thatiane lima sampaio hiv-1 production is dependent on the incorporation of dynamin-2 by nef
TRANSCRIPT
Thatiane Lima [email protected]
HIV-1 production is dependent on the incorporation of Dynamin-2 by Nef
HIV-1 PRODUCTION IS DEPENDENT ON THE INCORPORATION OF DYNAMIN-2 BY NEF
Thatiane Lima Sampaio1,2; Boris Matija Peterlin3, Sônia Nair Báo1
1University of Brasilia, DF 70910-900, Brazil.2Federal Institute of Brasilia, DF 70830-450, Brazil.3University of California, San Francisco, CA 94122, USA.
Atlanta, April 2015
e-mail: [email protected]
Lentivirus virus particles structure
http://www.brookscole.com
Introduction
HIV genomic organization
Introduction
SLIP region in RNAm de gag e pol
PAULUS et al., 2004 http://www.lookfordiagnosis.com/mesh_info.php?term=Fusion+Proteins%2C+Gag-Pol&lang=1
Introduction
Viral Protease
(LEE et al., 2012)
Introduction
Structure Molecular Model of Negative Factor (NEF)• Nef is a crucial factor
for virion infectivity and HIV-1 and SIVmac pathogenesis.
• 27-35 kDa.
Introduction
Role of Nef in primate lentiviral immunopathogenesis
Kirchhoff, F. Cell. Mol. Life Sci. 65 (2008) 2621 – 2636
Introduction
Jurkart Cells
Introduction
Introduction
Jurkart Cells
OBJECTIVE
To investigate the effects of the interaction between Dynamin-2 and Nef during HIV-1
production
MATHERIALS Antibodies: Primaries: anti-CA (NIH #1238), anti-RT
(NIH #6195), anti-IN (#757), anti-Nef (NIH #2949), anti-Clatrina HC (Santa Cruz Biotechnology), anti-Dinamia 2 (Sigma Aldrich), anti-GFP (Sigma Aldrich).
Células: Células Hek-293T e TZM-bl in DMEN (Sigma Aldrich).
RNAs de interferência (siRNA): for clathrin HC (AACCUGCGGUCUGGAGUCAAC) (Hinrichsen et al., 2003), for dynamin 2 (GACAUGAUCCUGCAGUUCA) (Pizzato et al., 2011), control (non-target #1) (Dharmacon).
MethodsTransient transfection in 293T cells
Iodixanol GradientMethods
0 1 2 3 4 5 6 7 8 9 10 11 HIV-1 WT + DNM2.GFP CA
BTop Bottom
Before After
+
+ +
++ +
1 2 3 4 5 6
The overexpression of Dynamin-2 allows for the enhancement of infectivity of HIV-1 wild type, but is not able to rescue HIV-1 Nef-deficient infectivity
0
100
200
300
Infe
ctiv
ity (R
LUx1
000) HIV-1 ΔNef
HIV-1 WT
Dinamin 2.GFP
Dinamin 2.K44A.GFP
+
+ +
+GFP vector+ +
Dinamin 2.GFP
Dinamin 2.K44A.GFP
GFP vector
A
%Ra
zão
CA/
Gag
B
C D
VLP
1 2 3 4 5 6
HIV-1 ΔNef HIV-1 WT
Fig. 1
1 2 3 4 5 6HIV-1 ΔNefHIV-1 WT
HIV-1 ΔNefHIV-1 WT
Results
1 2 3 4 5 6
Figure 1B. Clatrin HC and Dynamin 2 knockdown decrease HIV-1 wt and HIV-1 Δnef infectivity. (C) The siRNA-mediated depletion of clathrin HC and dynamin 2 in virus-producing 293T cells decreases the infectivity of nef –positive and nef-negative progeny virus. (D) The virus production level are equivalent during knockdown of HIV-1 wt and HIV-1ΔNef, except for lane 8. The siRNA assay follow the order (1 and 5) siRNA non-target, (2 and 6) siRNA Clathin HC, (3 and 7) siRNA Dynamin 2, (4 and 8) siRNA Dynamin 2 + siRNA Clathrin HC. Infectivities are the means of triplicate determinations of one experiment.
Clathrin HC and Dynamin 2 knockdown turned HIV-1 WT become as infectious as HIV-1ΔNef, while HIV-1ΔNef knockdown or double knockdown decreased significantly the viral infectivity.
Infectivity ELISA p24
A
B
HIV-1WT
HIV-1ΔNef
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
Fig. 2
WB: αClathrin LC
C
siRNA Clathrin LC +-Clathrin LC
Results
250150
100
50
75250
50
75
Dynamin 2 cleavage product is less incorporated in HIV-1 Δnef than in HIV-1 WT
Dinamin 2
Dinamin 2
Fig. 3. Purification of HIV-1 particles using a OptiprepTM gradient. HIV-1 WT and ΔNef were produced by transient transfection in human embryonic kidney 293T cells and purified as described in Materials and methods. The 100,000 x g HIV-1 pellet was overlaid on the Optiprep gradient. Western blotting of HIV-1 WT and HIV-1 ΔNef pellets using anti-dynamin2.
Optiprep fractions assay 3
Fig. 3 Results
0 1 2 3 4 5 6 7 8 9 10 11
WB: αDinamin 2
0 1 2 3 4 5 6 7 8 9 10 11
HIV-1 Δnef + DNM2.GFP
HIV-1 WT + DNM2.GFP
WB: α-CACA
CA
Top Bottom
HIV-1 Δnef + DNM2.GFP
HIV-1 WT + DNM2.GFP
Discussion
Dynamin 2 is expressed more in T cell lines than in other cells
The Dynamin 2 expression level on different cells lines should explain the difference of dynamin knockdown in Jukart cells (Pizzato) compared to the results for 293T.
Results
Dnm 2100
Β-actina
1 2 3 4 5 60
12
34
56
Molt 4
Jurka
rtHeL
aHeL
a CIITA
TZM-bl
293T
Rat
io β
-act
ina/
Dnm
2
Discussion
Molt 4
Jurkart
More Dynamin 2
293T
Less Dynamin 2
WT and ΔNef infectivity
difference is 40X.
WT and ΔNef infectivity
difference is 2-4X.
Pizzato et al., 2007 Sampaio et al., 2015
Model for cell type infectivity difference
CONCLUSIONS
This data suggests that Nef and Dynamin-2 interaction is an important factor for virus
infectivity and production
