1. Virology 5.2 2015  RNA Phage Gene Expression and Replication  MS2 & Q  : RNA Bacteriophages provide examples of several important RNA virus strategies

2. Review of Prokaryotic Gene Expression 1) Prokaryotic Cells have No Nuclei. 2) Transcription & Translation are Coupled. 3) Most mRNAs are Polystronic and Translation of Multiple ORFs Occurs During mRNA transcription. 4) Because transcription and translation are coupled, transcription can have a major role in regulating the timing and extent of protein synthesis.

3. Important Properties of Prokaryotic mRNAs 5’ End is Phosphorylated & 3’ End has No poly A tail. mRNAs are not spliced before translation. mRNAs have Multiple Open Reading Frames. Ribosomes can enter mRNAs internally depending on secondary structural interactions within the RNA.

4. Small RNA Bacteriophages 1) Two highly studied RNA phages: Levivirus Genus (MS2) Allolevivirus Genus (Q  2) Small genomes range <4500 nt. 3) All have same 5’ ends (pppG). 4) All have same 3’ bases (-- CCCA OH ). 5) RNAs are very highly structured. ~ 35 % base pairing in genomic RNA Structure regulates gene expression MS2 & Q  Provided Important Early Research Tools Description 1961 RNA Translation and Regulation of Protein Synthesis. RNase Protection to identify ribosome binding sites on mRNA. Provided first models for protein-RNA interactions. Characterization of first RNA-Dependent-RNA Polymerase.

5. Levivirus (Phage MS2) Genome Organization 3500 nt “A” or “Assembly” protein One Copy of 40 kD protein per virus particle. Binds to RNA & Required for RNA entry during capsid assembly. Recognizes bacterial pili & mediates RNA Entry during infection. Coat Protein (180 Copies of 14 kD CP protein/Virion). Forms Icosohedral Capsid & also represses replicase synthesis. Replicase (61 kD RNA-dependent RNA Polymerase protein). Complexes with three host proteins to make replicase holoenzyme. Lysis Protein (8 kD protein from an ORF that Overlaps CP ORF) Lyses bacterial cell at end of infection cycle. Lysis Protein is produced by a translational frameshift using a portion of the ribosomes that have translated the coat protein. Each of the proteins is multifunctional!!!!

6. RNA Phage Infections Require Bacterial Pili 1) Phage particles bind efficiently to host pili. Recognition of the pili determines the host range. 2) The “A” Protein functions to mediate binding to host pili. 3) During binding, the “A” protein is cleaved into 15 & 24 kDa subunits & the virions disassemble to release the viral RNA & “A” protein subunits. 4) The two “A” protein subunits remain associated with the viral RNA. The RNA and the subunits move along the pili to enter the cell. The RNA at this phase of infection is sensitive to RNase. 5) The RNA & “A” protein subunits penetrate the host cell membrane. 6) Naked RNA can be used to infect spheroplasts whose cell walls have been removed by enzyme treatments. This demonstrates that the viral RNA has all the information needed to infect cells. The host range can be expanded greatly by using this treatment to circumvent the pili. 7) Plasmids expressing pili proteins can also expand the host range to “nonpermissive” hosts that phage can not normally infect.

7. The first step after RNA entry is CP translation 1) The CP AUG is open and accessible to ribosomes. 2) The Polymerase (RdRp) AUG is inaccessible due to secondary structure and base pairing. Gene Expression during MS2 Infection

8. CP ORF Translation opens up the Polymerase AUG Ribosomes open up the Polymerase (Replicase) ORF as they traverse through the CP ORF. The CP & the Replicase accumulate early in replication. Ribosomes cause many secondary structural changes during translation of mRNAs that can regulate protein expression in the prokaryotes.

9. Accumulating CP Represses Replicase Synthesis CPi APi Ri 1) As CP accumulates, it forms regulatory complexes. 2) CP complexes bind strongly to Replicase AUG. 3) Ribosomes are unable to compete with the CP & Replicase synthesis is silenced. 4) CP synthesis continues throughout replication.

10. Translation of the “A” protein requires Replication 1) The “A” protein initiation site (GUG) of the full length Viral RNA is blocked by 2ndary structure and is inaccessible to ribosomes. 2) The replicase copies the viral RNA into a minus strand and this is then copied into progeny plus strands. The 2ndry structure changes continually during copying as new base pairs form during strand elongation. 3) As new plus strands are synthesized, the GUG initiation codon becomes available and ribosomes begin synthesis of the A protein. 4) The newly synthesized “A” protein binds to the progeny MS2 RNA molecules to facilitate entry into newly formed virus particles. 5) As the nascent progeny strands elongate, the conformation changes to block GUG initiation. So a “Window of Opportunity” for ribosome binding regulates the timing of “A” Protein synthesis.

11. The Lysis protein is produced by ribosomal frameshifting 1) The initiation codon for the Lysis gene is immediately downstream of two “out of frame” termination codons inside the Coat Protein gene. 2) A small proportion of the ribosomes translating the CP ORF terminate prematurely at these stop codons. 3) These ribosomes dissociate from the incomplete CP molecules & shift reading frames to begin translation of the Lysis ORF. The coat protein & the Lysis genes overlap. Ribosomes can not enter the Lysis initiation site due to base pairing.

12. The Qb RNA Dependent RNA Replicase Holoenzyme  The Q  replicase was the 1st RdRp to be isolated & characterized.  The Core or Holoenzyme consists of the viral RdRp plus three host proteins. The Replicase is very specific and can recognize the Q  RNAs but not other Levivirus RNAs or host mRNAs. The Replicase down regulates host protein synthesis late in replication.

13. Sol Speigelman, 1964 -Viral RNA plus RdRp & NTPs. -Conducted 20 min RXNs at various temps. -Products diluted & added to fresh RdRp & NTPs for new incubation. -75 RXNs carried under conditions designed to facilitate rapid replication. Q  RdRp Studies Show Darwinian Evolution in a Test Tube Incubate Many small Aliquot Transfers Incubate Transfer small aliquots after each Incubation. Analyze RNA samples after each Incubation. Biological Implications: - Provided understanding of mechanisms of nucleic acid evolution. - Enhanced knowledge about Kinetics & specificity of replication. - Resulted in a technique for making recombinant RNA molecules. - Helped in understanding of RNA recombination. Results: - Rapid evolution to smaller RNAs ( ~ 12% the size of Q  RNA). - Minimal sequence conservation but strong structural conservation.

14. RNA Phages Provide Examples of Four Important Themes 1) Secondary structure in viral RNA is important. 2) Viruses like to use multifunctional proteins. 3) Viruses often have overlapping genes. 4) Viral proteins often rely on host subunits.

15. RNA Bacteriophages Are Elegant They coordinate gene expression so that correct proteins are produced in the correct amounts and at the correct time. They do this with less than 5000 nucleotides of RNA.