Gene expression of viral genomes: Translation strategies Flashcards
Structure of eukaryotic mRNAs
5 ́cap: Binding of initiations factors
5 ́UTR: 50-100 bases
monocistronic: one ORF
3 ́UTR: PolyA
Structure of prokaryotic mRNA
no cap, but Shine-Dalgarno sequence polycistronic composition
5’ cap structure -> Functions
- marks the 5’-end of the first exon and aids in the splicing process
(i) essential for nucleo-cytoplasmic transport of mRNAs through interaction with nuclear cap-binding proteins
(ii) increases the efficiency of translation by targeting formation of the preinitiation complex (cytoplasmic cap-binding proteins)
(iii) protects the transcript from 5’→3’ exoribonucleolytic activities
(iv) Blocks recognition of viral RNAs by an antiviral defense mechanism
Enzymatic reactions required for mRNA 5’-capping
- most eukaryotic mRNAs are capped at the 5’ end during nuclear processing
- the terminal 5’ phosphate is first removed by a 5’ triphosphatase
- Guanyltransferase transfers GMP from GTP to the 5’ end of the mRNA to add the GpppN cap structure
- the 5’ terminal inverted G residue is the modified by methylation
- many RNA viruses replicate in the cytoplasm and must use a viral dependent capping mechanism supplied by the RNA-dependent-RNA Polymerase
- the Cap structure, m7GpppN, is most common in viral and mammalian mRNAs
Many viruses have capped genomic RNAs similar to eukaryotic host mRNAs
- Cap structures of viral mRNAs are made in a variety of ways:
1) de novo synthesis by cellular enzymes (Adenoviridae, Hepadnaviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Retroviridae, Polyomaviridae)
2) synthesis by viral enzymes (Reoviridae, Rhabdoviridae, Togaviridae, Poxviridae)
3) acquisition of preformed 5’cap structures from cellular pre-mRNAs or mRNAs (Bunyaviridae, Arenaviridae, orthomyxoviridae)
Unconventional viral capping pathways
- Covalent enzyme-pNp-RNA intermediate with the nacent viral RNA
- Transfer of GDP to the RNA 5‘ end
- First 2‘O-methylation, followed by methylation at the guanine-N7 position
- Covalent enzyme- m7G- Intermediate
- Transfer of m7G to the RNA 5‘ end
- No 2‘O- methylation
3’ processing of mRNA in eukaryotes
(1) An enzyme complex recognizes the polyadenylation signal (AAUAAA) and a less well conserved G-U rich sequence located 20-40 nucleotides downstream.
(2) An endonuclease cleaves the primary transcript 10-30 nucleotides downstream of the AAUAAA signal.
(3) A series of 80-250 A residues are added to the 3’-end of the cleaved transcript by polyadenylate polymerase.
Like the 5‘ cap-structure the 3’ poly (A) tail was first identified in a viral mRNA (reovirus mRNA)
Functions:
(1) stabilizes the mRNA
(2) increases the efficiency of translation
There are examples of mRNAs without a poly (A) tail
viral
- reoviral mRNA
- arenaviral mRNA
- flaviviral mRNA
- Pestiviral mRNA
- Hepaciviral mRNA
cellular
- histone mRNA
Viral strategies of translation
Animal viruses are completely dependent on the host cell machinery for translation of their mRNAs
Special feature of viral translation:
Many viruses use translation strategies to maximize the coding capacity of their genomes of limited size
- functional polycistronic mRNAs
- readthrough of stop codons
- polyproteins
Virus infection often results in modification of the host‘s translational machinery to translate selectively the viral mRNAs
-> „host cell shut-off“
Genetic Economy: one primary transcript - multiple translation products
- Polyprotein Processing
Initiation at different start codons
*Reinitiation
*Leaky scanning
*Internal ribosomal entry
*Ribosomal shunting
Changing the translational reading frame
*Splicing
*Ribosomal frameshifting
*RNA editing
- Translational read through of stop codons
Initiation
*Rate limiting step
*Requires hydrolysis of ATP and GTP
*Results in formation of a complex containing the mRNA, the ribosome and the initiator Met- tRNAi
A. 5’ end (Cap) dependent initiation
* The initiation complex binds to the 5’ cap structure and scans in a 5’ to 3’ direction until initiating AUG is encountered
B. Internal ribosome entry
* Initiation complex binds upstream of initiation codon (EMCV IRES, Poliovirus IRES or directly at the start codon (BVDV/HCV IRES; CrPV-IRES)
5’ end (cap) dependent initiation
- The first step is the recognition of the 5’ cap by eIF4F, which consists of three proteins, eIF4E, eIF4G and eIF4A.
- The cap binding protein eIF4E binds to the 5’ cap
- The N-terminus of eIF4G binds eIF4E and the C-terminus binds eIF4A
- The 40S subunit binds to eIF4G via eIF3
Cap-dependent initiation of protein synthesis in eukaryotes
An initiation complex forms at the cap with the 40S ribosomal subunit and other translation initiation factors.
The 40S complex then scans down the 5’ untranslated region to the first AUG codon.
A GTP hydrolysis step by eIF5 triggers GDP binding of eIF2 and release of initiation proteins.
The 60S subunit joins the complex and the 80S ribosome initiates translate the ORF.
Elongation
Ribosome selects aminoacylated tRNA
eEF1a and GTP are bound to aminoacylated tRNA
Ribosome catalyzes formation of a peptide bond
Translocation is dependent on eEF2 and GTP hydrolysis
Many ribosomes may translate mRNAs simultaneously on the same strand.
Termination
*Translation is terminated at one of three stop codons (UAA, UAG & UGA).
* Termination codon at the A site is recognized by the release factor instead of a tRNA
* The release factor binds the termination codon
* The peptide chain is then released followed by dissociation of the tRNA and the ribosome
Closed loop model
- The 5’ end dependent initiation is stimulated by the poly(A) binding protein Pabp1p, which interacts with eIF4G
- This interaction circularizes the mRNA and facilitates formation of the initiation complex
- Mechanism to ensure that only intact mRNA is translated
Direct effects on cellular translation factors during virus infection
- rotavirus NSP3 protein disrupts the PABP–eIF4G association
- Enteroviral, apthoviral, caliciviral, and retroviral proteases cleave the poly(A)-binding protein (PABP)
- Vesicular stomatitis virus (VSV), influenza virus, and adenovirus (Ad) decrease eIF4E phosphorylation
- Ad 100K protein displaces the kinase Mnk1, resulting in the accumulation of unphosphorylated eIF4E late in infection, stimulating selective late viral mRNA translation
- Cleavage of eIF4G by viral proteases, separating its (amino- terminal) eIF4E-interacting domain from its eIF4A- and eIF3- binding segment
- enterovirus 71 infection induces host micro-RNA (miRNA) miR-141 expression: reduction of eIF4E abundance
- Measles, rabies viruses impair cap-dependent translation via virus-encoded eIF3-interacting proteins
Inhibition of translation
- missing 5‘cap-structure
-strong RNA secondary structure in 5 ́NTR
(mRNAs with less or no secondary structure have a lower need for eIF4A helicase activity- for example during Alfalfa mosaic virus segment 4 translation) - long 5 ́NTR
- upstream AUG and open reading frames
- poor sequence context of the AUG codon (Kozak rule)
optimal: GCCACCAUGG
(only 5% of eukaryotic mRNAs have optimal sequence context around the initiator AUG- translatability contributes to appropriate protein levels in the cell)
Inhibition of host cell protein synthesis in Poliovirus infected Cells
- Complete shut off of cap-dependent translation of host cell mRNAs in cell infected with Poliovirus
- Poliovirus genomic RNA is efficiently translated under these conditions
molecular basis:
cleavage of eIF4G by Poliovirus encoded protease 2A
cap-independent translation of Poliovirus RNA
- IRES-mediated
- efficient translation after eIF4G cleavage
- use of the C-terminal cleavage product
Translation of Poliovirus ORF (PV)
- (+)-strand-RNA,
- no cap, 5‘ end linked to VPg protein
- long 5‘NTR
- extensive secondary structure of the 5‘end of the viral RNA - 7 AUG codons located in the 5‘NTR
In Poliovirus-infected cells:
- Efficient translation of PV RNA despite the inhibition of host cell translation by the virus (eIF4G cleavage)
Conclusion:
- 5‘-end-dependent initiation of translation with scanning to the initiator AUG are highly unlikely
- ORF B translation is dependent of (functional) IRES element upstream of ORF B -> circular RNA to demonstrate internal initiation of translation
IRES-mediated translation
IRES-mediated translation also makes use of general eukaryotic translation initiation factors
viral IRES elements can be separated in 4 different groups
- each IRES type has different requirements for translation initiation factors
The IRES groups also diverge in relation to the position of the authentic initiator AUG codon
- cardiovirus-aphthovirus IRES group and HAV: initiation of translation from the AUG codon at the immediate 3’ boundary of the IRES
- enteroviruses and rhinoviruses: initiate translation from an AUG codon located approx. 40 and 160 nt downstream from the 3’ IRES boundary – Scanning!!
- HCV: actual site of translation initiation resides approx. 40 nt upstream to the 3’ IRES boundary
Methionine-independent initiation of translation
- can assemble 80S ribosomes without any eIFs or Met-tRNAi
- RNA mimics tRNAi
- The turnip yellow mosaic virus (TYMV) contains a tRNA-like structural element that recruits ribosomes and places the translation factors proximal to the 5’ initiation site.
IRES-types in viruses
Typ II/EMCV (translations initiation factors: eIF4G-Ct, eIF4A, eIF3, eIF2/ assess. factors: PTB)
Typ III/HCV (eIF3, eIF2/PTB, PCBP2, La protein)
Typ IV/CrPV (none/protein 25 of small ribosomal subunit (RPS25), common to all IRES elements)
Conclusion:
- certain IRES elements are active in their tissue/cell-type of choice
- tissue-specific and selective gene expression
Viral IRESes
- Picornaviruses
- Flaviviruses (hepatitis C virus<)
- Pestiviruses (bovine viral diarrhea virus, classical swine fever virus)
- Retroviruses (SIV, MMLV, HTLV, FLV)
- Insect picorna-like viruses (cricket paralysis virus, Plautia stall intestine virus)
