P-A7
HIV-1 Vpu Antagonizes Rhesus Macaque and Chimpanzee BST-2
Through Cytoplasmic Domain Interactions
Takeshi Yoshida1, 2, 3, Yoshio Koyanagi 3, Hirofumi Akari 2, 3 and Klaus Strebel1
1.Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health
2. Primate Research Institute, Kyoto University, JAPAN 3. Institute of Virus Research, Kyoto University, JAPAN. (contact: [email protected])
Aims & Approaches
Aims
To gain further insights into the species-specificity of BST-2 antagonism by Vpu, we
focused on the physical interaction between Vpu and BST-2 and asked whether physical
interaction is important, necessary, or sufficient for the antagonism of BST-2 by Vpu.
- panel IApproaches
BST-2/Tetherin
- We took advantage of two phenomena (panel I).
human
rhesus
rhesus I48T chimpanzee
(i) Vpu of HIV-1DH12 is less species restricted
NOT
(ii)
antagonized antagonized antagonized antagonized
than HIV-1NL4-3 Vpu and antagonizes both
human and rhesus BST-2 (Shingai&Yoshida et al, JVI, 2011).
(i)
(ii) A single a.a. change in rhesus BST-2 (I48T)
antagonized
antagonized (not tested)
antagonized
renders the protein sensitive to HIV-1NL4-3 Vpu
(Yoshida et al. Frontiers in Microbiology, 2011).
- To assess the physical interaction between Vpu
and BST-2 we used BiFC assay (see Method section).
SIVcpz Vpu HIV-1
HIV-1
(MB897) DH12 Vpu NL4-3 Vpu
HIV-1 Vpu consists of the
Vpu TM cytoplasmic domain
cytoplasmic domain and the
important for CD4 degradation
transmembrane (TM) domain. Vpu has important for enhancement of virus release
two main functions; degradation of CD4 humanBST-2 (+), Vpu (-) humanBST-2 (+), Vpu (+)
and enhancement of virus release.
Human host protein, BST-2 (Tetherin)
was identified as a target of Vpu. BST-2
inhibits HIV-1 virus release but its
monkey BST-2 (+), Vpu (-) monkey BST-2 (+), Vpu (+)
inhibitory activity is antagonized by
Vpu. Further research showed
HIV-1NL4-3 Vpu cannot antagonize
monkey or murine BST-2. However,
mechanism of the species-specificity
; monkey BST-2
; human BST-2
is still not known.
; HIV-1 Vpu
; released virus
; tethered virus
Conclusions
Vpu
Background
NOT
NOT
antagonized (not tested) (not tested) antagonized
- Physical interaction between BST-2 and Vpu was observed in
live cells by BiFC assay (see Method section).
- Human and rhesus BST-2 interact with
NL4-3 Vpu via their TM and CYT domains, respectively.
How BST-2 interacts with HIV-1 Vpu
- Cytoplasmic interaction occurs
between chimpanzee BST-2 and
NL4-3 Vpu also.
Chim
-panzee
human
BST-2
- Physical interaction is necessary
but not sufficient for the BST-2
antagonism by HIV-1 Vpu.
rhesus
BST-2
lumen BST-2
cytoplasm
Vpu
Adapted from Strebel et al. 2009
Vpu
;physical interaction
Experiments & Results
Antagonized by
Yes
No
Yes
Can antagonize
huBST-2 rhBST-2
Yes
No
No
No
Yes
Yes
NL4-3Vpu tmDH12
Method
n.s.
n.s.
n.s.
interaction
100
NL4-3
NL4-3 Vpu
tmCL-7
NL4-3Vpu
tmDH12
75
50
25
0
0.000
0.025
Bst-2 DNA (ug)
each other
protein X
A
rhesus BST-2
protein Y
Adapted from Morell et al. 2008
0.050
B
rhesusBST-2CYThu
rhesus BST-2 CYThu
100
NL4-3
NL4-3 VputmDH12
tmCL-7
NL4-3Vpu
75
50
25
0
0.000
0.025
Bst-2 DNA (ug)
0.050
BST-2
I48T
rhesus
I48T
C rhesus
NL4-3
N4-3 Udel
100
75
50
25
0
0.0
0.1
Bst-2 DNA (ug)
0.2
rhesus BST-2 CYThuI48T
I48T
D rhesusBST-2CYThu
chimpBST-2
chimpBST-2CYThu
rhBST-2
75
50
25
0
0.0
0.1
0.2
Bst-2 DNA (ug)
NL4-3 VputmDH12 antagonizes rhesus BST-2 (A) but not rhesus BST-2CYThu (B).
NL4-3 Vpu antagonizes rhesus BST-2I48T (C) but not rhesus BST-2CYThu I48T (D).
We found that physical interaction is necessary for the BST-2 antagonism by HIV-1 Vpu.
MASTSYDYCRVPM-----EDGDKRCK-(huTM)
MASTLYDYCRVPMDDIWKKDGDKRCK-(chTM)
MASTSYDYCRVPM-----EDGDKRCK-(chTM)
MAPILYDYRKMPMDDIWKEDGDKRCK-(rhTM)
C
B
NL4-3
NL4-3 Udel
100
We hypothesized that rhesus
BST-2 interacts with Vpu via
its CYT domain because
rhesus BST-2 interaction is
CYThu
not dependent on TM domain
of Vpu in Fig. 2C.
** **
WB: anti-KGC
(A) We generated rhesus BST2 mutants carrying human
BST-2 CYT or TM domain.
(B) We confirmed similar amount of protein.
(C) We found that Vpu interacts with TM domain
of human BST-2 and cytoplasmic domain of
rhesus BST-2. ***; P<0.0001, **; P<0.01
The cytoplasmic domain of HIV-1 Vpu contributes to the
physical interaction with, and antagonism of, chimpanzee BST-2.
A huBST-2
Physical interaction is necessary for the BST-2 antagonism by Vpu.
% gag in virus (vs. 0 ug of Bst-2)
KGC
Fig. 4
% gag in virus (vs. 0 ug of Bst-2)
Fluorescence
Fig. 4
***; P<0.0001, n.s.; not significant
% gag in virus (vs. 0 ug of Bst-2)
BiFC (Bimolecular Fluorescence Complementation)
assay is based on the association of complementary
fragments of protein (KGC&KGN) similar to
Yeast-Two-Hybrid assay.
Interaction of the complementary BiFC fragments
causes fluorescence (Kusabira-Green like GFP)
that can be detected by microscopy and FACS.
**
(C) We found that mutation of I48T
in rhesus BST-2 is not critical for the
interaction with NL4-3 Vpu and that
TM of DH12 Vpu is not important
for the interaction with rhesus BST-2.
What is BiFC assay?
***
MFI of BiFC
***
***
WB: anti-KGC
(A) We generated BiFC-tagged
rhesus BST-2 I48T and NL4-3
VputmDH12. (B) After confirming
equal amount of protein, we
NL Vpu tmDH12
compared MFI of BiFC signal in
antagonized
NOT antagonized 293T cells by FACS.
***
MFI of BiFC
To assess the physical interaction between human
BST-2 and HIV-1NL4-3 Vpu, we transfected 293T cells
with BiFC-tagged plasmids (KGC-huBST-2 and NL4-3
Vpu-KGN) and analyzed cells with microscopy. We
observed strong BiFC signal between human BST-2
and NL4-3 Vpu (lower left panel).
***
WB: anti-KGN
250
MFI of BiFC
KGC-huBST-2
&
KGN-NL Vpu
Yes
Yes
Yes
B
200
tag only
NL4-3 Vpu
SIVcpz Vpu
SIVcpz Vpu cytNL
150
100
n.s.
50
0
chimpBST-2
chimpBST-2 CYThu
***, P < 0.01; n.s., P> 0.05.
% gag released
NL4-3 Vpu DH12 Vpu
KGC-huBST-2
&
KGN-tag
(negative control)
KGN
B
Human BST-2 interacts with NL4-3 Vpu via its TM domain.
Rhesus BST-2 interacts with NL4-3 Vpu via its CYT domain.
mock
KGC-huBST-2
KGC-rhBST-2
KGC-rhBST-2
CYThu
KGC-rhBST-2
TMhu
A
Fig. 3
mock
tag only
KGN-NL Vpu
KGN-NL VpuRD
KGN-NL
VputmDH12
bright field
Physical interaction is not sufficient for the BST-2 antagonism by Vpu.
% gag in virus (vs. 0 ug of Bst-2)
BiFC
Fig. 2
mock
KGC-huBST-2
KGC-rhBST-2
KGN-rhBST-2
I48T
Human BST-2 physically interacts
with HIV-1 Vpu in live cells.
***
Fig. 1
chimpanzee BST-2
15
10
5
0
0
1
2
3
4
time (hr)
no Vpu
SIVcpz Vpu
NL4-3 Vpu
SIVcpz Vpu cytNL
(A) Amino acid of the
cytoplasmic domains of
the BST-2 variants used
in this experiment are
shown. (B) We found
that the cytoplasmic
domain of HIV-1NL4-3
Vpu contributes to the
physical interaction
with chimpanzee BST-2.
(C) We found SIVcpz
VpucytNL antagonized
chimpanzee BST-2 but
not chimpBST-2CYThu
through the cytoplasmic
domain interactions.