Identification of an Arylnaphthalene Lignan Derivative as an Inhibitor against Dengue Virus Serotypes 1 to 4 (DENV-1 to -4) Using a Newly Developed DENV-3 Infectious Clone and Replicon.

Abstract

Dengue virus (DENV) is the most widespread arbovirus, causing symptoms ranging from dengue fever to severe dengue, including hemorrhagic fever and shock syndrome. Four serotypes of DENV (DENV-1 to -4) can infect humans; however, no anti-DENV drug is available. To facilitate the study of antivirals and viral pathogenesis, here we developed an infectious clone and a subgenomic replicon of DENV-3 strains for anti-DENV drug discovery by screening a synthetic compound library. The viral cDNA was amplified from a serum sample from a DENV-3-infected individual during the 2019 epidemic; however, fragments containing the prM-E-partial NS1 region could not be cloned until a DENV-3 consensus sequence with 19 synonymous substitutions was introduced to reduce putative Escherichia coli promoter activity. Transfection of the resulting cDNA clone, plasmid DV3syn, released an infectious virus titer of 2.2 × 102 focus-forming units (FFU)/mL. Through serial passages, four adaptive mutations (4M) were identified, and addition of 4M generated recombinant DV3syn_4M, which produced viral titers ranging from 1.5 × 104 to 6.7 × 104 FFU/mL and remained genetically stable in transformant bacteria. Additionally, we constructed a DENV-3 subgenomic replicon and screened an arylnaphthalene lignan library, from which C169-P1 was identified as exhibiting inhibitory effects on viral replicon. A time-of-drug addition assay revealed that C169-P1 also impeded the internalization process of cell entry. Furthermore, we demonstrated that C169-P1 inhibited the infectivity of DV3syn_4M, as well as DENV-1, DENV-2, and DENV-4, in a dose-dependent manner. This study provides an infectious clone and a replicon for the study of DENV-3 and a candidate compound for future development against DENV-1 to -4 infections. IMPORTANCE Dengue virus (DENV) is the most prevalent mosquito-transmitted virus, and there is no an anti-dengue drug. Reverse genetic systems representative of different serotype viruses are invaluable tools for the study of viral pathogenesis and antiviral drugs. Here, we developed an efficient infectious clone of a clinical DENV-3 genotype III isolate. We successfully overcame the instability of flavivirus genome-length cDNA in transformant bacteria, an unsolved issue for construction of cDNA clones of flaviviruses, and adapted this clone to efficiently produce infectious viruses following plasmid transfection of cell culture. Moreover, we constructed a DENV-3 subgenomic replicon and screened a compound library. An arylnaphthalene lignan, C169-P1, was identified as an inhibitor of virus replication and cell entry. Finally, we demonstrated that C169-P1 exhibited a broad-spectrum antiviral effect against the infections with DENV-1 to -4. The reverse genetic systems and the compound candidate described here facilitate the study of DENV and related RNA viruses.