Virology 5.1, 2015Virology 5.1, 2015

RNA Virus Gene Expression and RNA Virus Gene Expression and ReplicationReplication

Issues, Problems, Strategies for ss+ Issues, Problems, Strategies for ss+ RNA Viruses RNA Viruses

Terms and definitionsTerms and definitions

ReplicationReplication

Replicase (old name)Replicase (old name)

RdRpRdRp

Transcriptase (old name)Transcriptase (old name)

Initiation/termination codonsInitiation/termination codons

CistronCistron

ORFORF

Cis and transCis and trans

RF and RI (see cartoon)RF and RI (see cartoon)

A Cartoon of the Replication of + Viral RNAA Cartoon of the Replication of + Viral RNA - + -+

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- +**

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+

Genomic RNA(+ssRNA)

or

Synthesis of Minus-StrandBy RDRP

Synthesis of Minus-Strand IntermediatesBy RDRP

Replicative Form

RF

Replicative Intermediate

RI

Replicative Intermediate

Synthesis of Plus-Strand Progeny RNAs

Intermediates are double-stranded RNAs; Asymmetrical copying of RNA intermediates occurs.

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- +

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Plus-Strand RNA Viruses

Viruses with this genome strategy have been found in both prokaryotes and eukaryotes. They are among the most abundant plant and animal viruses.

Three different supergroups of plus strand viruses have been identified based on the phylogeny of their RNA dependent RNA polymerases (RdRP).

Supergroup 1: include picornaviruses and comoviruses that express their genomes as polyproteins that are cleaved into individual functional viral proteins.

Supergroup 2 viruses include flavi and tombus viruses whose RNAs are not capped and 3’ encoded ORFs are expressed from subgenomic mRNAs.

Supergroup 3 viruses are monopartite, bipartite and tripartite viruses whose RNAs are capped and 3’ encoded ORFs expressed from sg RNAs.

Virologists are thinking about virus evolution

General challenges for these virusesGeneral challenges for these virusesRNA viruses must encode many of their own

enzymes butGenetic Economy is still in force.

Viruses must accommodate the realities of the host cell environment.

Nonconventional methods for gene expressionand regulation.

More specific challenges

Preferential initiation of translationExpression of internal genes

Regulation: timing and fine tuning

Studies of TMV Provide Some Answers

Strains of TMV infect tomato and cause poor yield, distorted fruits, delayed fruit ripening and various fruit discoloration problems that

affect market values.

The TMV genome is a messenger-sense RNA that produces 4 proteins during virus infection. The RNA possesses a 7mG cap structure at the 5’-end and a tRNA-like structure charged with the amino acid histidine (His) at the 3’-end. Two polymerase-related proteins are produced. One protein possesses methyltransferase (MET, capping enzyme) and RNA helicase (HEL) domains. A second protein also contains an RNA-dependent RNA polymerase (RdRp) domain. Two other proteins, movement protein (MP) and capsid protein (CP), are produced from the viral RNA

Diagram of TMV RNA Suggests Some Strategies

Initiation of Translation-TMV RNAInitiation of Translation-TMV RNA

Initiation complex assembles at 5’ endInitiation complex assembles at 5’ end Ribosome strips away capsid proteins Ribosome strips away capsid proteins

as it translatesas it translates Cotranslational disassemblyCotranslational disassembly

Cap and UTRs Boost Translation of TMV RNAdRP The Omega () sequence in the 5’ untranslated region (UTR) of TMV RNA consists of 68 NT with 8 CAA repeats.

35S Promoter-driven plasmid

Both the 5’ sequence and a pseudoknot in the 3' UTR region increase the stabilty of synthetic mRNAs and translation of GUS or LUC reporter genes in plant and animal cells.

The sequence, the 3’ UTR and the p102 host protein interact synergistically to

enhance translation.

These results fit a model for translation of circular mRNAs.

Expression of 5’ TMV Genes

The 183K protein contains the 126K protein but also has a C- terminal extension.

Both proteins must be translated before viral RNA replication can begin-and the amounts of each protein must be regulated.

The 126K and 183K RDRP protein subunits are expressed by

direct translation of the infecting genomic RNA. Most of the ribosomes expressing the 126K subunit fall off of the mRNA when they reach the (UAG) termination codon. A small proportion (~5%) of the ribosomes continue

synthesis of protein to produce the 183K translational readthrough

protein. The stop codon is “leaky” due to a suppressor tRNA for Tyrosine

183K MP

CPCCA

126KAUG UAG UAG

The TMV Replicase is a Multifunctional Protein

CCA183K MP

CP

126K

UAG

Methyl-transferase

domainHelicase Polymerase

domain

3’-tRNA-like

binding

The TMV 126/183K protein is a multifunctional enzyme with several domains. The RdRP binds very strongly to the 3’ tRNA-like structure on the plus strand genomic RNA The RdRP also binds to an ordered structure at the 3’ end of the minus strand of the replicating RNA. The 126K & 183K subunits interact as heterodimers to form RdRP complexes.126K also has VSR activity

183K MP

CPCCA

126K

The MP and CP ORFs at the 3’ end of TMV RNA are expressed from subgenomic mRNAs

TMV gRNA

I1 RNA Function is unknown

MP

CPCCA

CPCCA

I2-RNA

LMC RNA

sg-m

RN

As

54KMP

CPCCAI1-RNA

The 3’ ORFs on the TMV genes are not expressed from the genomic RNA.Three subgenomic mRNAs (I1, I2 & LMC) are copied from the full length minus strand The I1 sgRNA is present in very low amounts and no protein has been detected.The 30K MP is translated from the I2 sgRNA but the CP ORF is not translated. The 17K CP is translated from the LMC sgRNA.

What Strategies Does TMV Use?

Initiation: 126 K, 183K, MP, CP

Internal cistrons: 183 K, MP, CP

Fine tuning: 126 K, 183K, MP, CP

Genetic Economy?

BROME MOSAIC VIRUSBROME MOSAIC VIRUS • BMV particles are Icosahedra

consisting of 180 coat protein subunits. • Type member of the Bromovirus

genus, family Bromoviridae. • Virions are nonenveloped icosahedral

(T=3), 26 nm in diameter, contain 22% nucleic acid and 78% protein.

• The BMV genome consists of three positive sense RNAs. RNA1 (3.2 kb) & RNA2 (2.9 kb), are encapsidated in separate particles. RNA3 (2.1 kb) & RNA4 (0.9 kb) are located in a third spherical particle.

RNA 1

RNA 2

RNA 3 + 4

Divided RNA Genome of Brome Mosaic VirusViruses with divided genomes can efficiently express

genes needed early in infection & can regulate the timing and amounts of late genes by synthesis of sgRNAs.Brome mosaic virus is a tripartite RNA virus. Four proteins are expressed from three genomic RNAsRNA 1 encodes the helicase subunit of the RDRP.

RNA 2 encodes the polymerase subunit of the RDRP.

RNA 3 is bicistronic and encodes the movement protein and the coat protein.

Ribosomes initiate at the 5’ m7G Cap of RNAs 1, 2 and 3 but can not initiate internally on RNA 3.

RNA 4 is a sg mRNA translated from an internal promoter on the minus strand of RNA 3.

Brome Mosaic Virus RNAs

5’ m7G

tRNA-like

Polymerase Subunit

Movement

Helicase Subunit

Coat

3’

3’

3’

3’

5’ m7G

5’ m7G

5’ m7G

Coat

3.2 kB

2.9 kB

2.1 kB

0.9 kB

P

Coronaviruses Use Another Mechanism for sg mRNA Synthesis

Subgenomic mRNAs

(+) Viral RNA

Genomic length (-) RNA

Jump

5’ Leader Sequence