The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA

1. The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA Hui Zhang, Bin Yang, Roger J. Pomerantz, Chune Zang, Shyamala C. Arunachalam, and Ling Gao A presentation by Alaric Smith

2. HIV-1 • From the Lentivirus category of retroviruses; lentiviri cause immunodeficiency in mammals • Reproduces via reverse transcriptase • Affects humans, leads to AIDS

3. Lentiviral hypermutability • In primate lentiviri, GA transitions common • HIV-1 Vif protein required for viral replication in nonpermissive cells (binds to viral RNA) • Is Vif counterdefensive against host nonpermissivity?

4. CEM15 • AKA: Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, or APOBEC3G • Expressed in nonpermissive cells • Has been shown to inhibit HIV-1 replication • Is a cytidine deaminase • Has been shown to act as a mutagen against E. Coli

5. Does CEM15 act on HIV-1 rtDNA? • Creation of ∆vif mutant HIV-1 • Incubation in permissive (SupT1) and nonpermissive (H9) cells • Analysis of newly synthesized viral DNA

6. CEM15: An HIV-1 mutagen? • ∆vif HIV-1 show significantly higher GA mutation rate in nonpermissive cells, and when compared to wild-type HIV-1 • ∆vif viruses passaged in semipermissive (C8166) cells show significantly higher GA substitution rate than wild-type HIV-1 in C8166 or ∆vif HIV-1 passaged in SupT1 cells

8. How does CEM15 affect GA substitution? • Infect 293T and SupT1 cells (both permissive) with both ∆vif and wild type HIV-1 in presence or absence of CEM15 • After 48h, allow newly synthesized virions to infect C8166 cells, sequence viral DNA • Conclusion: GA hypermutations much more common in ∆vif HIV-1 when CEM15 is present

10. How does CEM15 affect wild type HIV-1 over the long term? • Transduce CEM15 expression into SupT1 cells • Infect both wild-type and CEM15+ SupT1 cells with wild-type HIV-1 • Sequence viral DNA after four passages • Conclusion: Wild type HIV-1 will accumulate GA hypermutations over time when exposed to CEM15 • Also, CEM15 does not affect dNTP concentrations

12. Verification of CEM15 cytidine deaminase activity • Fuse CEM15 with glutathione S-transferase, purify from E. coli • (GST)-CEM15 has significantly higher conversion rate than GST alone, and can be inhibited by tetrahydrouridine

13. Zinc fingers: An essential domain? • In other cytidine deaminases, zinc finger domains are essential for conversion • CEM15 contains two such zinc finger domains • Create several cell lines with mutations in CEM15 zinc finger domains • Measure cytidine deaminase activity

15. At what level does CEM15 act? • Incubate PCR-amplified DNA, in vitro-transcribed RNA, and in vitro reverse-transcribed RNA-DNA duplexes with (GST)-CEM15 • Sequence resulting nucleic acids • Result: No discernible mutations were found upon sequencing • Conclusion: CEM15 must require other factors to perform cytidine deamination

16. Are CEM15 mutant cells able to defend against ∆vif HIV-1? • Incubate ∆vif HIV-1 in CEM15 wild type and mutant 293T cells, then use virions to infect C8166 and HLCD4-CAT cells • Analyze viral nucleotide squence • Conclusion: Cells expressing mutant CEM15 effect less GA substitution than cells expressing wild-type CEM15 in∆vif HIV-1

18. How exactly does CEM15 work? • Observation: Hypermutations preferentially occur in GpA or GpG dinucleotides, or in multiple G groups • Proposed mechanism: Direct deamination of dC in newly synthesized viral DNA, causing conversion to dU • Could lead to premature stop codons and/or decreased viral protein stability • Another possibility: Uracil-DNA glycosylase (UDG) may excise uracil from viral DNA, causing break points

20. Future Development • What is the biochemical mechanism of CEM15-induced hypermutation? (Is UDG involved?) • How does Vif neutralize CEM15? • Does hypermutation ever “backfire?” (i.e., confer added virulence to HIV-1)