Open Access

Differential gene expression analysis of in vitro duck hepatitis B virus infected primary duck hepatocyte cultures

  • Sajith Nair1,
  • Devaki S Arathy1,
  • Aneesh Issac1 and
  • Easwaran Sreekumar1Email author
Virology Journal20118:363

DOI: 10.1186/1743-422X-8-363

Received: 1 April 2011

Accepted: 23 July 2011

Published: 23 July 2011

Abstract

Background

The human hepatitis B virus (HBV), a member of the hepadna viridae, causes acute or chronic hepatitis B, and hepatocellular carcinoma (HCC). The duck hepatitis B virus (DHBV) infection, a dependable and reproducible model for hepadna viral studies, does not result in HCC unlike chronic HBV infection. Information on differential gene expression in DHBV infection might help to compare corresponding changes during HBV infection, and to delineate the reasons for this difference.

Findings

A subtractive hybridization cDNA library screening of in vitro DHBV infected, cultured primary duck hepatocytes (PDH) identified cDNAs of 42 up-regulated and 36 down-regulated genes coding for proteins associated with signal transduction, cellular respiration, transcription, translation, ubiquitin/proteasome pathway, apoptosis, and membrane and cytoskeletal organization. Those coding for both novel as well as previously reported proteins in HBV/DHBV infection were present in the library. An inverse modulation of the cDNAs of ten proteins, reported to play role in human HCC, such as that of Y-box binding protein1, Platelet-activating factor acetylhydrolase isoform 1B, ribosomal protein L35a, Ferritin, α-enolase, Acid α-glucosidase and Caspase 3, copper-zinc superoxide dismutase (CuZnSOD), Filamin and Pyruvate dehydrogenase, was also observed in this in vitro study.

Conclusions

The present study identified cDNAs of a number of genes that are differentially modulated in in vitro DHBV infection of primary duck hepatocytes. Further correlation of this differential gene expression in in vivo infection models would be valuable to understand the little known aspects of the hepadnavirus biology.

Introduction

The human hepatitis B virus (HBV) and the duck hepatitis B virus (DHBV), which are members of the same virus family, hepadnaviridae, share several features in common [1]. Unavailability of primary animal models susceptible to HBV infection, and inefficiency and unreliability of the infection process in in vitro systems [2] are major limitations in HBV research which restrain the study of this major human pathogen. But the establishment of the animal model with domestic duck employing the DHBV has helped greatly to overcome the shortcomings in HBV research [1, 3]. However, this model has its own limitations as revealed by the differences in the clinical manifestations of the disease in humans and birds infected by these viruses. This mainly pertains to the chronicity in DHBV infection without liver injury/hepatocellular carcinoma (HCC)/cirrhosis; spontaneous elimination of infection in adult ducks; and at the molecular level, the expression of only a cryptic X-protein [4]. A major lacuna in HBV biology is the lack of sufficient information on the molecular mechanisms involved in the development of HCC in chronic HBV patients, which has become a major medical challenge [5].

A few studies have been performed comparing the gene expression in HBV positive HCC and non-cancerous liver [6] and viral and non-viral HCC [7] in patient samples. However, no study has focused to identify the differential gene expression in infection with DHBV either in vivo or in vitro to facilitate a comparative analysis. A recent in vitro study has addressed the proteomic changes during DHBV infection, which has brought to light a number of genes that are involved in the infection process [8]. However, a purely proteome based approach might not reveal changes in the expression levels of many of the low abundant proteins due to technical limitations, which needs to be complemented by mRNA/cDNA differential expression based approaches. In this context, we carried out a subtractive hybridization cDNA library construction and screening to identify the differential gene expression during DHBV infection in primary duck hepatocytes (PDH) in culture. The protocol we followed identified 42 up-regulated and 36 down-regulated genes in DHBV infected PDH in culture.

Methods

Primary duck hepatocytes (PDH) were isolated from 27-day old embryonated, un-hatched, duck eggs free of duck hepatitis B virus (DHBV) infection as previously described [9] and maintained at 5 × 106 cells/ml in DMEM+F12 (Sigma) and 5% FBS supplemented with glucose (0.5 gm/l), dexamethasone (10-5 M) and insulin (1 μg/ml) (all from Sigma) at 37°C in a 5% CO2 atmosphere. DHBV stock was concentrated from LMH-D2 cell culture supernatant, a chicken hepatoma cell line that constitutively replicate DHBV, (a kind gift from Dr. William S Mason, Fox Chase Cancer Centre, California), by precipitation with 10% polyethylene glycol 8000 (USB, USA) [10]. The pellet was re-suspended in DMEM+F12 medium and this concentrated virus was used to infect PDH at an MOI of 103 genome equivalents per hepatocyte, as previously described [11] in presence of 1% DMSO (Sigma). DHBV infection was confirmed by PCR on the DNA obtained from the culture supernatant using DHBV specific primers P1F and D2R (Additional File 1, Table 1).
Table 1

List of cDNAs up-regulated during PDH infection with DHBV

No.

Name of the clone

Abundance Ratio

BLAST Result

BLAST/tBLASTx

Amplicon Size (bp)

e-value

GenBank Accession No.

1

F22

2.41

Cadherin 11, type 2, OB-cadherin (osteoblast) (CDH11)-Gallus gallus

BLAST

462

0

JG662697

2

F125

2.23

Pyruvate dehydrogenase E1-beta subunit variant 3-like-Taeniopygia guttata

BLAST

268

2.00E-85

JG662698

3

F106

2.08

Anas platyrhynchos female-specific sequence

tBLASTx

371

3.00E-04

JG662699

4

F8

2.04

Similar to SH3 domain containing 19-Taeniopygia guttata

tBLASTx

678

4.00E-07

JG662700

5

F71

2

Ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1)-Gallus gallus

BLAST

593

0

JG662701

6

F21

1.87

Zinc finger CCCH-type containing 13 (ZC3H13)-Gallus gallus

BLAST

681

0

JG662702

7

F13

1.77

Succinate-CoA ligase, GDP-forming, alpha subunit(SUCLG1)-Gallus gallus

BLAST

767

0

JG662703

8

F76

1.76

ElaC homolog 2 (E. coli) (ELAC2)-Gallus gallus

tBLASTx

218

1.00E-14

JG662704

9

F19

1.58

CWC22 spliceosome-associated protein homolog-Taeniopygia guttata

BLAST

680

0

JG662705

10

F70

1.55

Similar to KIAA2019 protein/AHNAK nucleoprotein 2-Gallus gallus

BLAST

544

1.00E-150

JG662706

11

F46

1.51

Filamin B, beta-Gallus gallus

BLAST

655

1.00E-140

JG662707

12

F77

1.45

Tumor necrosis factor receptor superfamily,member 6b, decoy (TNFRSF6B)-Gallus gallus

tBLASTx

527

5.00E-07

JG662708

13

F131

1.44

Nuclear protein Matrin 3 (MATR3)-Gallus gallus

BLAST

448

0

JG662709

14

F45

1.42

Heat shock transcription factor 2 (HSF2)-Gallus gallus

BLAST

755

0

JG662710

15

F74

1.42

High mobility group AT-hook 2 (HMGA2)-Gallus gallus

BLAST

209

2.00E-49

JG662711

16

F6

1.4

CLE7-Gallus gallus

BLAST

541

0

JG662712

17

F16

1.35

Cu/Zn superoxide dismutase (SOD1)-Melopsittacus undulatus

BLAST

346

8.00E-31

JG662713

18

F43

1.33

Exonuclease NEF-sp-Gallus gallus

BLAST

680

9.00E-143

JG662714

19

F26

1.32

Component of oligomeric golgi complex 3 (COG3)-Bos taurus

tBLASTx

308

0.002

JG662715

20

F42

1.31

CD9 protein-Anas platyrhynchos

BLAST

748

0

JG662716

21

F127

1.31

Junco hyemalis 164 gene, partial sequence

tBLASTx

234

5.00E-16

JG662717

22

F17

1.29

Quaking homolog, KH domain-Taeniopygia guttata

BLAST

678

0

JG662718

23

F44

1.29

Alanine-glyoxylate aminotransferase 2-Gallus gallus

tBLASTx

721

5.00E-21

JG662719

24

F135

1.27

Leucine-rich repeats and calponin homology (CH) domain containing 4-Oryctolagus cuniculus

tBLASTx

288

0.4

JG662720

25

F14

1.24

Clathrin, light chain A (CLTA)-Gallus gallus

BLAST

673

0

JG662721

26

F83

1.2

Sequestosome 1-Gallus gallus

BLAST

562

0

JG662722

27

F10

1.18

RAB 32, member of Ras oncogene-Gallus gallus

BLAST

743

0

JG662723

28

F30

1.16

Ribosomal protein L6 (RPL6)-Gallus gallus

BLAST

591

0

JG662724

29

F64

1.16

Holocytochrome c synthase (cytochrome c heme-lyase)-Gallus gallus

tBLASTx

421

8.00E-52

JG662725

30

F32

1.16

Lysosomal-associated membrane protein 1-Taeniopygia guttata

tBLASTx

591

4.00E-93

JG662726

31

F7

1.14

Serine protease 23-Gallus gallus

BLAST

740

3.00E-168

JG662727

32

F18

1.09

Beta-catenin isolate 3-Anas platyrhynchos

BLAST

710

0

JG662728

33

F52

1.09

Zebrafish DNA sequence from clone CH211-276C22 in linkage group 6

tBLASTx

218

2.2

JG662729

34

F25

1.08

Gallus gallus finished cDNA, clone ChEST457d18

tBLASTx

696

2.00E-27

JG662730

35

F59

1.07

Leucine proline-enriched proteoglycan (leprecan)1/prolyl 3-hydroxylase 1 (P3H1)-Gallus gallus

BLAST

581

0

JG662731

36

F12

1.07

Ribophorin I-Gallus gallus

BLAST

796

0

JG662732

37

F87

1.06

Gallus gallus finished cDNA, clone ChEST855m19

BLAST

505

3.00E-91

JG662733

38

F95

1.06

Spastic paraplegia 3A (autosomal dominant)-Gallus gallus

BLAST

316

3.00E-54

JG662734

39

F107

1.06

High-mobility group box 3-Taeniopygia guttata

BLAST

276

2.00E-136

JG662735

40

F1

1.05

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 (ATP5J2)-Gallus gallus

tBLASTx

199

9.00E-18

JG662736

41

F78

1.03

Ubiquitin specific peptidase 47 (USP47)-Gallus gallus

BLAST

695

0

JG662737

42

F88

1.03

No significant similarity found

tBLASTx

360

-

JG662738

2 μg of polyA RNA each from DHBV infected and uninfected PDH on zero and 4th day of infection was isolated using PolyATract mRNA isolation system-III (Promega, USA) and was used to construct forward and reverse subtracted cDNA libraries using Clontech PCR-Select cDNA subtraction kit (Clontech, USA), as per kit protocols. PCR amplification of a house-keeping gene GAPDH (Additional File 1, Table 1) from subtracted and un-subtracted samples was used for confirmation of the subtraction efficiency. The subtracted cDNAs were ligated with the pGEM-T (Easy) vector (Promega), competent JM109 Escherichia coli cells (Promega) were transformed and plasmids were isolated following standard molecular biology protocols to obtain 137 forward and 148 reverse subtracted clones.

Macroarrays of these plasmids were generated by vacuum transferring 100 ng each of the denatured clone plasmid in duplicate spots onto nylon membranes (Hybond-N+, Amersham Biosciences UK) using a dot-blot apparatus (Bio-Dot, Bio-Rad). The arrays were hybridized with α32 P labelled forward and reverse subtracted cDNA mixtures as radioactive probes in a reverse-northern procedure. The probes were radio-labelled in a 50 μl PCR reaction using [α32 P]-dCTP, dATP, dGTP, dTTP (0.2 mM each) and unlabelled dCTP (0.02 mM) using the nested PCR primers 1 and 2R (10 μM each) (Additional File 1, Table 1) and the Advantage 2 polymerase mix (Clontech). The adaptor regions common to both the probe and library clones were removed by digestion with RsaI restriction enzyme (NEB). The arrays were individually hybridized with both forward and reverse radio-labelled probes. Subsequent to a pre-hybridization of the membrane for 30 min in the hybridization solution(10% Polyethylene glycol, 1.5× SSPE and 7% sodium dodecyl sulphate), heat denatured probe solution containing 100 μl of RsaI digested radio-labelled probe, 250 μl of 10 mg/ml Herring sperm DNA(Promega) and 100 μl of 0.2N NaOH was added. The probe solution was removed after 16 hrs of hybridization at 65°C and the membrane was washed twice in 2× SSC and 0.1%SDS for 10 min at room temperature followed by two high stringency washes using 0.2× SSC and 0.1%SDS at 65°C for 10 min, and exposure to a phosphor screen for 30 min. The images were captured in Molecular Imager FX (Bio-Rad). The hybridization intensity was measured in the captured images by densitometry analysis of the signal on individual clones using VisionWorksLS image acquisition and analysis software (UVP, USA). The relative abundance ratio of gene expression was calculated using the following formulas.
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-363/MediaObjects/12985_2011_Article_1458_Equa_HTML.gif
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-363/MediaObjects/12985_2011_Article_1458_Equb_HTML.gif

All genes with an abundance ratio of more than one, a cut-off fixed arbitrarily, were then short-listed as the ones with true differential expression. These clones were subjected to automated DNA sequencing in an ABI Prism 310 sequencer (Applied Biosystems) with the Big Dye Terminator 3.0 kit (ABI Prism; Applied Biosystems) as per the manufacturer's directions using the primers TvectF and TvectR (Additional File 1, Table 1). The sequences thus obtained were analyzed using the BLAST online software (NCBI).

Three genes, randomly selected from the top five genes in Table 1 and 2 (with high abundance); one gene from the bottom (with lower abundance) of the table; and one gene, which was not short-listed, were used for real-time PCR analysis for validation of the short-listing procedure. Specific primers for these 10 genes (five from each of the up-regulated and down-regulated library) and primers for the house keeping gene GAPDH were designed (Additional File 1, Table 1) and used in the real-time PCR. cDNA was synthesized using total RNA from fresh sets of primary duck hepatocyte cultures either infected with DHBV or uninfected, as described above, using Avian Myeloblastosis Virus (AMV) reverse transcription system (Promega). Real-time PCR was carried out as previously described [12]. The experiments were repeated thrice, each in duplicates, and average fold change in gene expression was calculated for individual genes.
Table 2

List of cDNAs down-regulated during PDH infection with DHBV

No.

Name of the clone

Abundance Ratio

BLAST Result

BLAST/

tBLASTx

Amplicon Size (bp)

e-value

GenBank Accession No.

1

R73

1.52

Ferritin, heavy polypeptide 1 (FTH1)-Gallus gallus

BLAST

342

4.00E-143

JG662661

2

R90

1.41

Zinc finger CCCH-type, antiviral 1 (ZC3HAV1)-Gallus gallus

tBLASTx

328

1.00E-14

JG662662

3

R130

1.39

T-complex 1-Taeniopygia guttata

BLAST

400

6.00E-172

JG662663

4

R108

1.39

Y box binding protein 1-Gallus gallus

BLAST

486

0

JG662664

5

R96

1.34

MYST/Esa1-associated factor 6-Taeniopygia guttata

BLAST

646

1.00E-17

JG662665

6

R97

1.33

Similar to RGD-CAP-Gallus gallus

BLAST

743

0

JG662666

7

R111

1.31

PREDICTED: Gallus gallus similar to Ankycorbin

BLAST

646

0

JG662667

8

R134

1.3

No significant similarity found

tBLASTx

490

-

JG662668

9

R123

1.25

ATPase8, ATPase6 genes for F0-ATP synthase subunit 8, F0-ATP synthase subunit 6-Anas platyrhynchos

BLAST

462

0

JG662669

10

R133

1.24

Versican-Gallus gallus

BLAST

257

3.00E-98

JG662670

11

R103

1.22

Platelet-activating factor acetylhydrolase isoform Ib, alpha subunit 45kDa (PAFAH1B1)-Gallus gallus

BLAST

508

4.00E-145

JG662671

12

R16

1.18

UPF0308 protein-Gallus gallus

BLAST

593

0

JG662672

13

R100

1.17

TRAF interacting protein (TRAIP)-Gallus gallus

BLAST

631

0

JG662673

14

R84

1.16

Acid alpha-glucosidase-Macaca mulatta

tBLASTx

756

3.8

JG662674

15

R15

1.15

Microtubule-associated protein RP/EB family, member 1-Taeniopygia guttata

BLAST

438

5.00E-168

JG662675

16

R126

1.15

Catechol-O-methyltransferase-Gallus gallus

tBLASTx

395

1.00E-25

JG662676

17

R135

1.14

Chromosome 15 hypothetical ATG/GTP binding protein-Gallus gallus

tBLASTx

239

0.048

JG662677

18

R143

1.13

Ankyrin repeat domain 17 (ANKRD17)-Gallus gallus

BLAST

546

0

JG662678

19

R45

1.13

Splicing factor, arginine/serine-rich 18 (SFRS18)-Gallus gallus

tBLASTx

476

1.00E-145

JG662679

20

R141

1.11

Cytochrome oxidase subunit I (COI)-Anas platyrhynchos

BLAST

336

1.00E-152

JG662680

21

R10

1.11

Eukaryotic translation initiation factor 5 (EIF5)-Gallus gallus

BLAST

735

0

JG662681

22

R129

1.11

Beta-actin-Anas platyrhynchos

BLAST

664

0

JG662682

23

R106

1.1

Alpha enolase-Peking Duck

BLAST

381

0

JG662683

24

R93

1.1

No significant similarity found

tBLASTx

488

-

JG662684

25

R139

1.1

Similar to KIAA1824 protein/WD repeat domain 22-Gallus gallus

BLAST

279

7.00E-100

JG662685

26

R104

1.09

Caspase 3, apoptosis-related cysteine peptidase (CASP3)-Gallus gallus

BLAST

792

0

JG662686

27

R95

1.09

Gallus gallus finished cDNA, clone ChEST757h13

tBLASTx

793

5.00E-19

JG662687

28

R79

1.07

Ral guanine nucleotide dissociation stimulator-like 1 (RGL1)-Gallus gallus

BLAST

333

1.00E-132

JG662688

29

R105

1.06

Gallus gallus similar to MGC53471 protein

BLAST

646

7.00E-89

JG662689

30

R128

1.05

Hydroxyacyl glutathione hydrolase-like,transcript variant 2-Taeniopygia guttata

BLAST

382

4.00E-54

JG662690

31

R22

1.05

Proteasome (prosome, macropain) 26S subunit, ATPase,1 (PSMC1)-Gallus gallus

BLAST

324

4.00E-133

JG662691

32

R99

1.04

Gallus gallus hypothetical protein

BLAST

362

2.00E-86

JG662692

33

R2

1.04

Ribosomal protein L35a-Gallus gallus

BLAST

90

3.00E-08

JG662693

34

R36

1.04

Gallus gallus finished cDNA, clone ChEST191i5

tBLASTx

414

0.025

JG662694

35

R124

1.02

Gallus gallus BAC clone CH261-189F16 from chromosome z

BLAST

524

0

JG662695

36

R86

1.02

Transmembrane protein 30A-Taeniopygia guttata

BLAST

220

4.00E-72

JG662696

The threshold cycle (Ct) values obtained in the real-time PCR analysis were normalized with the expression of the house-keeping gene GAPDH, and the relative expression of individual genes in infected and uninfected cells were calculated by Pfaffl method [13] for Day 0 and Day 4 of infection using the equation:
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-363/MediaObjects/12985_2011_Article_1458_Equc_HTML.gif

The ratios for day 0 and day 4 infected samples were compared and analysed statistically by paired Student's t-test to validate the significance of gene expression changes. P-values < 0.05 were considered significant.

Results & Discussion

The infection of PDH with DHBV did not produce any visible changes on the cell monolayer (Figure 1A). The virus infection was confirmed by PCR detection of a 300 bp DHBV glycoprotein 1 (gp1) gene fragment in the DNA isolated from infected PDH culture supernatant (Figure 1B) and by sequence analysis. The establishment of a productive infection was indicated by the increasing PCR amplification intensity of the gene fragment with every successive day of culture for the total culture period of eight days. For RNA isolation for subtraction library construction, we selected an early time point of 4 days as described in previous studies [14]. Two libraries were generated- the forward subtracted or up-regulated genes and the reverse subtracted or down-regulated genes. The efficiency of subtraction procedure was indicated by a decrease in the intensity and appearance of discrete banding patterns in the lanes with subtracted products (Figure 1C) and was confirmed by PCR detection of the house-keeping gene GAPDH, the amplicons of which appeared at an earlier time point (25 cycles) in un-subtracted samples compared to a later time point (30 cycles) in both forward and reverse subtracted libraries (Figure 1D). Hybridization of macroarrays blotted with 137 up-regulated and 148 down-regulated clones (Figure 1E) and short-listing only the ones with an abundance ratio of more than 1, we obtained 42 non-redundant up-regulated clones and 36 non-redundant down-regulated clones (Tables 1 and 2). Real-time PCR done using the representative sets of short-listed clones gave results confirming the reliability of the short-listing procedure. Genes that topped the differential expression among the up-regulated genes (F22, F8, F71) showed a significant (P < 0.05) increase in expression at 4-days compared to the 0 day in infected PDH (Figure 2A), while the reverse was the case of the down-regulated genes (R73, R90, R130) (Figure 2B), all of whose expression decreased significantly (P < 0.05) at 4-day DHBV infection. F88 and R86, which were selected from the bottom end of the up-regulated and down-regulated gene-tables, respectively, also showed the expected modulation albeit at a lower fold. F62 and R47, picked from the genes left-out did not show any significant difference in their expression pattern.
https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-363/MediaObjects/12985_2011_Article_1458_Fig1_HTML.jpg
Figure 1

Subtractive hybridization cDNA library construction and screening. (A) PDH infected with DHBV, 8 days post-infection. (B) PCR Confirmation of DHBV infection. Upper lane shows the increase in amplification of a DHBV specific gene from days 1 through 8, while the amplification is missing from uninfected controls. (C) Comparison of subtracted and unsubtracted cDNAs on a 2% agarose gel. Individual lanes are marked. Lane 5 is a 100 bp DNA ladder. Lane 8 is a positive control provided with the kit. (D) Analysis of subtraction efficiency using PCR for GAPDH. (E) Macroarray screening by dot-blot hybridization. Each clone is spotted in duplicates. Membranes were hybridized with radio-labelled probes as indicated. The average densitometric intensities of each duplicate clone pair was read for relative abundance calculation.

https://static-content.springer.com/image/art%3A10.1186%2F1743-422X-8-363/MediaObjects/12985_2011_Article_1458_Fig2_HTML.jpg
Figure 2

Real-time PCR of representative genes in DHBV infected PDH, 0-day and 4-days post-infection. (A) Significant up-regulation of cDNAs (F22, F8, and F71) selected from the top of short-listed clones in the up-regulated gene table (Table-1). (B) Significant down-regulation of cDNAs (R73, R90, and R130) selected from the top of short-listed clones in the down-regulated gene table (Table-2). The Y-axis represents relative gene expression values obtained from the Pfaffl analysis (see Methods). Significant P-values (< 0.05) are indicated. The values in parenthesis indicate fold-change in expression.

Functional classification of the short-listed clones using gene ontology based on BLAST results grouped them mainly into those belonging to cellular processes such as cellular respiration, signal transduction, transcription/translation, ubiquitin/proteasome pathway and apoptosis besides those coding for membrane and cytoskeletal proteins (Table 3). Among them, the category that was maximum up-regulated were the ones involved in transcription/translation (19%), whereas the ones maximum down regulated (11%) belonged to cytoskeletal proteins. The former included the HMG Box proteins and Y-box binding proteins. Previous studies have implicated the Y-box binding protein1, Platelet-activating factor acetylhydrolase isoform 1B (PAFAH1B1), Ribosomal Protein L35a, Ferritin, α-enolase, Caspase 3, CuZn Superoxide Dismutase (CuZnSOD), Filamin B, Pyruvate dehydrogenase 1-β, β-catenin, prolyl-3-hydroxylase 1, β-actin, acid α-glucosidase, and clathrin, the cDNAs of which were identified to be up-regulated, with chronic HBV infections and HCC development [6, 1526]. In comparison to the earlier report based on proteome analysis in DHBV infected PDH [8], except for β-actin and α-enolase, all the cDNAs identified in the present study represented new genes. The difference could be due to multiple reasons, and importantly it might include the selective enrichment/elimination of some of the cDNAs during the process of RT-PCR amplification and cloning as part of the subtraction library construction. Nevertheless, our data provides a new set of candidate genes worth further investigation in hepadnaviral infection.
Table 3

Categorization of genes according to the reported function available from literature

 

FORWARD

REVERSE

Membrane proteins

Cadherin 11

Transmembrane protein 30A

 

Lysosomal-associated membrane protein 1

 
 

CD9 protein

 
 

Leucine-rich repeats and calponin homology (CH) domain containing 4

 

Cellular Respiration

Pyruvate dehydrogenase E1-beta subunit

Alpha enolase

 

Succinate-CoA ligase, GDP-forming, alpha subunit(SUCLG1)

Hydroxyacyl glutathione hydrolase-like

 

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2(ATP5J2)

ATPase8, ATPase6 genes for F0-ATP synthase subunit 8, F0-ATP synthase subunit 6

  

Cytochrome oxidase subunit I (COI)

Cytoskeletal

Filamin B, beta

Beta-actin

  

T-complex 1

  

Microtubule-associated protein RP/EB family, member 1

  

Similar to Ankycorbin

Signal Transduction

Beta-catenin isolate 3

TRAF interacting protein (TRAIP)

 

Quaking homolog, KH domain

 

Transcription and Translation

High mobility group AT-hook 2 (HMGA2)

Splicing factor, arginine/serine-rich 18 (SFRS18)

 

High-mobility group box 3

MYST/Esa1-associated factor 6

 

Heat shock transcription factor 2 (HSF2)

Y box binding protein 1

 

CWC22 spliceosome-associated protein homolog

Ribosomal protein L35a

 

Ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1)

Eukaryotic translation initiation factor 5 (EIF5)

 

Nuclear protein Matrin 3 (MATR3)

 
 

Ribosomal protein L6 (RPL6)

 
 

Ribophorin I

 

Ubiquitin-proteasome

Ubiquitin specific peptidase 47 (USP47)

Proteasome (prosome, macropain) 26S subunit, ATPase,1 (PSMC1)

 

Sequestosome 1

 

Apoptosis

Tumor necrosis factor receptor superfamily, member 6b, decoy (TNFRSF6B)

Caspase 3, apoptosis-related cysteine peptidase (CASP3)

Others

Anas platyrhynchos female-specific sequence

No significant similarity found

 

Junco hyemalis 164 gene, partial sequence

UPF0308 protein

 

Gallus gallus finished cDNA, clone ChEST457d18

No significant similarity found

 

No significant similarity found

Gallus gallus finished cDNA, clone ChEST757h13

 

Gallus gallus finished cDNA, clone ChEST855m19

Gallus gallus finished cDNA, clone ChEST191i5

 

Zebrafish DNA sequence from clone CH211-276C22 in linkage group 6

Gallus gallus BAC clone CH261-189F16 from chromosome z

 

Similar to SH3 domain containing 19

Gallus gallus hypothetical protein

 

Zinc finger CCCH-type containing 13 (ZC3H13)

Gallus gallus similar to MGC53471 protein

 

ElaC homolog 2 (E. coli) (ELAC2)

Ferritin, heavy polypeptide 1 (FTH1)

 

Similar to KIAA2019 protein/AHNAK nucleoprotein 2

Zinc finger CCCH-type, antiviral 1 (ZC3HAV1)

 

CLE7

Similar to RGD-CAP

 

Cu/Zn superoxide dismutase (SOD1)

Versican

 

Exonuclease NEF-sp

Platelet-activating factor acetylhydrolase isoform Ib, alpha subunit 45kDa (PAFAH1B1)

 

Component of oligomeric golgi complex 3 (COG3)

Catechol-O-methyltransferase

 

Clathrin, light chain A (CLTA)

Chromosome 15 hypothetical ATG/GTP binding protein

 

RAB 32, member of Ras oncogene

Ankyrin repeat domain 17 (ANKRD17)

 

Holocytochrome c synthase (cytochrome c heme-lyase)

Similar to KIAA1824 protein/WD repeat domain 22

 

Serine protease 23

Ral guanine nucleotide dissociation stimulator-like 1 (RGL1)

 

Leucine proline-enriched proteoglycan (leprecan)1/prolyl 3-hydroxylase 1 (P3H1)

Acid alpha-glucosidase

 

Spastic paraplegia 3A (autosomal dominant)

 
 

Alanine-glyoxylate aminotransferase 2

 

An interesting observation in this study was the inverse pattern of differential expression of ten of these genes in in vitro DHBV infected cells as against the reports on HCC clinical samples [6, 1520]. The mRNAs for the Y-box binding protein1, PAFAH1B1, Ribosomal Protein L35a, Ferritin, α-enolase, acid alpha-glucosidase and Caspase 3 were shown to be down-regulated during in vitro DHBV infection, whereas those of CuZnSOD, Filamin B and Pyruvate dehydrogenase were shown to be up-regulated, where as the reverse was the trend in human HCC. This observation may be purely coincidental owing to the fact that the experimental method we used was an in vitro system, and the changes in primary hapatocytes during culture itself, such as de-differentiation, might have led to these alterations in gene expression.

Conclusions

In summary, the present study identified cDNAs of a number of genes that are differentially modulated in cultured PDH, in vitro infected with DHBV. cDNAs of both novel as well as already reported genes/proteins associated with HBV/DHBV infection or HCC were identified in the library. The genes short-listed here could be valuable leads for further studies in animal models, which might help to understand the pathology of chronic HBV infections and pathogenesis of HCC.

Declarations

Acknowledgements

The financial support by the Department of Biotechnology, Government of India (Grant No. BT/PR8930/GBD/27/39/2006) is gratefully acknowledged.

Authors’ Affiliations

(1)
Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology (RGCB)

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© Nair et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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