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.

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.

NLRC5 restricts dengue virus infection by promoting the autophagic degradation of viral NS3 through E3 ligase CUL2 (cullin 2).

NLRC5 has been reported to be involved in antiviral immunity; however, the underlying mechanism remains poorly understood. Here, we investigated the functional role of NLRC5 in the infection of a flavivirus, dengue virus (DENV). We found that the expression of NLRC5 was strongly induced by virus infection and IFNB or IFNG stimulation in different cell lines. Overexpression of NLRC5 remarkably suppressed DENV infection, whereas knockout of NLRC5 led to a significant increase in DENV infection. Mechanistic study revealed that NLRC5 interacted with the viral nonstructural protein 3 (NS3) protease domain and mediated degradation of NS3 through a ubiquitin-dependent selective macroautophagy/autophagy pathway. We demonstrated that NLRC5 recruited the E3 ubiquitin ligase CUL2 (cullin 2) to catalyze K48-linked poly-ubiquitination of the NS3 protease domain, which subsequently served as a recognition signal for cargo receptor TOLLIP-mediated selective autophagic degradation. Together, we have demonstrated that NLRC5 exerted an antiviral effect by mediating the degradation of a multifunctional protein of DENV, providing a novel antiviral signal axis of NLRC5-CUL2-NS3-TOLLIP. This study expands our understanding of the regulatory network of NLRC5 in the host defense against virus infection.

Development and Characterization of a Genetically Stable Infectious Clone for a Genotype I Isolate of Dengue Virus Serotype 1.

Dengue virus (DENV) is primarily transmitted by the bite of an infected mosquito of Aedes aegypti and Aedes albopictus, and symptoms caused may range from mild dengue fever to severe dengue hemorrhagic fever and dengue shock syndrome. Reverse genetic system represents a valuable tool for the study of DENV virology, infection, pathogenesis, etc. Here, we generated and characterized an eukaryotic-activated full-length infectious cDNA clone for a DENV serotype 1 (DENV-1) isolate, D19044, collected in 2019. Initially, nearly the full genome was determined by sequencing overlapping RT-PCR products, and was classified to be genotype I DENV-1. D19044 wild-type cDNA clone (D19044_WT) was assembled by four subgenomic fragments, in a specific order, into a low-copy vector downstream the CMV promoter. D19044_WT released the infectious virus at a low level (1.26 × 103 focus forming units per milliliter [FFU/mL]) following plasmid transfection of BHK-21 cells. Further adaptation by consecutive virus passages up to passage 37, and seven amino acid substitutions (7M) were identified from passage-recovered viruses. The addition of 7M (D19044_7M) greatly improved viral titer (7.5 × 104 FFU/mL) in transfected BHK-21 culture, and virus infections in 293T, Huh7.5.1, and C6/36 cells were also efficient. D19044_7M plasmid was genetically stable in transformant bacteria after five transformation-purification cycles, which did not change the capacity of producing infectious virus. Moreover, the D19044_7M virus was inhibited by mycophenolic acid in a dose-dependent manner. In conclusion, we have developed a DNA-launched full-length infectious clone for a genotype I isolate of DENV-1, with genetic stability in transformant bacteria, thus providing a useful tool for the study of DENV-1.

Identification of Ascomycin against Zika virus infection through screening of natural product library.

Zika virus (ZIKV) infection could lead to Guillain-Barré syndrome in adults and microcephaly in the newborns from infected pregnant women. To date, there is no specific drug for the treatment of ZIKV infection. In this study, we sought to screen inhibitors against ZIKV infection from a natural product library. A ZIKV replicon was used to screen a library containing 1680 natural compounds. We explored the antiviral mechanism of the compound candidate in vitro and in vivo infection models. Ascomycin, a macrolide from Streptomyces hygroscopicus, was identified with inhibitory effect against ZIKV in Vero cells (IC50 = 0.11 µM), hepatoma cell Huh7 (IC50 = 0.38 µM), and glioblastoma cell SNB-19 (IC50 = 0.06 µM), far below the cytotoxic concentrations. Mechanistic study revealed that Ascomycin suppressed ZIKV RNA replication step during the life cycle and the regulation of calcineurin-NFAT pathway maybe involved in this inhibitory effect, independent of innate immunity activation. Moreover, we found that Ascomycin also inhibited the infection of other Flaviviridae members, such as hepatitis C virus and dengue virus. Ascomycin reduced ZIKV load in blood by up to 3500-fold in A129 mice. Meanwhile, the infection in the mice brain was undetectable by immunohistochemistry staining. Together, these findings reveal a critical role of Ascomycin in the inhibition of ZIKV and related viruses, facilitating the development of novel antiviral agents.