Reverse transcrip-on and integra-on Lecture 9 Biology W3310/4310 Virology Spring 2013 “One can’t believe impossible things,” said Alice. “I dare say you haven’t had much pracce,” said the Queen. “Why, some=mes I’ve believed as many as six impossible things before breakfast.” --LEWIS CARROLL, Alice in Wonderland 1 Some history • 1908 - Discovery oF chicken leukemia virus, Bang & Ellerman • 1911 - Discovery oF Rous sarcoma virus, Peyton Rous (Nobel Prize 55 years later) • Called tumor viruses 2 Tumor viruses • Found to have RNA genomes • How could these viruses transForm cells? • 1970 - Discovery oF retroviral reverse transcriptase, Temin & BalZmore; Nobel Prize 1975 3 Reverse transcriptase • Enzyme that countered Central Dogma: DNA => RNA => protein • Retroviruses got their name because oF their ability to reverse the flow oF geneZc inFormaon 4 retroviruses hepaZs B virus reovirus Poliovirus Sindbis virus Influenza virus VSV 5 Balmore and Temin independently discovered RT in virions of RNA tumor viruses Listen to TWiV #100 (BalZmore) and TWiV #66 (Reverse transcripZon) For more insight 6 Temin’s insight • Retroviral DNA was integrated into host genome • Became permanent part oF host DNA • Provirus 7 Lessons from lambda 8 9 10 11 Reverse transcriptase Base P OH 5’- Primer -3’OH 3’- -5’ Direction Of Chain Growth • Primer can be DNA or RNA • Primer must have paired 3’-OH terminus • Template can be RNA or DNA • Only dNTPs, not rNTPs, are incorporated • Correct incoming dNTP is selected by base pairing 12 RT • Some strains oF myxobacteria, E. coli, and Archaea have RT acZvity • ThereFore RT evolved beFore the separaon oF Archaea, prokaryotes and eukaryotes • RT might be the bridge between early RNA world and modern DNA world 13 Sequence relaonships 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 RNAse H: A second ac-vity of RT • Cleaves RNA only when in duplex Form • RNA can be in RNA:RNA or RNA:DNA duplexes • Makes endonucleolyZc cleavages • Produces short oligonucleoZdes with 5’-phosphate, 3’-OH 15 DNA synthesis is slow (4 h per 9 kb genome) and error prone (1 misincorporaon per 104 to 106 nt) 16 Coated with NC protein 50-100 molecules RT per virion 17 RNA dimer • Explains why retroviruses are relavely resistant to UV and ionizing radiaon • Two copies oF all genes • Copy-choice rebuilds a FuncZonal genome 18 19 DNA synthesis: cytoplasmic 20 ppt = polypurine tract 21 22 23 24 25 26 PreFerence For integraon into DNA sequences that are bent or underwound by being wrapped around a nucleosome 27 Retrovirus transcrip-on •A strong DNA promoter (the LTR) is built during reverse transcripZon •The integrated DNA copy then directs the host transcripZon machinery to synthesize many copies oF viral mRNA •The viral mRNA is translated into viral proteins OR encapsidated into inFecZous virus parZcles. There is no DNA replica0on and no RNA replica0on 28 29 Provirus is a permanent part of host genome • No mechanism For precise excision oF integrated provirus • Only way out oF genome is transcripZon by host RNA pol II • Endogenous = in germline DNA • Genomes are lijered with ancient and modern retroelements 30 Retroelements • Retrovirus-related sequences in cellular genomes • When integrated into the germline = endogenous proviruses • Oken replicaon-deFecZve • Nearly 50% oF human genome comprises mobile geneZc elements, including endogenous proviruses and other retroelements 31 32 Rescue of an endogenous human retrovirus • HERV-K, inFected human ancestors <1 Myr ago • Repaired mutaons ReconsZtuZon oF an inFecZous human endogenous retrovirus. Lee YN, Bieniasz PD. PLoS Pathog. 2007 Jan;3(1):e10. 33 34 hjp://www.virology.ws/2012/10/11/museum-pelts-help-date-the-koala-retrovirus/ 35 36 No genome integraon 37 38 39 40 41 Unexpected endogenous viruses hjp://www.virology.ws/2010/12/10/unexpected-endogenous-viruses/ 42.