Highly conserved nucleotide sequence and its deduced amino acids of the 5′-noncoding region and the capsid protein of a Bangkok isolate dengue-3 virus

Abstract

The dengue-3 virus genome encodes an uninterrupted open reading frame (ORF) flanked by 5′ and 3′ non-coding regions. The order of proteins encoded in dengue-3 virus ORF, as with other flaviviruses, is : Cap 5′-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5- 3′. The nucleotide sequence of the 5′-noncoding region and the capsid protein of dengue-3virus (a Bangkok isolate; CH53489 isolated by USAMC-AFRIMS in 1973) has been analyzed in both forward and reverse directions. The PCR-based cycle sequencing technique by the enzymatic method of Sanger et al (1977) using a sequencing primer 5′-end labeled with γ-32P-ATP is the method of our choice for sequencing analysis. One cDNA template was prepared by RT-PCR technique starting from the 5′-end nucleotide 1-465 of the dengue-3 genome. In our cycle sequencing experiments, the substitution of 7-deaza-dG was used for dG in DNA eliminated much of the secondary structures that produced gel artifacts. The final sequence result of this cDNA template was established from its sequence data determined on both strands in opposite directions. Alignment between the newly established nucleotide sequence as well as its deduced amino acid sequence of the Bangkok dengue-3 virus and the published sequence data of the dengue-3 prototype (H87) was manipulated by the PC-DOS-GIBIO-DNASIS TM 06-00 (Hitachi Software). According to the deduced amino acid sequence of the Bangkok dengue-3 virus, its C protein was found to be highly positively charged because of large numbers of lysine and arginine. The homology of the nucleotide sequence between the two dengue-3 virus revealed 97%. The deduced amino acid sequences from the nucleotides 95-465 of the two viruses showed the same indicating highly conserved capsid proteins. Multiple alignment of the nucleotide sequences as well as the deduced amino acid sequences among the Bangkok dengue-3 virus and other dengue 3 viruses also confirmed the highly conserved 5′-noncoding regions and the capsid proteins.