Effect Of Gene Mutantion Of Capsid Protein On Biological Characteristics Of Dengue Virus

Dengue (DEN) viruses are enveloped RNA viruses that contain a single-stranded, positive-sense, capped RNA genome of approximately 11 kb. A single viral polypeptides is co-translationlly processed by viral and cellular proteases to generate three structural protein (C, M and E) and at least seven non-structural proteins (NS). The 5'-UTR-C-prM/M-E-NSl-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'-UTR is its gene structure (untranslated region, UTR). Capsid (C) protein within the virus particle is involved to assembly the nucleocapsid core (NC), consisting of multiple copies of C protein that surround a single copy of the viral RNA genome, thus to play an important role in NC formation and infectious particles assembly.Study of novel attenuated dengue vaccine based on the reverse genetic systems highlights the field with the development of infectious clone method since the 1990's. The target sequence to be mutated focused on the UTRs and prM-E of genomic RNA. And the capsid protein of flaviviruses is generally thought to be indispensable. But the study done with another member TBE showed that recovered virus could still be viable, highly attenuated and immunogenicity when 4-21 amino acid residues of the internal hydrophobic domain in its capsid protein were deleted, suggesting its functional flexibility, meanwhile, the capsid proteins of the flaviviruses families have conserved structure, all of which exist internal hydrophobic sequence. In the process of the assembly of nucleocapsid (NC), the formed hydrophobic cleft may combined with the enveloped protein E, and deletion of special internal hydrophobic sequence of capsid protein might affect its normal assembly, as a result, an attenuated, high immunogenic recovered virus was acquired. In this study, we obtained recovered virus of capsid protein deletion mutant based on the construction of the infectious full-length cDNA clone of DEN2-43 virus, and elucidated the biological effect of the gene mutation of capsid protein on the dengue virus, which provided new evidences in clarifying the relationship of the structure of genomic RNA and its function and established new method to develop dengue vaccines.To construct infectious full-length cDNA clone of dengue virus type 2 isolated in China (DEN2-43), approximate 11kb full-length cDNAs were amplified by long RT-PCR and overlap-PCR according to the published nucleotide sequence of DEN2-43. Then full-length cDNAs were cloned into a low copy number vector...