Significance: Infection with Zika virus can cause severe consequences in a subset of the patient population. When infection occurs during pregnancy, congenital abnormalities can occur. Additionally, the onset of Guillain–Barré syndrome, encephalitis, and myelitis are associated with Zika virus infection. We have identified several small molecules with favorable clinical profiles, including the five-lipoxygenase–activating protein inhibitor, MK-591, and the tetracycline antibiotic, methacycline, as inhibitors of Zika virus infection, the latter of which reduced neurological deficits in a Zika virus mouse model. These compounds have the potential to be used as prophylactics or for the treatment of the neurological complications of Zika virus infection.

Neurological disorders have been reported in a large number of coronavirus disease 2019 (COVID-19) patients, suggesting that this disease may have long-term adverse neurological consequences. COVID-19 occurs from infection by a positive-sense single-stranded RNA virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The membrane fusion protein of SARS-CoV-2, the spike protein, binds to its human host receptor, angiotensin-converting enzyme 2 (ACE2), to initiate membrane fusion between the virus and host cell. The spike protein of SARS-CoV-2 contains the furin protease recognition site and its cleavage enhances the infectivity of this virus. The binding of SARS-CoV-2 to the ACE2 receptor has been shown to downregulate ACE2, thereby increasing the levels of pathogenic angiotensin II (Ang II). The furin protease cleaves between the S1 subunit of the spike protein with the binding domain toward ACE2 and the S2 subunit with the transmembrane domain that anchors to the viral membrane, and this activity releases the S1 subunit into the blood circulation. The released S1 subunit of the spike protein also binds to and downregulates ACE2, in turn increasing the level of Ang II. Considering that a viral particle contains many spike protein molecules, furin-dependent cleavage would release many free S1 protein molecules, each of which can downregulate ACE2, while infection with a viral particle only affects one ACE2 molecule. Therefore, the furin-dependent release of S1 protein would dramatically amplify the ability to downregulate ACE2 and produce Ang II. We hypothesize that this amplification mechanism that the virus possesses, but not the infection per se, is the major driving force behind COVID-19-associated neurological disorders.

Mosquito-borne dengue viruses (DENVs) have evolved to four serotypes with 69%–78% amino acid identities, resulting in incomplete immunity, where one serotype’s infection does not cross-protect against secondary infections by other serotypes. Despite the amino acid differences, structural and nonstructural (NS) proteins among serotypes play similar functions. NS3 is an enzyme complex: NS3 has N-terminal protease (PRO) and C-terminal helicase (HEL) activities in addition to 5’ RNA triphosphatase (5’RTP), which is involved in the RNA capping process. In this study, the effects of NS3 replacements among serotypes were tested. The replacement of NS3 full-length (FULL), PRO or HEL region suppressed viral replication in BHK-21 mammalian cells, while the single compensatory mutation improved the viral replications; P364S mutation in HEL revived PRO (DENV3)-replaced DENV1, while S68T alteration in NS2B recovered HEL (DENV1)-replaced DENV2. The results suggest that the interactions between PRO and HEL as well as HEL and NS2B are required for replication competence. Lower-frequency mutations also appeared at various locations in viral proteins, although after infecting C6/36 mosquito cells, the mutations’ frequencies changed, and/or new mutations appeared. In contrast, the inter-domain region (INT, 12 amino acids)-replaced chimera quickly replicated without mutation in BHK-21 cells, although extended cell culture accumulated various mutations. These results suggest that NS3 variously interacts with DENV proteins, in which the chimeric NS3 domain replacements induced amino acid mutations, irrespective of replication efficiency. However, the viral sequences are further adjusted for replication efficiency, to fit in both mammalian cells and mosquito cells. IMPORTANCE: Enzyme activities for replicating DENV 5’ cap positive (+) sense RNA have been shown to reside in NS3 and NS5. However, it remains unknown how these enzymes coordinately synthesize negative (-) sense RNA, from which abundant 5’ cap (+) sense RNA is produced. We previously revealed that NS5 dimerization and NS5 methyltransferase(MT)–NS3HEL interaction are important for DENV replication. Here, we found that replication incompetence due to NS3PRO or HEL replacement was compensated by a mutation at HEL or NS2B, respectively, suggesting that the interactions among NS2B, NS3PRO, and HEL are critical for DENV replication.

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.

Neurological disorders have been reported in a large number of coronavirus disease 2019 (COVID-19) patients, suggesting that this disease may have long-term adverse neurological consequences. COVID-19 occurs from the infection by a positive-sense single-stranded RNA virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The membrane fusion protein of SARS-CoV-2, the spike protein, binds to its human host receptor, angiotensin-converting enzyme 2 (ACE2), to initiate membrane fusion between the virus and host cell. The spike protein of SARS-CoV-2 contains the furin protease recognition site and its cleavage enhances the infectivity of this virus. The binding of SARS-CoV-2 to the ACE2 receptor has been shown to downregulate ACE2, thereby increasing the levels of pathogenic angiotensin II (Ang II). The furin protease cleaves between the S1 subunit of the spike protein with the binding domain toward ACE2 and the S2 subunit with the transmembrane domain that anchors to the viral membrane, and this activity releases the S1 subunit into the blood circulation. The released S1 subunit of the spike protein also binds to and downregulates ACE2, in turn, increasing the level of Ang II. Considering that a viral particle contains many spike protein molecules, furin-dependent cleavage would release many free S1 protein molecules each of which can downregulate ACE2, while the infection with a viral particle only affects one ACE2 molecule. Therefore, the furin-dependent release of S1 protein would dramatically amplify the ability to downregulate ACE2 and produce Ang II. We hypothesize that this amplification mechanism that the virus possesses, but not the infection per se, is the major driving force behind COVID-19 associated neurological disorders.