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ORIGINAL RESEARCH article

Front. Microbiol.
Sec. Virology
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1533062

Dual EMCV-IRES-integrated dengue virus can express an exogenous gene; Cellular Mdm2 integration suppresses the dengue viral replication

Provisionally accepted
Tadahisa Teramoto Tadahisa Teramoto *
  • Georgetown University, Washington, United States

The final, formatted version of the article will be published soon.

    Flaviviruses transmit through a wide range of vertebrate and arthropod hosts, while the other genera in Flaviviridae replicate in a limited set of vertebrate hosts. Flaviviruses possess a 5' cap in their genome RNA for translation, while the other genera utilize their internal ribosome entry site (IRES) sequences instead of a 5' cap. In this study, the translational modification to add an IRES sequence was examined. An IRES sequence derived from encephalomyocarditis (EMCV) was inserted into dengue virus serotype 2 (DENV2); a non-structural (NS) polyprotein was translated by IRES separately from 5' cap-induced structural polyprotein translation. It was revealed that the IRES-integrated DENV2 is prevented from replicating in C6/36 mosquito cells, suggesting that the 5' cap is an advantageous mechanism for flavivirus translation in invertebrate species. I further created dual IRES-integrated DENV2, in which a non-viral gene can be expressed by the flanking IRESs. The insertion of eGFP fluorescently visualized the virus spread.The renilla luciferase (Rluc) integration enabled the viral replication quantification. It was also revealed that a cellular gene, Mdm2, which antagonizes Tumor suppressor protein p53 (TP53), could terminate the viral replication in BHK21 cells. Thus, the modifications of the DENV genome with IRES and the subsequent foreign gene could be utilized for controlling viral replications.I artificially created the translation-modified DENV2, in which viral NS genes as well as additional non-viral genes were translated by EMCV-IRES independently from the 5' cap in the viral genome.The replication of eGFP or Rluc gene-integrated DENV2 could be traced with these markers in BHK21 and HEK293 mammalian cells while both viruses were suppressed in C6/36 mosquito cells. Moreover, the integrated cellular Mdm2 gene suppressed the viral replication in BHK21 cells, suggesting the potential creation of a replication-controllable virus by IRES insertion and a cellular gene integration with the viral genome.

    Keywords: DENV: Dengue Virus, NS: non-structural, IRES: internal ribosome entry site, TP53: tumor suppressor protein 53, Mdm2: mouse double minute 2, RT-qPCR: reverse transcriptase quantitative PCR, eGFP: enhanced green fluorescent protein, Rluc: renilla luciferase IRES-X-IRES-E73-DENV2 gene expression system Dengue virus

    Received: 23 Nov 2024; Accepted: 06 Jan 2025.

    Copyright: © 2025 Teramoto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Tadahisa Teramoto, Georgetown University, Washington, United States

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