cDNA-AFLP analysis of differential gene expression in human hepatoma cells (HepG2) upon dengue virus infection.
In infectious diseases, the disease pathogenesis is the outcome of the interaction between the genome of the host and the genome of the pathogen. Despite the wide distribution of dengue infections in the world, and the large number of annual infections, few studies have investigated how the dengue genome alters the global transcriptional profile of the host cell. To investigate alterations in the liver cell transcriptome in response to dengue virus infection, liver cells (HepG2) were infected with dengue serotype 2 at MOI 5 and at 3 days post-infection RNA extracted and analyzed by cDNA-AFLP in parallel with mock-infected cells. From 73 primer combinations over 5,000 transcription-derived fragments (TDFs) were observed, of which approximately 10% were regulated differentially in response to infection. Sixty-five TDFs were subsequently cloned and sequenced and 27 unique gene transcripts identified. Semi-quantitative reverse transcription (RT)-PCR was used to validate the expression of 12 of these genes and 10 transcripts (CK2, KIAA509, HSP70, AK3L, NIPA, PHIP, RiboS4, JEM-1, MALT1, and HSI12044) were confirmed to be differentially regulated, with four transcripts (HSP70, NIPA, RiboS4, and JEM-1) showing a greater than twofold regulation. These results suggest that the expression of a large number of genes is altered in response to dengue virus infection of liver cells, and that cDNA-AFLP is a useful tool for obtaining information on both characterized and as yet uncharacterized transcripts whose expression is altered during the infection process.
Ekkapongpisit
Maneerat
M
Molecular Pathology Laboratory, Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Nakorn Pathom, Thailand.
Wannatung
Tirawat
T
Susantad
Tharinee
T
Triwitayakorn
Kanokporn
K
Smith
Duncan R
DR
D048868:Adaptor Proteins, Signal Transducing; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D003715:Dengue / Q000378:metabolism*; D003716:Dengue Virus; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; C105528:BLZF1 protein, human; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018797:Cell Cycle Proteins; D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D011506:Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C477288:ZC3HC1 protein, human