1. Virology 5.1, 2015 RNA Virus Gene Expression and Replication Issues, Problems, Strategies for ss+ RNA Viruses

2. Terms and definitions Replication Replicase (old name) RdRp Transcriptase (old name) Initiation/termination codons CistronORF Cis and trans RF and RI (see cartoon)

3. A Cartoon of the Replication of + Viral RNA - + - + * * * * - + * * * * + Genomic RNA (+ssRNA) or Synthesis of Minus-Strand By RDRP Synthesis of Minus-Strand Intermediates By 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. - - - + + + +

4. 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.

5. Virologists are thinking about virus evolution

6. General challenges for these viruses RNA viruses must encode many of their own enzymes but Genetic Economy is still in force. Viruses must accommodate the realities of the host cell environment. Nonconventional methods for gene expression and regulation. More specific challenges Preferential initiation of translation Expression of internal genes Regulation: timing and fine tuning

7. 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.

8. 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

9. Initiation of Translation-TMV RNA  Initiation complex assembles at 5’ end  Ribosome strips away capsid proteins as it translates  Cotranslational disassembly

10. 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.

11. 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 CP CCA 126K AUG UAG

12. The TMV Replicase is a Multifunctional Protein CCA 183K MP CP 126K UAG Methyl- transferase domain Helicase 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

13. 183K MP CP CCA 126K The MP and CP ORFs at the 3’ end of TMV RNA are expressed from subgenomic mRNAs TMV gRNA I 1 RNA Function is unknown MP CP CCA CP CCA I 2 -RNA LMC RNA sg-mRNAs 54K MP CP CCA I 1 -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 I 2 sgRNA but the CP ORF is not translated.  The 17K CP is translated from the LMC sgRNA.

14. 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?

15. BROME 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

16. Divided RNA Genome of Brome Mosaic Virus Viruses 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 RNAs RNA 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’ m 7 G 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’ m 7 G tRNA-like Polymerase Subunit Movement Helicase Subunit Coat 3’ 5’ m 7 G Coat 3.2 kB 2.9 kB 2.1 kB 0.9 kB P

17. Coronaviruses Use Another Mechanism for sg mRNA Synthesis Subgenomic mRNAs (+) Viral RNA Genomic length (-) RNA Jump 5’ Leader Sequence