gualtiero alvisi, henna kuusisto, ivan k.h. poon, kylie m. wagstaff and david a. jans
DESCRIPTION
TUMOR CELL-SPECIFIC NUCLEAR TARGETING BY CAV VP3 (APOPTIN): PROMISES FOR ANTI- CANCER THERAPY ? Nuclear Signalling Lab. Dept. Biochem. & Mol. Biol. Monash University (Melbourne, Australia). Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff and David A. Jans. - PowerPoint PPT PresentationTRANSCRIPT
TUMOR CELL-SPECIFIC NUCLEAR TARGETING BY
CAV VP3 (APOPTIN): PROMISES FOR ANTI- CANCER THERAPY ?
Nuclear Signalling Lab.
Dept. Biochem. & Mol. Biol.Monash University
(Melbourne, Australia)
Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff
and David A. Jans
Anti-Cancer Approaches
– Cancer is a growing problem world-wide
– We have drugs that kill tumor (but also normal) cells
efficiently (ultimately the patient)
– There is an urgent need to be able to deliver drugs
specifically to tumor cells, and yet retain efficiency in
anti-cancer therapy
Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50
Viral/non-viral mechanisms of drug delivery: Cellular barriers to bioactive
molecules
Anti-Cancer Approaches– Cancer is a growing problem world-wide
– We have drugs that kill tumor (but also normal) cells
– There is an urgent need to be able to deliver drugs specifically to tumor cells, and
yet retain efficiency in anti-cancer therapy
– Cancer cells derive from normal cells; the idea of a magic bullet to
combat cancer (a single property of a cancer cell that can be targeted
specifically) may be naïve (Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50)
– VP3/apoptin – a viral gene-product with tumor cell-specific activity
– ssDNA virus
– Causes immunosuppression /severe
anaemia in young chicks by inducing
apoptosis in thymocytes/ erythroid
progenitors
– 3 viral proteins (VPs): VP1, VP2 and VP3
Chicken Anemia Virus (circovirus)
http://www.tanglewoodfarms.net/img/poulet-rouge/Cou-nus-young1.jpg
VP3/Apoptin
VP3
Nucleus
Cytoplasm
Tumor/transformed cells Normal/untransformed cells
• Nuclear localization
• Induces apoptosis
• Cytoplasmic localization
• No apoptosis
Non-isogenic cell comparisons (eg. SAOS-2 human osteosarcoma line v. VH10 foreskin
fibroblasts Transformed human embryonic kidney v. Rat primary
hepatocytes)
CYTOPLASM NUCLEUS
NLSNLS
Importin 1-Mediated Nuclear Import
NESNES
NLS
NUCLEARENVELOPE
NPC
MULTIPLE SIGNAL-DEPENDENT PATHWAYS FOR TRANSPORT THROUGH
THE NPC
Importin 1-Mediated Nuclear Import
IMPx (exportin)-Mediated Nuclear Export
c. 12 distinct EXPs
RanGTP
RanGTP
IMP1
IMP IMP
Exportin
Exportin
NLS
NLS, nuclear localisation
signal (generally
Lys/Arg-rich)
Nuclear Pore Complex(nups – nucleoporins)
RanGTP
RanGTP
CYTOPLASM NUCLEUS
NLSNLS
NLS
NUCLEARENVELOPE
NPC
MULTIPLE SIGNAL-DEPENDENT PATHWAYS FOR TRANSPORT THROUGH
THE NPC
IMP1
IMP IMP
NLS
NLS, nuclear localisation
signal (generally
Lys/Arg-rich)
RanGTP
RanGTP
NESRanGTP
Exportin
NES
Exportin Exportin 1 (CRM1)-
Mediated Nuclear Export
NES, nuclear export signal
(generally hydrophobic) Ran
GDP
Importin 1-Mediated Nuclear Import
Importin 1-Mediated Nuclear Import
CYTOPLASM NUCLEUS
NLSNLS
NLS
NUCLEARENVELOPE
NPC
MULTIPLE SIGNAL-DEPENDENT PATHWAYS FOR TRANSPORT THROUGH
THE NPC
IMP1
IMP IMP
NLS
NLS, nuclear localisation
signal (generally
Lys/Arg-rich)
RanGTP
RanGTP
NESRanGTP
Exportin
NES
Exportin Exportin 1 (CRM1)-
Mediated Nuclear Export
RanGDP
Importin 1-Mediated Nuclear Import
Importin 1-Mediated Nuclear Import
X X
LMB, leptomycin B:
CRM1-specific inhibitor
VP3/Apoptin
VP3
Nucleus
Cytoplasm
Tumor/transformed cells Normal/untransformed cells
• Nuclear localization
• Induces apoptosis
• Cytoplasmic localization
• No apoptosis
Nuclear Targeting Signals in VP3
IRIGIAGITITLSL
LRS NLS1 NES NLS2
1 33 46 82 88 97 105 111 121
KPPSKKR SCDPSEYR VSKLKESLI TT T108 PS RPRTAKRRIKL
N C
NLS: Nuclear Localization Sequence
NES: Nuclear Export Sequence
Is VP3 recognised by Importins or Exportins ?
AlphaScreen Binding Assay
S bIMPNi2
Acceptor
beads
Donor
beads
.O2 Emission:
620nm
Ni2Donor
beads
.O2
S Acceptor beads
Binding
No binding
VP3-H6
VP3-H6
No Emission
Excitation:
680nm
bImps: biotinylated Imps
VP3-His6: hexa-His-tagged GFP-VP3-HMG
S: streptavidin
b: biotin
680nm
(Amplified Luminescence Proximity Homogeneous Assay)
bIMP
200 nm
Wagstaff and Jans (2005) Anal. Biochem. 348, 49-56
GFP-VP3(1-121)
IMP (nM)
0 10 20 30 40 50 60
% M
axim
al B
ind
ing
0
20
40
60
80
100+ IMP
Kd = 9.7 nM
+ IMP
+ IMP
Wagstaff and Jans (2005) Anal. Biochem. 348, 49-56
VP3 is recognised with high affinity by IMP1; modulated by intramolecular
masking
GFP1 33 46 82 88 97 105 111 121
LRS NLS1 NES NLS2 1-121
GFP-VP3(74-121)
IMP (nM)
0 10 20 30 40 50 600
20
40
60
80
100+ IMP
Kd = 3.7 nM
+ IMP
+ IMP
74-121GFP NLS1 NES NLS2
Nuclear Targeting Signals in VP3
IRIGIAGITITLSL
LRS NLS1 NES NLS2
1 33 46 82 88 97 105 111 121
KPPSKKR SCDPSEYR VSKLKESLI TT T108 PS RPRTAKRRIKL
N C
NLS: Nuclear Localization Sequence
NES: Nuclear Export Sequence
Thr108: phosphorylated specifically in tumor cells
Rohn et al. (2002) 277, 50820
1
Importin 1
Wagstaff and Jans (2006) Anal. Biochem. 348, 49-56
Exportin 1
Poon et al. (2005) Cancer Res. 65, 7059-7064
* Goodrich et al. (1991) Cell 67:293-302
Untransformed Transformed
A) Isogenic cell line pair African Green Monkey Kidney
CV-1 COS-7 (CV-1 transformed with SV40)
B) Human osteosarcoma isogenic cell pair*
SR40 SAOS-2
(SAOS-2 with WT Rb) (Rb mutant)
C) Human skin fibroblast isogenic cell pair**
1br3 1br3N
(1br3 transformed using SV40 T-ag)
ISOGENIC CELL LINES
** From A. Lehmann (Uni. Sussex) (1999)
Curr. Biol. 9, 699-704
GFP-VP3(1-121)GFP
SAOS-2 (Transformed)
Cytoplasm
Nucleus
GFP1 33 46 82 88 97 105 111 121
LRS NLS1 NES NLS2 1-121
VP3 nuclear localization is dependent on the C-terminus
Poon et al. (2005) Cancer Res. 65,
7059-7064
GFP-VP3(1-121)GFP GFP-VP3(74-121) GFP-VP3(1-73)
GFP-VP3(1-89) GFP-VP3(1-111)
Cytoplasm
Nucleus
1-89
1-111
1-73
74-121
GFP
GFP
GFP
GFP
LRS NLS1 NES
LRS NLS1
LRS
NLS1 NES NLS2
SAOS-2 (Transformed)
VP3 nuclear localization is dependent on the C-terminus
Poon et al. (2005) Cancer Res. 65,
7059-7064
GFP-VP3(1-121)GFP GFP-VP3(74-121) GFP-VP3(1-73)
GFP-VP3(1-89) GFP-VP3(1-111) GFP-VP3(NLS1m) GFP-VP3(NLS2m)
Cytoplasm
Nucleus
GFP LRS NES NLS2
GFP LRS NLS1 NES
NLS1m
NLS2m
SAOS-2 (Transformed)
VP3 nuclear localization is dependent on NLS1 and NLS2
Poon et al. (2005) Cancer Res. 65,
7059-7064
GFP1 33 46 82 88 97 105 111 121
LRS NLS1 NES NLS2
GFP-VP3(1-121)GFP GFP-VP3(74-121) GFP-VP3(1-73)
GFP-VP3(1-89) GFP-VP3(1-111) GFP-VP3(NLS1m) GFP-VP3(NLS2m)
SR40 (Untransformed)
VP3 can accumulate in non-tumor cells dependent on NLS1 & NLS2
GFP1 33 46 82 88 97 105 111 121
LRS NLS1 NES NLS2
Poon et al. (2005) Cancer Res. 65,
7059-7064
IMAGE ANALYSIS USING A “WORK WINDOW”:Nuclear localization of VP3(LRSm) in SR40 cells
SOFTWARE (Public Domain): “IMAGE J” (NIH)http://rsb.info.nih.gov/ij/
Trapani et al. (1996) J. Biol. Chem. 271, 4127-4133
Fn
Fn
Fc
Fn
Fb
SR40 cells – VP3(LRSm)
Fn/c = (Fn – Fb)
(Fc – Fb)
0
10
20
30
40
50
60
GFP
GFP-VP3(
1-12
1)
GFP-VP3(
74-1
21)
GFP-VP3(
1-73
)
GFP-VP3(
1-89
)
GFP-VP3(
1-11
1)
GFP-VP3(
NLS1m
)
GFP-VP3(
NLS2m
)
SAOS-2 (Transformed)
SR40 (Untransformed)
p < 0.0001
p < 0.0001
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
GFP1 33 46 82 88 97 105 111 121
LRS NES NLS2 1-121
Dependent on NLS1 & NLS2, VP3 accumulates to a greater extent in tumor
than in non-tumor cells
Poon et al. (2005) Cancer Res. 65,
7059-7064 GFP97 105 111 121
NES NLS2 74-121NLS174 88
NLS1
82
Subcellular Localization of VP3 is Independent of Duration of Expression
DAY 1 DAY 6Fn/c
SAOS-2
SR40p = 0.0083
p = 0.0176
0
10
20
30
40
50
60
70
80
GFP
GFP-VP3(
1-12
1)
0
10
20
30
40
50
60
70
80
GFP
GFP-VP3(
1-12
1)
Is the increased nuclear accumulation in tumor cells due to more efficient nuclear
import or reduced nuclear export ?
- LMB + LMB
GFP-Rev(2-116)
SAOS-2 (Transformed)
GFP
GFP-VP3(1-121)
Lack of VP3 Nuclear Export Activity in Transformed Cells
LMB, leptomycin B: exportin 1 (CRM1)
inhibitor
0
10
20
30
40
50
60
70
80p < 0.0001
- LMB
+LMB
SAOS-2
GFP LRS NLS1 NES NLS2 1-121
(Transformed)
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
Lack of VP3 Nuclear Export Activity in Transformed Cells
Differential Nuclear Export Activity in Transformed and Untransformed Cells
- LMB + LMB
GFP-Rev(2-116)
- LMB + LMB
SR40 (Untransformed)
GFP
GFP-VP3(1-121)
SAOS-2 (Transformed)
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
60
70
80
p < 0.0001
p < 0.0001
- LMB
+ LMB
- LMB
+ LMB
SR40
Differential Nuclear Export Activity in Transformed and Untransformed Cells
GFP LRS NLS1 NES NLS2 1-121
(Untransformed)SAOS-2
(Transformed)
p < 0.0001
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
VP3 nuclear export is mediated via the C-terminal NES not the LRS
- LMB - LMB
GFP LRS NLS1 NES NLS2 1-121
GFP NLS1 NES NLS2 LRSm
GFP LRS NLS1 NLS2 NESm
p < 0.0001
SR40(Untransformed)
SAOS-2(Transformed)
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)0
10
20
30
40
50
60
GFP-VP3(
1-12
1)
GFP-VP3(
LRSm)
GFP-VP3(
NESm)
p < 0.0001
p < 0.0001
0
10
20
30
40
50
60
GFP-VP3(
1-12
1)
GFP-VP3(
LRSm)
GFP-VP3(
NESm)
Exportin 1
Poon et al. (2005)
Cancer Res. 65, 7059-
7064
Localization of VP3 to PML NBs
Anti-PML
SAOS-2 (Transformed)
SR40 (Untransformed) SR40
SAOS-2
GFP-VP3(1-121) Anti-PML Merge
GFP LRS NLS1 NES NLS2 1-121
Poon et al. (2005) Cancer Res. 65, 7059-7064; Janssen et al. (2006) Oncogene 26, 1557-1566
Deletion/mutation of the LRS abolishes PML NB localisation
Saos2
SR40
GFP-VP3(LRSm)GFP-VP3(1-121) GFP-VP3(74-121)
LRS NLS1 NES2 NLS21 33 46 82 88 97 105 111 121
N C
LRS NLS1 NES2 NLS21 33 46 82 88 97 105 111 121
N CX
NLS1 NES2 NLS274 82 88 97 105 111 121
C
VP3 FL wt
VP3 74-121
VP3 FL LRSm
VP3 nuclear export is mediated via the C-terminal NES not the LRS
- LMB - LMB
GFP LRS NLS1 NES NLS2 1-121
GFP NLS1 NES NLS2 LRSm
GFP LRS NLS1 NLS2 NESm
p < 0.0001
SR40(Untransformed)
SAOS-2(Transformed)
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)0
10
20
30
40
50
60
GFP-VP3(
1-12
1)
GFP-VP3(
LRSm)
GFP-VP3(
NESm)
p < 0.0001
p < 0.0001
0
10
20
30
40
50
60
GFP-VP3(
1-12
1)
GFP-VP3(
LRSm)
GFP-VP3(
NESm)Promyelocytic
leukemia (PML) nuclear bodies
Exportin 1
Poon et al. (2005)
Cancer Res. 65, 7059-
7064
Cytoplasm NucleusNuclear envelope
NPC
NPC
β1
Importin β1VP3
LRS NESNLS1 NLS2
β1GTP
Ran
GDP
RanRanGAP1
GTP
Ran
CRM1
CR
M1
GTP
Ran
Normal cell
Tumor cell
Differential Nuclear Export Activity Mediated by the VP3 NES in Normal and Tumor Cells
Testing the role of Thr108 in modulating VP3 nuclear transport
GFP LRS NLS1 NES NLS2 Glu108
GFP LRS NLS1 NES NLS2 Ala108
Ala108
Glu108
GFP LRS NLS1 NES NLS2 1-121
Thr108
Thr108: phosphorylated specifically in tumor cells
Rohn et al. (2002) 277, 50820
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
60
70
80
- LMB
+LMB
- LMB
+LMB
GFP-VP3(Ala108) - non-phosphorylatable VP3
GFP-VP3(Glu108) - mimic phosphorylated VP3
Phosphorylation at Thr108 Inhibits VP3 Nuclear Export in Tumor Cells
SR40(Untransformed)
SAOS-2(Transformed)
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
p = 0.032
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
Nuc
lear
to c
ytop
lasm
ic fl
uore
scen
ce (
Fn/
c)
Poon et al. (2005) Cancer Res. 65,
7059-7064
Tumor cell-specific nuclear targeting of VP3 is effected by a phosphorylation-
regulated NES Cytoplasm
Nucleus
Nuc
lea
r en
velo
pe
NPC
GDP
Ran RanGAP1
GTP
Ran
NPC
1
Importin 1
VP3 1
GTP
Ran
PML
PML
PML Nuclear Body
Exportin 1
Exp
ort
in 1
GTP
Ran
LRS NESNLS1 NLS2
Tumor cell
Normal cell
P
Poon et al. (2005) Cancer Res. 65, 7059-7064
Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50
Viral/non-viral mechanisms of drug delivery
Nucleus Cytoplasm
Tumor/transformed cells Normal/untransformed cells
DNA damage
Modular Recombinant Transporter
Tumour Cell-specific Drug Targeting ?
MSHT-ag NLSHMPDTox MRT
HMP, drug (PS) binding domainDtox, Diphtheria toxin T-domain (endosomolytic)
MSH, α-Melanocyte-stimulating hormone (Melanoma cell specific targeting/uptake)
Rosenkranz et al. (2003) FASEB J. 17, 1121-1124
MSHVP3 tNLSHMPDToxNew MRT
Sites of photoactivation (DHFA) MRT
+ DTox
- DTox
Concentration (nM)
MRT-p6
p6
% V
iab
le c
ells
The VP3 tNTS (aa 74-121) is sufficient for tumour cell-enhanced nuclear
accumulation SR40 SAOS-2 - LMB
+ LMB
:dependent on NES activity as regulated by
negative charge at Thr108
Nucleus Cytoplasm
Tumor/transformed cells Normal/untransformed cells
DNA damage
Modular Recombinant Transporter
Tumour Cell-specific Drug Targeting ?
MSHT-ag NLSHMPDTox MRT
HMP, drug (PS) binding domainDtox, Diphtheria toxin T-domain (endosomolytic)
MSH, α-Melanocyte-stimulating hormone (Melanoma cell specific targeting/uptake)
Rosenkranz et al. (2003) FASEB J. 17, 1121-1124
MSHVP3 tNLSHMPDToxNew MRT
Sites of photoactivation (DHFA) MRT
+ DTox
- DTox
Concentration (nM)
MRT-p6
p6
% V
iab
le c
ells
Anti-Cancer Approaches– Growing problem world-wide, with a need for efficient and specific anti-
cancer therapies
– Cancer cells derive from normal cells; the idea of a magic bullet to
combat cancer (a single property of a cancer cell that can be targeted
specifically) is naïve (Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50)
– VP3/apoptin – tumor cell-hyperactivity (NOT all or nothing)
– Need to combine multiple tumor cell-hyperactive components/strategies
to be able to deliver drugs to cancer cells specifically (MULTIPLICATIVE)
Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50
Non-viral approaches to achieve tumor cell-specific drug delivery
?
VP3
tNTS
MSH
Her2
c-Met
Cancer cell-specific
signallingPhosphorylatio
n
Anti-Cancer Approaches– Growing problem world-wide, with a need for efficient and specific anti-cancer therapies
– Cancer cells derive from normal cells; the idea of a magic bullet to combat cancer (a single property of a cancer cell that
can be targeted specifically) is naïve (Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50)
– VP3/apoptin – tumor cell-hyperactivity (NOT all or nothing)
– Need to combine multiple tumor cell-hyperactive components/strategies to be able to deliver drugs to
cancer cells specifically (MULTIPLICATIVE)
– VP3/apoptin – tumor cell-specific anti-cancer activities
Nuclear Signalling Lab., Dept. Biochem. & Mol.
Biol., Monash University, Clayton
IVAN POON
Cristina Oro
Gualtiero Alvisi Henna Kuusisto
Manisha Dias Kylie Wagstaff
Jingpu Zhang (Beijing)
Dept. of Mol. Genetics of Intracellular Transport,
Institute of Gene Biology, Russian Academy of
Sciences AND Dept. Biophysics, Moscow State
University
Alex Sobolev
Andrey Rosenkranz
Enhancement of cytotoxicity through cell-specific intranuclear delivery of the
photosensitiser bacteriochlorin p6 in B16-F1 melanoma cells
Concentration (nM)
p6
MRT-p6
% V
iab
le c
ells
Sites of photoactivation (DHFA) MRT
+ DTox
- DTox
MSHT-ag NLSHMPDTox MRT
HMP, drug (PS) binding domainDtox, Diphtheria toxin T-domain (endosomolytic)
MSH, α-Melanocyte-stimulating hormone (Melanoma cell specific targeting/uptake)
Rosenkranz et al. (2003) FASEB J. 17, 1121-1124
VP3/Apoptin
VP3
Nucleus
Cytoplasm
Tumor/transformed cells Normal/untransformed cells
• Nuclear localization
• Induces apoptosis
• Cytoplasmic localization
• No apoptosis
Non-isogenic cell comparisons (eg. human osteosarcoma line versus foreskin
fibroblasts)
The distinct subcellular localization of VP3 is determined by differential nuclear import/export of VP3 in tumor and normal
cells
VP3 Nuclear Accumulation is Independent of Cellular Expression Levels
GFP-VP3(74-121)
Fn/c
GFP-VP3(1-121)
0
5
10
15
20
25
30
35
40
45
50
< 20 20-40 > 40
Expression Level
Low Medium High
SAOS-2
SR40
0
5
10
15
20
25
30
35
40
45
50
< 20 20-40 > 40
Expression Level
Low Medium High
SAOS-2
SR40
Poon et al. (2005) J. Virol. 79, 1339-1341
1br3/n(transf.)
1br3
- LMB + LMB
0
55
110
165
220
Fn/c
1br3 1br3/n(transf.)
- LMB
+ LMB
Differential Nuclear Export Activity in Isogenic Transformed
and Untransformed Cells (Fibroblasts)
Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50
VP3-Induced Apoptosis
NPCNPC
NP
CN
PC
NucleusCytoplasm
Mitochondria
VP3*
NE
Transcriptionalmachinery
Apoptosis facilitating products
Hippi
Hippi Hip-1
Cytochrome crelease
Downstream caspases
Apoptosis
DNA
HipK2PML NB
PML
IMP α/β1
P
APC1APC/C
VP3*
P
ααβ1
IMPβ1
IRIGIAGITITLSL
LRS NLS1 NES NLS2
1 33 46 82 88 97 105 111 121
KPPSKKR SCDPSEYR VSKLKESLI TTT108PS RPRTAKRRIKL
N-terminus C-terminus
1 59Hippi binding
1
121
DNA binding
PML NB localization 731
80
66 DNA binding
80 1211 69Apoptosis induction Apoptosis induction
69Multimerization29
VP3
82 121APC1 binding
Domain Organization of VP3
Alvisi & Jans (2006) Drug Resistance Updates 6, 40-50
Properties of VP3/Apoptin
VIRAL PROTEIN IMPORT REGULATION OF IMPORTANCE/ROLE IN INFECTION /EXPORT IMPORT/EXPORT
SV40 T-ag IMP dsDNA-PK/CK2 enhances import CK2 site mutants have 50% reduced viability/slowed
replication kinetics/plaque formation
Cdk site reduces import Essential for polymerase activity
Dengue NS5 IMP Hyperphospho-form in the nucleus ?? Modulate transcription/apoptosis ?? IMP1 Dengue NS3 competes IMP1 binding CRM1 CK2 reduces accumulation Blocking export early in infection increases virus production
(accelerated infection kinetics)
RSV M IMP1 Accumulation facilitated by Inhibition of transcription DNA binding ?
CRM1 CK2 enhances nuclear export Blocking export early in infection reduces virus production
CAV VP3 IMP1 Tumour cell-specific accumulation ??? Enhanced by nuclear retention
CRM1 Negative charge (phos.) at Thr108 ??? prevents export
HIV Tat Nups Cytoplasmic retention requires Nuclear localisation essential for ATP hydrolysis virus replication
? IMP1 ? Accumulation involves nuclear retention
HIV Vpr Nups Accumulation involves nuclear Role in PIC nuclear import
retention ? IMP3 ? HIV MA IMP1 Cytoplasmic retention mechanism ? Role in PIC nuclear import
REGULATION OF VIRAL PROTEIN NUCLEAR IMPORT/EXPORT
– Causes immunosuppression and severe
anemia in young chicks by inducing
apoptosis in thymocytes/erythroid
progenitors
– 3 viral proteins – VP1, VP2 and VP3
Cancer– Growing problem world-wide, with a need for
efficient and specific anti-cancer therapies
– VP3/apoptin – tumor cell-specific anti-cancer
activities
Chicken Anemia Virus (circovirus)
Properties of VP3/Apoptin
VP3
Nucleus
Cytoplasm
Tumor/transformed cells Normal/untransformed cells
• Nuclear localization
• Induces apoptosis
• Cytoplasmic localization
• No apoptosis
Transformed human embryonic kidney Rat primary hepatocytesHuman osteosarcoma cells (SAOS-2) VH10 Human foreskin fibroblasts