Jeffrey Altenburg, Ph.D. Methodist Research Institute Phone: (317) 962-2750

1. Mutations in the C-terminal BBXB domains of CXCL12γ restore chemotactic activity and enhance anti-HIV effects Jeffrey Altenburg, Ph.D. Methodist Research Institute Phone: (317) 962-2750 Email: jaltenbu@clarian.org

2. Introduction • CXCL12γ is an isotype of CXCL12 that contains four overlapping heparin sulfate binding sequences (BBXB) in the carboxy terminus • CXCL12γ is the strongest inhibitor of X4-tropic HIV entry and the weakest inducer of chemotaxis • We mutated the BBXB domains to determine if they are the reason for the changes in function

3. CXCL12γ has the most potent anti-HIV activity and least potent chemotaxis activity Chemotaxis X4-mediated fusion Altenburg JD et al. J Virol. 2007 Aug;81(15):8140-8.

4. Mutations of the BBXB domains were expressed and purified from bacteria

5. Mutation of the C-terminal BBXB domains restores chemotaxis activity and enhances HIV inhibition Chemotaxis X4-mediated fusion

6. Binding to both CXCR4 and heparin sulfate is increased with CXCL12γ CXCR4 binding – flow method CXCR4 binding – I-125 method HSPG binding

7. CXCR4 internalization is increased upon treatment with CXCL12γ

8. Inhibition pattern is identical in HSPG- cells compared to HSPG+ cells Sog9 LM(tk-) HSPG HSPG

9. Conclusions • CXCL12γ binds with greater affinity to CXCR4 and heparin sulfate than CXCL12α • CXCL12γ induces a greater amount of CXCR4 internalization than CXCL12α = Our proposed mechanism • Increased binding to heparin sulfate may be the mechanism behind chemotaxis reduction for CXCL12γ but is not the mechanism behind its increased anti-HIV activity • Optimal CXCL12 antiviral activity might require a human-specific factor

10. Acknowledgements IUSM Lab: Ghalib Alkhatib, PhD Q Jin, MD; X Lu, MD; L Agrawal, PhD; IV Nicolescu, MD; Z VanHorne-Ali, K Grunden, J Gil MRI Lab: Rafat Siddiqui, PhD K Harvey; C Antalis, PhD; S Natarjan, PhD; A Hudson; J Korte; C Walker