Dimerization of differentially phosphorylated Hepatitis C Virus nonstructural protein 5A NS5A

1
Dimerization of differentially phosphorylated
Hepatitis C Virus non-structural protein 5A (NS5A)

• JULIAN AVILA

2
Hepatitis C Virus

• Affects about 3 of the worlds population, 80
  of the infections become chronic.
• Cirrhosis and cancer.
• Limited treatments.

3
Hepatitis C Virus
ss() RNA
5 NTR
3NTR
9600nt
Flaviviridae
4
NS5A
36
198
447 (2419)
1 (1973)
Tellinghuisen et al., Nature (2005) 435375-379
5
NS5A
BASAL PHOSPHORYLATION (2200-2250)
BASAL PHOSPHORYLATION (2350-2419)
447 (2419)
1 (1973)
49KDa
HYPERPHOSPHORYLATION (S2197, S2201, S2204)
REPLICON SYSTEM
32P labeling
Interferes with RNA replication
HIGH VIRAL RNA REPLICATION.
6
Hypothesis
Hyperphosphorylation of NS5A interferes with its
dimerization.
?
BASALLY PHOSPHORYALATED DIMERIC REPLICATING FORM
HYPERPHOSPHORYLATED
7
First specific Aim

• Does hyperphosphorylation affect NS5A
  dimerization in vitro?

Express NS5A-His in E. coli (wt and NS5A S2204I)
Phosphorylate in vitro using tagged zebrafish
CKI-a expressed and purified in E. coli and
32P-?-ATP.
8
Does hyperphosphorylation affect NS5A
dimerization in vitro?
NS5A-HIS
NS5A-S2204I
p58
p56
SDS PAGE
CKI-a - - - - 32P-ATP - -
- -
Phosphorimaging
p58
p56
p58
p56

S2204I
9
Equilibrium sedimentation
49
40 µM
15,000 r.p.m.

12,000 r.p.m.
A (280 nm)
8,000 r.p.m.
radius (cm)
10
Equilibrium sedimentation

40 µM
20 µM
4 µM
15,000 r.p.m.

12,000 r.p.m.
A (280 nm)
8,000 r.p.m.
radius (cm)
radius (cm)
radius (cm)
11
Equilibrium sedimentation

40 µM
20 µM
4 µM
15,000 r.p.m.

12,000 r.p.m.
A (280 nm)
8,000 r.p.m.
radius (cm)
radius (cm)
radius (cm)
12
Second specific aim

• In vivo interaction of phosphorylated NS5A

Huh-7 cells
S2204I
1
Jc1 - NS5A-RFP
Jc1- NS5A S2204I-GFP-His
S2204I
1
Adapted from Schaller et al., (2007) J Virol.
814591-4603
13
Second specific aim

• In vivo interaction of phosphorylated NS5A

1
Huh-7 cells
1
Jc1 - NS5A-RFP
Jc1- NS5A S2204I-GFP-His
S2204I
1
Adapted from Schaller et al., (2007) J Virol.
814591-4603
14

• In vivo interaction of phosphorylated NS5A

Precipitated
Precipitated
wce
wce
85
83
a-RFP
85
83
a-GFP
S2204I
S2204I
S2204I
S2204I
1
Jc1 - NS5A-RFP
Jc1- NS5A S2204I-GFP-His
S2204I
1
Adapted from Schaller et al., (2007) J Virol.
814591-4603
15
Future directions

• Identify domains necessary for NS5A dimerization.
• Evaluate impact of phosphorylation of NS5A on RNA
  binding.
• Evaluate mobility of NS5A and cellular
  localization of phosphorylated forms.
• Investigate differential binding of phosphoforms
  of NS5A to viral and cellular proteins.

16
Quintavalle et al., 2006
Schaller, et al. 2007
17
Sednterp estimates
Using gtgi29365458gbAAO53246.1 non-structural
5A Hepatitis C virus