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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 1 Some RNA history • • • • • • 1935 -‐ Stanley crystallizes TMV • • 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 • RNA genome must be copied end to end with no loss of nucleoDde sequence • ProducDon of viral mRNAs that can be efficiently translated by cellular protein syntheDc machinery 3 retroviruses hepaDDs B virus reovirus Poliovirus Sindbis virus Influenza virus VSV 4 Terminology • Replicase: enzyme that copies viral RNA to produce genomes • Transcriptase: enzyme that produces mRNA • Transcrip-on: copying of DNA into RNA • Promoter: sequences controlling transcripDon of DNA templates 5 Iden5fica5on of RNA polymerases Assays: cell extracts incubated with NTPs 6 Iden5fica5on of RNA polymerases • • Assays: cell extracts incubated with NTPs • • Virion polymerase discovered in (-‐) strand viruses • 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 7 Nature of the RNA template • (-‐) strand RNA genomes: coated with protein; ready to begin RNA synthesis upon entry • (+) strand RNA genomes: naked, ready to be translated upon entry (excepDon: retroviral, coronaviral) • dsRNA genomes: cannot be copied into mRNA by the cell; virions contain RNA polymerase 8 VSV N protein bound to RNA 9 10 Universal rules for RNA-‐directed RNA synthesis • RNA synthesis iniDates and terminates at specific sites on the template • RdRp may iniDate synthesis de novo (like cellular DdRp) or require a primer • • Other viral and cell proteins may be required • Non-‐templated RNA synthesis RNA is synthesized by template-‐directed stepwise incorporaDon of NTPs, elongated in 5’-‐3’ direcDon 11 NTP 12 Base P 5’- Primer OH -3’OH 3’- -5’ Direction Of Chain Growth 13 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 14 F A B D E C 15 D328 16 Product Template 17 18 19 20 cis-‐acDng RNA element cellular polyadenylated RNAs not copied 21 22 23 24 Vesicle forma5on in poliovirus-‐infected cells Uninfected HeLa cell PV-‐infected HeLa cell 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 Reoviridae: reovirus, rotavirus 40 41 42 Each dsRNA segment is acached to pol via the 5’-‐ cap 43 • RNA replicaDon of bacteriophage ϕ6 (3 ds RNA segments) is semiconservaDve • RNA replicaDon of reoviruses (10-‐12 ds RNA segments) is conservaDve 44 RNA synthesis as a source of viral diversity • Lack of proofreading acDvity in RNA dependent RNA polymerase: high error frequencies (1 minincorporaDon/103 -‐ 104 nt polymerized) • Single amino acid change in poliovirus RdRp causes increased fidelity • This mutaDon reduces viral fitness and virulence under selecDve pressure 45 46 RNA ediDng (non-‐templated): measles, mumps virus 47 48 RNA edi5ng produces mRNA for Ebola virus glycoprotein 49