HIV-1 Assembly in the Endocytic Pathway: Opportunities for the Identification of Novel Anti-HIV

1. HIV-1 Assembly in the Endocytic Pathway: Opportunities for the Identification of Novel Anti-HIV Drug Targets Éric A. Cohen Unité de rétrovirologie humaine Institut de Recherches Cliniques de Montréal Symposium on Novel Targets for Drug Development XVI International AIDS Conference Toronto, August 14, 2006

2. Gag-Driven HIV-1 Particle Production

3. HIV-1 Gag p6 CA (p24) p2 NC p1 MA(p17) M I L • M domain mediates Gag association to lipid membranes • I domain mediates Gag multimerization • L domain mediates the last step of viral particle morphogenesis- fission of the viral particle- by interacting with Tsg101, a host cell protein involved in the formation of internal vesicles in MVB.

4. Mechanism of HIV-1 Budding HIV-1 Gag co-opts a host cell machinery devoted to the formation of internal vesicles in multivesicular bodies (MVB) to mediate viral budding

5. Sites of HIV-1 Assembly and Release • HIV-1 assembles primarily at the plasma membrane in primary T lymphocytes, as well as in many tumor cells lines including Jurkat, HeLa, 293T and Cos cells. • HIV-1 buds from intracellular compartments in some cell types, particularly in macrophages. • These intracellular compartments express late endosomal / MVB markers including MHC-II, CD63, Lamp1 and LBPA. Retroviral assembly

6. Mechanisms that controls the choice of virus assembly and budding sites remain poorly understood. • What is the route(s) by which Gag reaches its cell surface or MVB steady-state accumulation? • What are the host cell factors that control the choice of HIV buddings sites?

7. Late endosome / MVB What is the route by which Gag reaches its PM or MVB steady-state accumulation? Plasma membrane (PM) Gag Cytoplasm Nucleus p55 p25 p24

8. Late endosome / MVB What is the route by which Gag reaches its PM or MVB steady-state accumulation? Plasma membrane (PM) Gag Cytoplasm Nucleus p55 p25 p24

9. Late endosome / MVB What is the route by which Gag reaches its PM or MVB steady-state accumulation? Plasma membrane (PM) Gag Cytoplasm Nucleus p55 p25 p24

11. 5% PNS 20% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Adapted from Ira Mellman (Sheff et al., 1999) Subcellular Fractionation on a Continuous Iodixanol (Optiprep) Gradient (0-20%) Disrupt cells Pellet nuclei

12. Characterization of Light Density Fractions

13. Characterization of High Density Fractions

14. p24 Virus released Mock 0 0.5 2 5 hs Supernatant Trafficking of HIV-1 Gag-Associated Products in HEK 293T Cells

15. Filipin, an Inhibitor of Endocytosis, Prevents Gag Internalization from the PM to MVB Effect of Filipin on Transferrin and Cholera-toxin internalization Filipin (4µg/mL) Transferrin + (clathrin dependent) Cholera-toxin + (caveolae dependent) Inhibition (+) Internalization (- )

16. Internalization of Gag from the PM to MVB Involves an Endocytosis Process that is Clathrin-independent Effect of Chlorpromazin on Transferrin and Cholera-toxin internalization Chlorpromazin (10 µg/mL) Transferrin ++ (clathrin dependent) Cholera-toxin - (caveolae dependent) Inhibition (+) Internalization (- )

17. Clathrin independent endocytosis X X Late endosome / MVB Route by which Gag reaches its PM or MVB steady-state accumulation in HEK 293T cells Plasma membrane (PM) Gag Cytoplasm Nucleus p55 p25 p24

18. Questions • What is the route by which Gag reaches its cell surface or endosomal steady-state accumulation? • What are the host cell factors that control the site of viral assembly and budding?

19. MHC Class II Molecules • MHC-II molecules present peptides to CD4+ T cells. MHC-II and HIV • HLA-DR is incorporated into HIV-1 particles (Cantin et al., 1996; Poon et al., 2000; Martin et al., 2005). • HIV-1 Nef protein modulates MHC-II cell surface expression (Stumptner-Cuvelette et al., 2003). • MHC-II molecules, which are expressed in macrophages and activated T cells can induce the formation/maturation of endocytic MHC-II-like structures analogous to MVB in HEK293 cells (Calafat et al., J. Cell Biol., 126: 966-77, 1994). • The cytoplasmic tails/transmembrane domain of MHC-II molecules are required for the formation/maturation of these compartments.

20. Andrés Finzi Yong Xiao Goal • To determine whether MHC-II could relocate HIV-1 Gag from the cell surface to intracellular compartments in 293T cells.

21. Gag staining Gag staining Diffuse Diffuse Punctuate Punctuate Dose-dependent Gag relocalization by HLA-DR Gag localization HLA-DR Induces Gag Accumulation in Intracellular Compartments in 293T Cells

22. Gag Accumulates in MVB Upon HLA-DR Expression in HEK 293T Cells Finzi et al., J. Virol., In Press

23. HIV-1 Assembles and Buds Within Intracellular Compartments Upon HLA-DR Expression

24. HIV-1 Assembles at the Plasma Membrane in Absence of HLA-DR

25. HIV-1 release (n=8) HLA-DR Expression Decreases HIV-1 Release Finzi et al., J. Virol., In Press

26. Cell lysis and infection of HeLa β-Gal HxBc2 +/- HLA-DR MAGI assay Wash cells -DR IVS 10µM Hours post-transfection 0 16 24 48 72 +DR Mature Virus Immature Virus MVB containing mature virus MVB containing immature virus Analysis of Cell-Associated Infectivity

27. DR- DR+ HLA-DR Promotes Intracellular Accumulation of Infectious Virus Particles b-Hexosaminidase activity Finzi et al., J. Virol., In Press

28. Conclusions (I) • Newly synthesized Gag is primarily targeted to the plasma membrane in HEK 293 T cells. A concomitant direct targeting of newly synthesized Gag to MVB is also detected but represents a minor fraction of total Gag • Mature Gag products were found to accumulate into MVB over time by a process of internalization from the cell surface that is independent of clathrin mediated-endocytosis. • HIV-1 appears to have adapted to exploit multiple host cell transport pathways to reach and accumulate into MVB

29. Conclusions (II) • HLA-DR expression promotes HIV-1 accumulation to MVB by a process that strictly relies on the cytoplasmic tails of the a and b chains of classical MHC-II molecules. • Intracellular virions produced in presence of HLA-DR remain stable and infectious, indicating that the stable sequestration of infectious virions within cytoplasmic compartments may represent an additional mechanism of viral persistence in HIV-1 infected individuals. • Overall, these results suggest that MHC-II molecules may represent a cellular determinant promoting HIV-1 accumulation into MVB in MHC-II expressing cells such as macrophages.

30. Implications for Drug Development • Fomation and sequestration of virions into MVB protects HIV from the humoral immune response and is likely to facilitate transfer of virus to target cells. • A better understanding of MVB exocytosis may suggest ways by which MVB could be prevented from releasing their content or perhaps encouraged to deliver it to lysosomes. • Given that MVB exocytosis is an essential host cell pathway, effective antiviral agents will need to specifically target interaction of HIV Gag with the endocytic pathway without perturbing the normal host-cell trafficking network.

31. Acknowledgements Laboratory of Molecular Immunology Université de Montréal Laboratory of Human Retrovirology, IRCM Andrés Finzi Alexandre Brunet Alexandre Orthwein Dr. Jacques Thibodeau Yong Xiao Johanne Mercier

32. Pr55Gag Association to Membranes WT Myr- Gag association to membranes (time 0) : SVC21 G2A % of signal % of signal Membranes Cytosol Membranes Cytosol

33. Inhibition of Transferrin uptake in cell expressing the Dynamin K44A DN mutant

34. HLA-DR-induced HIV-1 Gag accumulation in MVB is reduced when endocytosis is inhibited Gag localization (n=5) DR- DR+ DR- DR+ Dyn WT Dyn K44A

35. Filipin 4 ug/mL (09-08-06)

36. Stable HLA-DR Expression in HeLa Cells Induces Gag Accumulation in MVB

37. HIV-1 release HIV-1 Gag localization (n=2) MHC-II-Related Molecules Do Not Modify HIV-1 Release Nor Gag Localization Finzi et al., J. Virol., In Press

38. Late endosome / MVB What is the route by which Gag reaches its PM or MVB steady-state accumulation? Plasma membrane Gag Cytoplasm Nucleus p55 p25 p24