Differential Transcription of Fathead Minnow Immune-Related Genes

Following Infection with Frog virus 3 (FV3)

Kwang Cheng, B. Lynn Escalon, Natalia Garcia-Reyero, and V. Gregory

Chinchar

Immune Competence Determines Survival in Xenopus

• FV3 infection of Xenopus laevis tadpoles and immunocompromised adults triggers severe systemic disease and high mortality.

• In contrast, FV3 infection of immunocompetent adults is confined to the kidney and associated with high levels of survival.

Cellular vs Viral Responses Determine Survival

• Cellular genes contribute to anti-viral immunity and viral clearance.

– Immunocompetent adults display protective innate (IFN, pro-inflammatory) and acquired (Antibody, CTLs) responses that resolve infection.

• In contrast, viral immune evasion genes antagonize cellular immune responses.

– For example ranavirus vIF2α is thought to block PKR-mediated translational shut-off.

Goal

• Identify cellular immune-related molecules that are differentially expressed in FHM cells after FV3 infection.

Approach

• Monitor the expression of cellular immune-related genes using a 60K feature fathead minnow (FHM) microarray.

Q. When are immune-related genes expressed?

M 4h 8h 16h 24h 48h

MCP MCP

Mx

actin

Methodology

• Sham infect or infect replicate (6) cultures of FHM cells with wt FV3 or the 18K KO mutant, that was shown to be attenuated in vivo.

• Prepare total RNA at 8 hr after infection.

• Monitor cellular gene expression by microarray analysis

• Validate microarray results by qRT-PCR

Confirmation of a Productive Viral Infection

M FV3 ∆18K

MCP

18K

M WT Δ18K

MCP

18K

18

K-1

18

K-2

18

K-3

18

K-5

WT-

2

WT-

4

WT-

5

WT-

1

WT-

3

M-1

M-3

M-2

.1

M-2

.2

M-5

Heat map

analysis of

replicate

cultures of

mock-infected,

wt FV3-

infected, and

∆18K knock

mutant-infected

FHM cells

Gene Name (abbreviation WT ∆18K

TNFRSF14 151 51

Interleukin 8 (IL8) 145 70

EIF2S2 96 24

NFIL3 66 13

ICLP2 62 11

NFKBIA 52 23

GILT 30 9

TRAF3 20 12

CAV1 14 8

POLR2A 14 2

Interferon (IFNα) 11 4

Differential Gene Expression: WT FV3 and ∆18K versus Mock-

infected

Additional Upregulated Genes

Gene Name WT ∆18K

Interleukin 1β (IL1B) 9 7

Mx 9 ND

IRF3 5 2

MHC class I 5 4

RIGI 3 3

IRF1 3 2

CCNT2 3 -2

ADAR 1* 4

Genes down regulated after FV3 infection

Gene Name WT ∆18K

HSPB8 -3 -5

G6PD -3 -3

POLB -2 ND

POLA2 -3 -2

C9 -3 ND

ATG7 -4 -3

Pathways Affected by FV3 Infection

• IFN induction and regulation – IFN, IRF-1, IRF-2, IRF-3, IRF-7, RIGI

• Pro-Inflammatory immune response– IL8, IL3, IL1β

• Immune activation/ anti-viral activity– NFKBIA, TNFRSF14, MxD, ADAR

• Antigen Presentation– MHC class I, ICLP2

• Metabolism– EIF2S2 (eIF-2β), cyclin T2a, HSP70 family members,

POLR2A, CAV1

qRT-PCR Validation of Microarray Results

0

50

100

150

200

250

Mock WT 18K

Rela

tive G

ene E

xpre

ssio

n

IL-8

0

10

20

30

40

50

60

70

80

Mock WT 18K

Rela

tive G

ene E

xpre

ssio

n

INF

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

Mock WT 18K

Rela

tive G

ene E

xpre

ssio

n

Cyt2a

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Mock WT 18K

Rela

tive G

ene E

xpre

ssio

n

IRF3

-5

-4.5

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

Mock WT 18K

Rela

tive G

ene E

xpre

ssio

n

HSPB8

Q. Do qualitative/quantitative differences in gene expression

explain why 18K KO mutants were attenuated in vivo?

Pathway – Apoptosis – WT vs. Mock

Red = UPGreen = Down

Pathway – Apoptosis – 18K vs. Mock

Red = UPGreen = Down

Conclusions

• Observation: Numerous immune-related genes, affecting both innate and acquired immunity, were upregulated in FV3-infected cells.

• Hypothesis: Immune-related molecules impair virus replication and lead to long term survival.

Conclusions

• Transcripts induced following infection with wt FV3 and the 18K KO are qualitatively similar.

• However, quantitative differences may reflect the role that the 18K protein plays in viral replication and the modulation of cellular gene expression.

Future Plans

• Use FHM and Xenopus microarrays to analyze global host expression following infection with wt and KO mutants in vivo and in vitro.

• Determine whether specific cellular gene products are critical for a protective immune response.

Acknowledgements

• Kwang Cheng, UMMC (Jackson, MS)

• Lynn Escalon, ERDC (Vicksburg, MS)

• Natalia Garcia-Reyero, Mississippi State University (Starkville, MS)

• Jacques Robert/Guangchun Chen (U. Rochester School of Medicine)

• US ARMY Corps of Engineers

• National Science Foundation