Background
Methods
Cell Culture
Construction of DV2 deletion mutants
In vitro transcription and RNA transfection
Focus assay
Primary Virus Screen
Secondary Screen
Infection and purification of selected mutants
Expression and Processing of DV2 mutant proteins
Preparation of virus inoculum for mouse studies
RT-PCR analysis of mutant viruses
Focus reduction neutralization test (FRNT)
Experimental design for determination of neutralizing antibody titers
Transmission Electron Microscopy
Results
Production of Dengue virus TMD mutants
SEQUENCE E-T1 Domain | Foci | Critical AA Vero | Critical AA C6/36 | |||
---|---|---|---|---|---|---|
Vero | C6/36 | |||||
WT DV2 | 452SWTMKILIGVIITWIG467 | ++ | ++ | |||
1 | ΔIG466-67 |
SWTMKILIGVIITW--
|
-
|
-
|
2aa
| |
2 | ΔW465 |
SWTMKILIGVIIT-IG
| + | - | ||
3 | ΔT464 |
SWTMKILIGVII-WIG
| + | - |
T
464
| |
4 | T464A |
SWTMKILIGVIIAWIG
|
+
|
+
| ||
5 | ΔIT463-64 |
SWTMKILIGVI--WIG
| + | - |
2aa
| |
6 | ΔIIT462-64 |
SWTMKILIGV---WIG
|
-
|
-
|
T
464
| |
7 | ΔVIIT461-64 |
SWTMKILIG----WIG
| + | - |
T
464
| |
8 | ΔGVIIT461-64 |
SWTMKILI-----WIG
| + | - |
5aa
|
5aa
|
9 | ΔI463 |
SWTMKILIGVI-TWIG
| + | - |
I
463
| |
10 | ΔII462-63 |
SWTMKILIGV--TWIG
| + | - |
2aa
| |
11 | ΔVII461-63 |
SWTMKILIG---TWIG
|
-
|
-
| ||
12 | ΔGVII460-63 |
SWTMKILI----TWIG
|
+
|
+
| ||
13 | ΔIGVI459-64 |
SWTMKIL----ITWIG
| + | - | ||
14 | ΔIGVII459-63 |
SWTMKIL-----TWIG
| + | - |
5aa
|
5aa
|
15 | ΔV461 |
SWTMKILIG-IITWIG
|
+
|
+
| ||
16 | ΔGV460-61 |
SWTMKILI--IITWIG
| + | - |
2aa
| |
17 | ΔIGV459-61 |
SWTMKIL---IITWIG
|
+
|
+
| ||
18 | ΔLIGV458-61 |
SWTMKI----IITWIG
| + | - | ||
19 | ΔILIGV457-61 |
SWTMK-----IITWIG
|
-
|
-
|
5aa
|
5aa
|
20 | ΔG460 |
SWTMKILI-VIITWIG
|
+
|
+
| ||
21 | ΔIG459-60 |
SWTMKIL--VIITWIG
| + | - |
2aa
| |
22 | ΔLIG458-60 |
SWTMKI---VIITWIG
|
+
|
+
| ||
23 | ΔILIG457-60 |
SWTMK----VIITWIG
|
+
|
+
| ||
24 | ΔILI457-59 |
SWTMK---GVIITWIG
|
+
|
+
| ||
25 | ΔIL457-58 |
SWTMK--IGVIITWIG
|
-
|
-
|
2aa
| |
26 | ΔI457 |
SWTMK-LIGVIITWIG
| + | - |
I
457
| |
27 | ΔKI456 |
SWTM--LIGVIITWIG
| + | - |
2aa
| |
28 | ΔK456 |
SWTM-ILIGVIITWIG
|
-
|
-
|
K
456
|
K
456
|
29 | K456A |
SWTMAILIGVIITWIG
|
+
|
+
| ||
30 | ΔMK455-56 |
SWT--ILIGVIITWIG
| + | - |
2aa
| |
31 | ΔM455 |
SWT-KILIGVIITWIG
|
-
|
-
|
M
455
|
M
455
|
32 | ΔTM454-55 |
SW--KILIGVIITWIG
|
+
|
-
|
2aa
| |
33 | ΔT454 |
SW-MKILIGVIITWIG
|
+
|
+
| ||
34 | ΔW453 |
S-TMKILIGVIITWIG
|
+
|
+
|
Analysis of the DV2 deletion mutants
SEQUENCE M-T1 DOMAIN | Foci | Critical AA Vero | Critical AA C6/36 | |||
---|---|---|---|---|---|---|
Vero | C6/36 | |||||
WT DV2
|
245
PGFTMMAAILAYTIG
259
|
++
|
++
| |||
1 | ΔA251 |
PGFTMM-AILAYTIG
|
+
|
+
| ||
2 | ΔAA251-52 |
PGFTMM--ILAYTIG
|
-
|
-
| AA251-52 | AA251-52 |
3 | ΔAAI251-53 |
PGFTMM---LAYTIG
|
-
|
-
| AAI251-53 | AAI251-53 |
4 | ΔAAIL251-54 |
PGFTMM----AYTIG
|
+
|
+
| ||
5 | ΔAAILA251-55 |
PGFTMM-----YTIG
|
-
|
-
| 5aa | 5aa |
6 | ΔM250 |
PGFTM-AAILAYTIG
|
+
|
+
| ||
7 | ΔMM249-50 |
PGFT--AAILAYTIG
| + | - | M249 | |
8 | ΔTMM248-50 |
PGF---AAILAYTIG
| + | - | M249 | |
9 | ΔFTMM247-50 |
PG----AAILAYTIG
|
-
|
-
| 5aa | 5aa |