Reverse transcripon and integraon

Lecture 9 Biology W3310/4310 Spring 2015

“One can’t believe impossible things,” said Alice. “I dare say you haven’t had much pracce,” said the Queen. “Why, somemes I’ve believed as many as six impossible things before breakfast.” --LEWIS CARROLL, Alice in Wonderland Some history • 1908 - Discovery of leukemia , Bang & Ellerman • 1911 - Discovery of Rous sarcoma virus, Peyton Rous (Nobel Prize 55 years later) • Called tumor

Vilhelm Ellerman Oluf Bang Peyton Rous Tumor viruses

• Found to have RNA • How could these viruses transform cells? • 1970 - Discovery of retroviral , Temin & Balmore; Nobel Prize 1975 Reverse transcriptase

• Enzyme that countered Central Dogma: DNA => RNA => protein

• got their name because of their ability to reverse the flow of genec informaon retroviruses

hepas B virus

reovirus Poliovirus Sindbis virus Influenza virus VSV

©Principles of Virology, ASM Press Balmore and Temin independently discovered RT in virions of RNA tumor viruses

Listen to TWiV #100 (Balmore) for more insight Temin’s insight

• Retroviral DNA was integrated into host • Became permanent part of host DNA • Provirus ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press 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 RT • Bacteria and Archaea have RT acvity • Therefore RT evolved before the separaon of Archaea, prokaryotes and • RT might be the bridge between early RNA world and modern DNA world • RT also in HBV, Sequence relaonships among polymerases

• Gly-Asp-Asp in (+) strand RNA polymerases • Asp-Asp in RT, segmented (-) strand polymerases • Gly-Asp-Asn in nonsegmented (-) strand polymerases

©Principles of Virology, ASM Press RNAse H: A second acvity of RT

• Cleaves RNA only when in duplex form • RNA can be in RNA:RNA or RNA:DNA duplexes • Makes endonucleolyc cleavages • Produces short oligonucleodes with 5’-phosphate, 3’-OH DNA synthesis is slow (4 h per 9 kb genome) and error prone (1 misincorporaon per 104 to 106 nt)

©Principles of Virology, ASM Press Go to:

m.socrave.com room number: virus Reverse transcriptase has revoluonized molecular biology. Which statement about the enzyme is not correct?

1. RT is unique to retroviruses 2. RT is packaged in the parcle 3. The RT protein also has RNAse H acvity 4. The name of the enzyme comes from its ability to reverse the flow of genec informaon 5. Might have bridged the ancient RNA world and the DNA worlds 1 Coated with NC protein 50-100 molecules RT per virus parcle

©Principles of Virology, ASM Press RNA dimer

• Explains why retroviruses are relavely resistant to UV and ionizing radiaon • Two copies of all genes • Copy-choice rebuilds one funconal genome

©Principles of Virology, ASM Press ©Principles of Virology, ASM Press DNA synthesis: cytoplasmic

©Principles of Virology, ASM Press ppt = polypurine tract

©Principles of Virology, ASM Press ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press Go to:

m.socrave.com room number: virus

Which of the following steps occur during reverse transcripon of retroviral genomic RNA?

1. Priming of (-) DNA synthesis by tRNA 2. Two template exchanges 3. Degradaon of the viral RNA by RNAse H 4. Generaon of two LTRs 5. All of the above

2 ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press Preference for integraon into DNA sequences that are bent or underwound by being wrapped around a nucleosome

©Principles of Virology, ASM Press Retrovirus transcripon

•One DNA produced from two by RT •Strong promoter (the LTR) built during RT •Proviral DNA directs the host transcripon machinery to synthesize many copies of viral mRNA •Viral mRNA is translated into viral proteins OR encapsidated into virus parcles

There is no DNA replicaon and no RNA replicaon ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press Go to:

m.socrave.com room number: virus Integraon of retroviral DNA has which of the following properes?

1. Catalyzed by a viral enzyme 2. Occurs on all chromosomes but preferenally at sites that are bent or under-wound because of being wrapped around a nucleosome 3. Leads to the formaon of a provirus 4. Leads to producon of viral mRNAs by host II 5. All of the above 3 Provirus is a permanent part of host genome

• No mechanism for precise excision of integrated provirus • Only way out of genome is transcripon by host RNA pol II • Genomes are liered with ancient and modern retroelements

©Principles of Virology, ASM Press Retroelements

• Retrovirus-related sequences in cellular genomes • When integrated into the germline = endogenous proviruses, ERV • Oen replicaon-defecve • ~42% of human genome comprises mobile genec elements, including endogenous proviruses and other retroelements ©Principles of Virology, ASM Press Rescue of an endogenous human retrovirus

• HERV-K, infected human ancestors <1 Myr ago • Repaired mutaons

Reconstuon of an infecous human . Lee YN, Bieniasz PD. PLoS Pathog. 2007 Jan;3(1):e10. hp://www.virology.ws/2012/10/11/museum-pelts-help-date-the-koala-retrovirus/ Placenta 33 (2012) 663-671 A retrovirus makes chicken eggshells blue

http://www.virology.ws/2013/09/11/a-retrovirus-makes-chicken-eggshells-blue/ Unexpected endogenous viruses

hp://www.virology.ws/2010/12/10/unexpected-endogenous-viruses/ hp://www.twiv.tv/2014/09/21/twiv-303/ ©Principles of Virology, ASM Press Go to:

m.socrave.com room number: virus

Which of the following statements about retroelements is not correct?

1. There are many copies in eukaryoc genomes 2. They are currently entering the Koala germline 3. Those in the human genome produce infecous viruses 4. They can be beneficial 5. None of the above

4

©Principles of Virology, ASM Press No genome integraon ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press ©Principles of Virology, ASM Press Yellow = in virus parcle

©Principles of Virology, ASM Press