Transcript Powerpoint Slides 5.2

Virology 5.2 2015
 RNA
 MS2
Phage Gene Expression and
Replication
& Qb: RNA Bacteriophages
provide examples of several
important RNA virus strategies
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.
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.
Small RNA Bacteriophages
1)Two highly studied RNA phages:
Levivirus Genus (MS2)
Allolevivirus Genus (Qb)
2) Small genomes range <4500 nt.
3) All have same 5’ ends (pppG).
4) All have same 3’ bases (-CCCAOH).
5) RNAs are very highly structured.
~35 % base pairing in genomic RNA
Structure regulates gene expression
MS2 & Qb 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.
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!!!!
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.
Gene Expression during MS2 Infection
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.
CP ORF Translation opens up the Polymerase AUG
Ribosomes cause
many secondary
structural
changes during
translation of
mRNAs that can
regulate protein
expression in the
prokaryotes.
Ribosomes open up the Polymerase (Replicase)
ORF as they traverse through the CP ORF. The CP
& the Replicase accumulate early in replication.
Accumulating CP Represses Replicase Synthesis
Ri
Ri
APi
CPi
CPi
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.
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.
The Lysis protein is produced by ribosomal frameshifting
The coat protein & the
Lysis genes overlap.
Ribosomes can not enter
the Lysis initiation site
due to base pairing.
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 Qb RNA Dependent RNA Replicase Holoenzyme
The Qb 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 Qb RNAs but not
other Levivirus RNAs or host mRNAs.
The Replicase down regulates host protein synthesis late in replication.
Qb RdRp Studies Show Darwinian Evolution in a Test Tube
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.
Results:
Many
small
Aliquot
Transfers
Incubate
Incubate
Transfer small aliquots after each Incubation.
Analyze RNA samples after each Incubation.
- Rapid evolution to smaller RNAs
(~ 12% the size of Qb RNA).
- Minimal sequence conservation but strong structural conservation.
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.
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.
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.