40S ribosome independent of iniaon proteins

©Principles of Virology, ASM Press ©Principles of Virology, ASM Press Maximizing coding capacity of the viral genome

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

• Subgenomic mRNAs (Rhabdoviridae, Paramyxoviridae, Togaviridae)

• Segmented genome (Orthomyxoviridae, Reoviridae)

• RNA Splicing (Orthomyxoviridae, Adenoviridae, Polyomaviridae)

• Internal iniaon (IRES) (Picornaviridae, Flaviviridae)

• Leaky scanning (Retroviridae, Paramyxoviridae)

• Re-iniaon of translaon (Orthomyxoviridae, Herpesviridae)

• Suppression of terminaon (Retroviridae, Togaviridae)

• Ribosomal frameshiing (Retroviridae) Polyprotein synthesis

©Principles of Virology, ASM Press Leaky scanning

©Principles of Virology, ASM Press Suppression of terminaon

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

Stop codons may be recognized by charged tRNA - misreading, or charged suppressor tRNA (e.g. selenocysteine for UGA)

©Principles of Virology, ASM Press Suppression of terminaon

©Principles of Virology, ASM Press Ribosomal frameshiing

©Principles of Virology, ASM Press Model for -1 frameshiing

©Principles of Virology, ASM Press Regulaon of translaon in virus-infected cells

• Iniaon • Elongaon • Terminaon ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press PKR and cellular anviral response

• PKR induced and acvated by virus infecon • Leads to inhibion of host translaon, apoptosis • Different viral mechanisms have evolved to inacvate the PKR pathway Adenovirus VA RNA I prevents acvaon of PKR

PKR

inactive

VA RNA I dsRNA

P P

inactive active

no phosphorylation eIF2α subunit of eIF2α subunit phosphorylation

active protein protein synthesis synthesis inhibited Vaccinia virus encoded pseudosubstrates mimic eIF2α Targets of viral inhibitors of eIF2α phosphorylaon Cleavage of eIF4G

©Principles of Virology, ASM Press ©Principles of Virology, ASM Press

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 miRNA

• >1000 human miRNAs, regulate ~60% of protein- coding genes • Control gene expression post-transcriponally via either repression of translaon or mRNA degradaon • Mainly target 3’-UTR of RNA miRNA

• miRNA funcon in a complex, the miRNPs, composed of Ago, Dicer, and TRBP • Complementarity to 3’-UTR determines whether the mRNA is degraded or translaon is repressed Two ways that miRNAs regulate translaon

©Principles of Virology, ASM Press Viruses and cellular miRNAs

• miR-122 is a liver-specific miRNA required for HCV replicaon • miRNAs target viral or cellular genes needed for viral replicaon • miR-141 induced during enterovirus infecon inhibits translaon of eIF4E mRNA Virus-encoded miRNAs

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