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RNA Synthesis
Lecture 6
Biology W3310/4310
Virology
Spring 2013
Truth is ever to be found in the simplicity, and not in the mul7plicity and confusion of things
-­‐-­‐SIR ISAAC NEWTON
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Some RNA history
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1935 -­‐ Stanley crystallizes TMV
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By 1959, RNA was idenDfied in many animal viruses
1936 -­‐ TMV crystals contain 5% RNA
1944 -­‐ DNA is geneDc material
1952 -­‐ Hershey-­‐Chase experiment
1953 -­‐ Structure of DNA
1956 -­‐ TMV nucleic acid is infecDous; first demonstraDon that RNA can be geneDc material
1960s -­‐ studies on viral RNA replicaDon begin 2
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RNA genome must be copied end to end with no loss of nucleoDde sequence
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ProducDon of viral mRNAs that can be efficiently translated by cellular protein syntheDc machinery
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retroviruses
hepaDDs B virus
reovirus
Poliovirus
Sindbis virus
Influenza virus
VSV
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Terminology
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Replicase: enzyme that copies viral RNA to produce genomes
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Transcriptase: enzyme that produces mRNA
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Transcrip-on: copying of DNA into RNA
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Promoter: sequences controlling transcripDon of DNA templates
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Iden5fica5on of RNA polymerases
Assays: cell extracts incubated with NTPs
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Iden5fica5on of RNA polymerases
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Assays: cell extracts incubated with NTPs
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Virion polymerase discovered in (-­‐) strand viruses
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Crystal structures of many RNA polymerases have been determined
ActD resistant cytoplasmic RNA synthesis in mengovirus/poliovirus infected cells
IdenDficaDon by sequence alignments (GDD), expression of recombinant proteins
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Nature of the RNA template
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(-­‐) strand RNA genomes: coated with protein; ready to begin RNA synthesis upon entry
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(+) strand RNA genomes: naked, ready to be translated upon entry (excepDon: retroviral, coronaviral)
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dsRNA genomes: cannot be copied into mRNA by the cell; virions contain RNA polymerase
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VSV N protein bound to RNA
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Universal rules for RNA-­‐directed RNA synthesis
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RNA synthesis iniDates and terminates at specific sites on the template
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RdRp may iniDate synthesis de novo (like cellular DdRp) or require a primer
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Other viral and cell proteins may be required
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Non-­‐templated RNA synthesis
RNA is synthesized by template-­‐directed stepwise incorporaDon of NTPs, elongated in 5’-­‐3’ direcDon
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NTP
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Base
P
5’-
Primer
OH
-3’OH
3’-
-5’
Direction Of Chain Growth
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Sequence rela5onships among polymerases
Gly-­‐Asp-­‐Asp
• Gly-­‐Asp-­‐Asp in all (+) strand RNA polymerases
• Asp-­‐Asp in RT, segmented (-­‐) strand polymerases
• Gly-­‐Asp-­‐Asn in nonsegmented (-­‐) strand polymerases; birnaviruses have Ala-­‐Asp-­‐Asn
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F
A
B
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C
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D328
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Product
Template
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cis-­‐acDng RNA element
cellular polyadenylated RNAs not copied
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Vesicle forma5on in poliovirus-­‐infected cells
Uninfected HeLa cell
PV-­‐infected HeLa cell
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Reoviridae: reovirus, rotavirus
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Each dsRNA segment is acached to pol via the 5’-­‐
cap
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RNA replicaDon of bacteriophage ϕ6 (3 ds RNA segments) is semiconservaDve
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RNA replicaDon of reoviruses (10-­‐12 ds RNA segments) is conservaDve
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RNA synthesis as a source of viral diversity
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Lack of proofreading acDvity in RNA dependent RNA polymerase: high error frequencies (1 minincorporaDon/103 -­‐ 104 nt polymerized)
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Single amino acid change in poliovirus RdRp causes increased fidelity
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This mutaDon reduces viral fitness and virulence under selecDve pressure
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RNA ediDng (non-­‐templated): measles, mumps virus
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RNA edi5ng produces mRNA for Ebola virus glycoprotein
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