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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

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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



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.


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


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



Late endosome / MVB budding sites remain poorly understood.

What is the route by which Gag reaches its PM

or MVB steady-state accumulation?

Plasma membrane (PM)

Gag

Cytoplasm

Nucleus

p55

p25

p24


Late endosome / MVB budding sites remain poorly understood.

What is the route by which Gag reaches its PM

or MVB steady-state accumulation?

Plasma membrane (PM)

Gag

Cytoplasm

Nucleus

p55

p25

p24


Late endosome / MVB budding sites remain poorly understood.

What is the route by which Gag reaches its PM

or MVB steady-state accumulation?

Plasma membrane (PM)

Gag

Cytoplasm

Nucleus

p55

p25

p24


5% budding sites remain poorly understood.

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


Characterization of Light Density Fractions budding sites remain poorly understood.


Characterization of High Density Fractions budding sites remain poorly understood.


p24 budding sites remain poorly understood.

Virus released

Mock

0 0.5 2 5 hs

Supernatant

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


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 (- )


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 (- )


Clathrin Endocytosis Process that is Clathrin-independent

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


Questions
Questions Endocytosis Process that is Clathrin-independent

  • 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 Class II Molecules Endocytosis Process that is Clathrin-independent

  • 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.


Andrés Finzi Endocytosis Process that is Clathrin-independent

Yong Xiao

Goal

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


Gag staining Endocytosis Process that is Clathrin-independent

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


Gag Accumulates in MVB Upon HLA-DR Expression Endocytosis Process that is Clathrin-independent

in HEK 293T Cells

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


HIV-1 Assembles and Buds Within Intracellular Compartments Endocytosis Process that is Clathrin-independent

Upon HLA-DR Expression


HIV-1 Assembles at the Plasma Membrane in Absence of HLA-DR Endocytosis Process that is Clathrin-independent


HIV-1 release Endocytosis Process that is Clathrin-independent

(n=8)

HLA-DR Expression Decreases HIV-1 Release

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


Cell lysis Endocytosis Process that is Clathrin-independent

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


DR- Endocytosis Process that is Clathrin-independent

DR+

HLA-DR Promotes Intracellular Accumulation

of Infectious Virus Particles

b-Hexosaminidase activity

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


Conclusions (I) Endocytosis Process that is Clathrin-independent

  • 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


Conclusions (II) Endocytosis Process that is Clathrin-independent

  • 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.


Implications for Drug Development Endocytosis Process that is Clathrin-independent

  • 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.


Acknowledgements Endocytosis Process that is Clathrin-independent

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


Pr55 Endocytosis Process that is Clathrin-independentGag Association to Membranes

WT

Myr-

Gag association to membranes (time 0) : SVC21 G2A

% of signal

% of signal

Membranes

Cytosol

Membranes

Cytosol


Inhibition of Transferrin uptake in cell expressing Endocytosis Process that is Clathrin-independent

the Dynamin K44A DN mutant


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


Filipin 4 ug/mL when endocytosis is inhibited

(09-08-06)


Stable HLA-DR Expression in HeLa Cells Induces when endocytosis is inhibited

Gag Accumulation in MVB


HIV-1 release when endocytosis is inhibited

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


Late endosome / MVB when endocytosis is inhibited

What is the route by which Gag reaches its PM or MVB

steady-state accumulation?

Plasma membrane

Gag

Cytoplasm

Nucleus

p55

p25

p24


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