HIV-1 Assembly in the Endocytic Pathway:

Opportunities for the Identification of Novel Anti-HIV Drug Targets

Éric A. CohenUnité de rétrovirologie humaine

Institut de Recherches Cliniques de Montréal

Symposium on Novel Targets for Drug Development XVI International AIDS Conference

Toronto, August 14, 2006

Gag-Driven HIV-1 Particle Production

HIV-1 Gag

• 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.

p6CA (p24) p2 NC p1

M I L

MA(p17)

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

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 inmacrophages. These intracellular compartments express late endosomal / MVB markers including MHC-II, CD63, Lamp1 and LBPA.

Sites of HIV-1 Assembly and Release

Retroviral assembly

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?

Nucleusp55

p25

p24

Plasma membrane (PM)

Cytoplasm

What is the route by which Gag reaches its PM or MVB steady-state accumulation?

Gag

Late endosome / MVB

Nucleusp55

p25

p24

Plasma membrane (PM)

Cytoplasm

What is the route by which Gag reaches its PM or MVB steady-state accumulation?

Gag

Late endosome / MVB

Nucleusp55

p25

p24

Plasma membrane (PM)

Cytoplasm

What is the route by which Gag reaches its PM or MVB steady-state accumulation?

Gag

Late endosome / MVB

Subcellular Fractionation on a Continuous Iodixanol (Optiprep) Gradient (0-20%)

Disrupt cellsPellet nuclei

PNS

1 2 3 4 5 6 7 8 9 10 11 12 13 14

5%5%

20%20%

Adapted from Ira Mellman(Sheff et al., 1999)

Characterization of Light Density Fractions

Characterization of High Density Fractions

Trafficking of HIV-1 Gag-Associated Products in HEK 293T Cells

p24

Moc

k

0 0.5 2 5 hs

Virus released

Supernatant

Filipin, an Inhibitor of Endocytosis, Prevents Gag Internalization from the PM to MVB

Filipin (4µg/mL)

Transferrin + (clathrin dependent)

Cholera-toxin + (caveolae dependent)

Inhibition (+) Internalization (- )

Effect of Filipin on Transferrin and Cholera-toxin internalization

Internalization of Gag from the PM to MVB Involves an Endocytosis Process that is Clathrin-independent

Chlorpromazin (10 µg/mL)

Transferrin ++ (clathrin dependent)

Cholera-toxin - (caveolae dependent)

Inhibition (+) Internalization (- )

Effect of Chlorpromazin on Transferrin and Cholera-toxin internalization

p55

p25

p24

Route by which Gag reaches its PM or MVB steady-state accumulation in HEK 293T cells

Nucleus

Plasma membrane (PM)

Cytoplasm

Gag

Late endosome / MVB

Clathrinindependentendocytosis

X

X

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?

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).

MHC-II molecules present peptides to CD4+ T cells.

MHC Class II Molecules

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).

The cytoplasmic tails/transmembrane domain of MHC-II molecules are required for the formation/maturation of these compartments.

To determine whether MHC-II could relocate HIV-1 Gag from the cell surface to intracellular compartments in 293T cells.

Goal

Andrés Finzi Yong Xiao

Gag localization

Diffuse

Punctuate

Gag staining

HLA-DR Induces Gag Accumulation in Intracellular Compartments in 293T Cells

Diffuse

Punctuate

Gag staining

Dose-dependent Gag relocalization by HLA-DR

Gag Accumulates in MVB Upon HLA-DR Expression in HEK 293T Cells

Finzi et al., J. Virol., In Press

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

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

HLA-DR Expression Decreases HIV-1 Release

HIV-1 release(n=8)

Finzi et al., J. Virol., In Press

HxBc2 +/- HLA-DR

0 16 24 48 72Hours post-transfection

IVS 10µMWash cells

Cell lysisand infection of HeLa β-Gal MAGI

assay

-DR

+DR

Mature Virus

Immature Virus

MVB containing mature virus

MVB containing immature virus

Analysis of Cell-Associated Infectivity

HLA-DR Promotes Intracellular Accumulation of Infectious Virus Particles

-Hexosaminidase activityDR- DR+

Finzi et al., J. Virol., In Press

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

HIV-1 appears to have adapted to exploit multiple host cell transport pathways to reach and accumulate into MVB

Conclusions (I)

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.

HLA-DR expression promotes HIV-1 accumulation to MVB by a process that strictly relies on the cytoplasmic tails of the and 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.

Conclusions (II)

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.

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.

Implications for Drug Development

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.

Acknowledgements

Laboratory of HumanRetrovirology, IRCM

Yong Xiao

Alexandre Brunet

Dr. Jacques Thibodeau

Andrés Finzi

Laboratory of Molecular ImmunologyUniversité de Montréal

                                           

Alexandre Orthwein

Johanne Mercier

Gag association to membranes (time 0) : SVC21 G2AWT Myr-

Pr55Gag Association to Membranes

% o

f s

ign

al

% o

f s

ign

al

Membranes Cytosol Membranes Cytosol

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

DR- DR+ DR- DR+

Dyn WT Dyn K44A

Gag localization(n=5)

HLA-DR-induced HIV-1 Gag accumulation in MVB is reduced when endocytosis is inhibited

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

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

MHC-II-Related Molecules Do Not Modify HIV-1 Release Nor Gag Localization

HIV-1 release

HIV-1 Gag localization(n=2)

Finzi et al., J. Virol., In Press

Nucleusp55

p25

p24

Plasma membrane

Cytoplasm

What is the route by which Gag reaches its PM or MVB steady-state accumulation?

Gag

Late endosome / MVB