Figure 1. - PowerPoint Presentation

Slide1

Figure 1.

Schematic

of

picornavirus

genome

organization

. The positive-

strand

RNA has the viral

protein

VPg

covalently

linked

to the 5′ end of the

genome

.

Both

the 5′ and 3′

noncoding

regions

are

highly

structured

and

contain

RNA

secondary

structural

elements

required

for

enterovirus

translation initiation (

Internal

Ribosome Entry Site (IRES)) and RNA

replication

.

Picornaviruses

have a

genome-encoded

poly(A) tract

at

the 3′ terminus of

their

RNA and express a single

polyprotein

that

is

proteolytically

processed

into

precursor

and mature viral

proteins

,

required

for

replication

of the virus, by the

two

proteinases

2A and 3Cpro. The

polyprotein

is

segregated

into

three

major

regions

. The

capsid

proteins

are

encoded

in the P1

region

, and the

nonstructural

proteins

(viral

proteins

required

for modification of the host

cell

environment

,

protein

processing

, and RNA

replication

) are

encoded

in the P2 and P3

regions

(

adapted

from

the

Swiss

Institute of

Bioinformatics

website

/ http://

viralzone.expasy.org

).Slide2

TD21

TD7

0

28

TD30

TD49

800

ng

of

in vitro

transcribed RNA

HeLa S10 Cytoplasmic extract ATP

6 hours

of incubation

at 30°C

Visualization of proteins synthesized on polyacrylamide gel

[35S]-methionine

Wild-type

strains

Terminaly

d

eleted

strains

3CD

VP3

2A

3AB

3Dpol

VP1

2C

Figure 2.

In vitro

translation assay. After 6h of incubation at 30°C of viral RNA in the presence of a cytoplasmic extract of

HeLa

S10 cells and

35

S-labeled methionine, synthesized viral proteins (most significant are indicated to the right of the gel) are observed after migration on a SDS-Page gel.

Slide3

rLuciferase

activity

B- HCM

C- HL-1

cells

Control

Control

Wild-type

strains

Deleted

strains

P1

region (capside proteins)  Rluc

Inserting deletionsA

Figure 3. Construction and transfection of luciferase replicons into two types of cardiac myocytes. (A) The replicon consists of the viral genome in which the region encoding the capsid proteins (P1) was replaced with the gene encoding Renilla luciferase (kindly provided by Dr. F.J. Van Kuppeveld

, Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, the Netherlands). Deletions of 7 to 49 nucleotides have been inserted at the 5 'end of the viral genome. Replicons were then transfected (B) in primary human cardiac myocytes or (C) in a continuous cell line of murine cardiac myocytes from an atrial tumor. Luciferase activity was measured at 2 hours post-transfection. Full-length replicons are indicated in blue and deleted replicons in red. Slide4

rLuciferase

activity

HCM

Control

2 Logs

HL-1

Control

2 Logs

Translation

-RNA

+RNA

3D

pol

Transfected

RNA

Luciferase

signal amplification

A

B

C

Wild-type

viruses

Deleted

viruses

Figure 4

. Luciferase activity compared between full-length and deleted replicons. Non-deleted replicons in blue and deleted replicon in red were transfected (A) in primary human cardiac

myocytes

or (B) in murine

cardiomyocytes

. Luciferase activity was measured from 2 to 8 hours post-transfection. The results presented are the product of three independent experiments. (C) Schematic representation of luciferase signal amplification mechanism by replicating transfected viral RNA.

Slide5

PV1

TD21

TD7

0

28

TD30

TD49

Positive RNA

Double-

stranded

RNA

+

-Deleted strains

Wild-type strainsPositive controlFigure 5. In Vitro

replication assay of the viral RNA. Six hours after in vitro translation in the absence of [35S]-methionine, the reaction mixture consisting of viral RNA (400 ng for the CVB3 wild-type strains and poliovirus; 400 and 800 ng for the deleted CVB3 strains) and

HeLa S10 cytoplasmic extract was incubated for further 2h at 34C in the presence of α-[32P] CTP. The purified RNA was then loaded on an agarose gel at 1.1%. Poliovirus 1 (PV1) is used here as a positive control. Slide6

rLuciferase

activity

A- HCM

2 Logs

B- HL-1

2 Logs

C- HCM

Control

D- HL-1

Control

TD7

TD30

TD21

TD49

CV-B3 0

CV-B3 28

Without

guanidine hydrochloride With

guanidine hydrochlorideWild-type strains

Deleted

strainsFigure 6.

Impact of guanidine hydrochloride treatment on the luciferase activity measured after transfection of human and murine cardiac myocytes

with deleted and not deleted replicons. Full-length (A and B) and deleted (C and D) luciferase replicons were transfected into human (HCM) (A and C) and murine (HL-1) (B and D) cardiac myocytes. Guanidine hydrochloride at a final concentration of 3 mM was added or not to the cell culture medium 30 minutes after transfection of the replicons. Luciferase activity was then measured from T0 to T8H post-transfection in the GuHCl

treated (in red) or untreated (blue) cells. The results presented here are the product of three independent experiments. Slide7

28

TD7

TD21

TD30

TD49

28

TD7

TD21

TD30

TD49

28

TD7TD21TD30TD49

+

strand- strand

Viral load in genomic RNA copy/ml of cell medium65552.4

1.5231.4

1.41.53.224h PI48h PI0h PI

Figure 7. Quantification by one step RT-qPCR of positive- and negative-strands of viral RNA in a kinetics of infection of primary human cardiac myocytes. The full-length CVB3-28 and CVB3 viruses deleted of 7, 21, 31 and 49 nucleotides used to achieve this infection were produced on human

hepatocarcinoma cells Huh7.5 defective for type I interferon response. Cardiomyocytes infected in triplicate were collected at 0, 24 and 48 hours post-infection in 2 ml of cell culture medium after three cycles of freezing/thawing. Load of positive- (blue) and negative-strand (orange) of viral RNA is shown on the y-axe as the number of RNA copies per ml of cell culture medium. Slide8

3’ end

2C

A

B

Figure 8.

Schematic representation of cellular proteins (PCBP2,

hnRNPC

) and viral proteins (2C, 3AB, 3CDpro, 2BC) binding (A) the 5’ end of the viral genome in order to prime the synthesis of the negative-strand

antigenomic

RNA and (B) the 3' end of the

antigenomic

negative-strand for the synthesis of positive-strand genomic RNA. Slide9

28

0

TD30

PV1

Positive control

TD7

TD49

250

500

1000

ng

of PCBP2RNA alonePV1

1

2

3

4123

41234

14

123

412

3

4

1

34

Figure 9. RNA mobility shift assay of full-length (0 and 28) and deleted positive-strand viral RNA (TD7, TD31 and TD49) in the presence of the cellular protein PCBP2. A RNA fragment of 110 nucleotides located at the 5’ end of the genomic positive-strand viral RNA (stem-loop I or clover-leaf) was transcribed with

32P-CTP and incubated in the absence (free probe=FP; lines 1)

or in the presence of 250 (lines 2), 500 (lines 3) and 1000

ng (lines 4) of

the protein PCBP2. The formation of a ribonucleoprotein complex (indicated by the black arrows) results in a migration delay of the radioactive nucleic acid probe on the

agarose

gel. The stem-loop IV of poliovirus 1 IRES was used as positive control for PCBP2 experiments. Slide10

Figure 10.

RNA mobility shift assay of full-length (strain 28) and deleted positive-strand viral RNA (TD7, TD21, TD31 and TD49) in the presence of the viral protein 3CD. A RNA fragment of 110 nucleotides located at the 5’ end of the genomic positive-strand viral RNA (stem-loop I or clover-leaf) was transcribed with

32

P-CTP and incubated in the absence (free probe=FP) or in the presence of 50, 100, 150, 250, 500 and 1000

ng

of the viral protein 3CD. The formation of a

ribonucleoprotein

complex (indicated by the black arrows) results in a migration delay of the radioactive nucleic acid probe on the

agarose

gel.

501501002

50

5001000

FP50150

100250500

1000FP50150

100250

5001000

FP

50

150

100

250500

10

00

FP

50

150

10

02505001000

FP28TD30TD7TD49

TD21Slide11

Figure 11.

RNA mobility shift assay of full-length (strain 28) and deleted negative-strand viral RNA (TD7, TD21, TD31 and TD49) in the presence of the cellular protein

hnRNPC

. A RNA fragment of 750 nucleotides corresponding to the 3’ end of the

antigenomic

negative-strand viral RNA was transcribed with 32P-CTP and incubated in the absence (free probe=FP) or in the presence of 100, 250 and 500 of the recombinant protein

hnRNPC. The formation of a ribonucleoprotein

complex (indicated by the black arrows) results in a migration delay of the radioactive nucleic acid probe on the agarose gel.

28

TD30

TD7

TD49

TD21

Free Probe100250

500

Free Probe100

250500

Free Probe100250

500Free Probe

100

250

5

00Free Probe

100

250

500