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Current seasonal influenza vaccines have suboptimal effectiveness, especially in seasons dominated by viruses that do not match the vaccine. Therefore, finding new approaches to improve the immunogenicity and efficacy of traditional influenza vaccines is of high priority for public health. Licensed live attenuated influenza vaccine (LAIV) is a promising platform for designing broadly protective vaccines due to its ability to induce cross-reactive T-cell immunity. In this study, we tested the hypothesis that truncation of the nonstructural protein 1 (NS1) and the replacement of the nucleoprotein (NP) of the A/Leningrad/17 master donor virus with a recent NP, i.e., switching to 5:3 genome composition, could improve the cross-protective potential of the LAIV virus. We generated a panel of LAIV candidates differing from the classical vaccine by the source of NP gene and/or by the length of NS1 protein. We showed that NS1-modified LAIV viruses had reduced viral replication in the respiratory tract of mice, indicating a more attenuated phenotype compared to the LAIVs with full-length NS1. Most importantly, the LAIV candidate with both NP and NS genes modified induced a robust systemic and lung-localized memory CD8 T-cell response targeting more recent viruses, and better protected immunized mice against lethal challenge with a heterosubtypic influenza virus than the control LAIV variant. Overall, these data indicate that the 5:3 LAIVs with truncated NS1 may be beneficial for protection against heterologous influenza viruses and warrant further preclinical and clinical development.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged as a global health threat. The rise in ZIKV infections has driven an increased incidence of neonates born with microcephaly or other neurological malformations. Therefore, screening for ZIKV infection can considerably impact pregnant women, especially during the first trimester. The majority of ZIKV infections are mild or asymptomatic, and clinical diagnosis is inaccurate. Moreover, given the high level of cross-reactivity among flaviviruses, serological approaches to distinguish ZIKV from dengue virus (DENV) infections are complicated. We used the combination of DENV and ZIKV nonstructural protein 1 (NS1) IgG enzyme-linked immunosorbent assay (ELISA) and ZIKV NS1 blockade-of-binding (BOB) ELISA to test the convalescent sera of non-flavivirus, primary DENV, secondary DENV, and ZIKV infections. Our findings indicate that primary testing using a ZIKV NS1 IgG ELISA, the test of choice for large-scale ZIKV serosurvey studies, provided relatively high sensitivity. Moreover, the confirmation of positive ELISA results using the ZIKV NS1 BOB ELISA increased average specificity to 94.59% across serum samples. The combined use of two simple ELISAs for ZIKV serosurveys and the monitoring of ZIKV infection during pregnancy can elucidate the epidemiology, pathogenesis, and complications of ZIKV in DENV-endemic areas.

Non-structural protein 1 (Nsp1) is a virulence factor found in all beta coronaviruses (b-CoVs). Recent studies have shown that Nsp1 of SARS-CoV-2 virus interacts with the nuclear export receptor complex, which includes nuclear RNA export factor 1 (NXF1) and nuclear transport factor 2-like export factor 1 (NXT1). The NXF1–NXT1 complex plays a crucial role in the transport of host messenger RNA (mRNA). Nsp1 interferes with the proper binding of NXF1 to mRNA export adaptors and its docking to the nuclear pore complex. We propose that drugs targeting the binding surface between Nsp1 and NXF1–NXT1 may be a useful strategy to restore host antiviral gene expression. Exploring this strategy forms the main goals of this paper. Crystal structures of Nsp1 and the heterodimer of NXF1–NXT1 have been determined. We modeled the docking of Nsp1 to the NXF1–NXT1 complex, and discovered repurposed drugs that may interfere with this binding. To our knowledge, this is the first attempt at drug-repurposing of this complex. We used structural analysis to screen 1993 FDA-approved drugs for docking to the NXF1–NXT1 complex. The top hit was ganirelix, with a docking score of −14.49. Ganirelix competitively antagonizes the gonadotropin releasing hormone receptor (GNRHR) on pituitary gonadotrophs, and induces rapid, reversible suppression of gonadotropin secretion. The conformations of Nsp1 and GNRHR make it unlikely that they interact with each other. Additional drug leads were inferred from the structural analysis of this complex, which are discussed in the paper. These drugs offer several options for therapeutically blocking Nsp1 binding to NFX1–NXT1, which may normalize nuclear export in COVID-19 infection.

The 1918 influenza A virus (IAV) caused the worst flu pandemic in human history. Non-structural protein 1 (NS1) is an important virulence factor of the 1918 IAV and antagonizes host antiviral immune responses. NS1 increases virulence by activating phosphoinositide 3-kinase (PI3K) via binding to the p85β subunit of PI3K. Intriguingly, unlike the NS1 of other human IAV strains, 1918 NS1 hijacks another host protein, CRK, to form a ternary complex with p85β, resulting in hyperactivation of PI3K. However, the molecular basis of the ternary interaction between 1918 NS1, CRK, and PI3K remains elusive. Here, we report the structural and thermodynamic bases of the ternary interaction. We find that the C-terminal tail (CTT) of 1918 NS1 remains highly flexible in the complex with p85β. Thus, the CTT of 1918 NS1 in the complex with PI3K can efficiently hijack CRK. Notably, our study indicates that 1918 NS1 enhances its affinity to p85β in the presence of CRK, which might result in enhanced activation of PI3K. Our results provide structural insight into how 1918 NS1 hijacks two host proteins simultaneously.

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses of public health relevance. Both viruses circulate in the same endemic settings and acute infections generally manifest similar symptoms. This highlights the importance of accurate diagnosis for clinical management and outbreak control. One of the commonly used acute diagnostic markers for flaviviruses is nonstructural protein 1 (NS1). However, false positives due to antigenic cross-reactivity have been reported between DENV and ZIKV infections when using DENV NS1 antigen (NS1 Ag) detection assays in acute cases. Therefore, we investigated the lowest detectable virus titres and cross-reactivity of three commercial dengue NS1 Ag rapid assays and two ELISAs for different flaviviruses. Our results showed that substantially high viral titres of ZIKV, Kunjin virus (KUNV) and yellow fever virus (YFV) are required to give false-positive results when using DENV NS1 rapid detection assays. Commercial DENV NS1 ELISAs did not react with ZIKV and YFV. In comparison, tested assays detected DENV at a significantly low virus titre. Given the relatively low viral loads reported in clinical samples, our findings suggest that commercially available dengue NS1 Ag detection assays are less likely to generate false-positive results among clinical samples in areas where multiple flaviviruses cocirculate.

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling.

Current seasonal influenza vaccines have suboptimal effectiveness, especially in seasons dominated by viruses that do not match the vaccine. Therefore, finding new approaches to improve the immunogenicity and efficacy of traditional influenza vaccines is of high priority for public health. Licensed live attenuated influenza vaccine (LAIV) is a promising platform for designing broadly protective vaccines due to its ability to induce cross-reactive T-cell immunity. In this study, we tested the hypothesis that truncation of the nonstructural protein 1 (NS1) and the replacement of the nucleoprotein (NP) of the A/Leningrad/17 master donor virus with a recent NP, i.e., switching to 5:3 genome composition, could improve the cross-protective potential of the LAIV virus. We generated a panel of LAIV candidates differing from the classical vaccine by the source of NP gene and/or by the length of NS1 protein. We showed that NS1-modified LAIV viruses had reduced viral replication in the respiratory tract of mice, indicating a more attenuated phenotype compared to the LAIVs with full-length NS1. Most importantly, the LAIV candidate with both NP and NS genes modified induced a robust systemic and lung-localized memory CD8 T-cell response targeting more recent viruses, and better protected immunized mice against lethal challenge with a heterosubtypic influenza virus than the control LAIV variant. Overall, these data indicate that the 5:3 LAIVs with truncated NS1 may be beneficial for protection against heterologous influenza viruses and warrant further preclinical and clinical development.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged as a global health threat. The rise in ZIKV infections has driven an increased incidence of neonates born with microcephaly or other neurological malformations. Therefore, screening for ZIKV infection can considerably impact pregnant women, especially during the first trimester. The majority of ZIKV infections are mild or asymptomatic, and clinical diagnosis is inaccurate. Moreover, given the high level of cross-reactivity among flaviviruses, serological approaches to distinguish ZIKV from dengue virus (DENV) infections are complicated. We used the combination of DENV and ZIKV nonstructural protein 1 (NS1) IgG enzyme-linked immunosorbent assay (ELISA) and ZIKV NS1 blockade-of-binding (BOB) ELISA to test the convalescent sera of non-flavivirus, primary DENV, secondary DENV, and ZIKV infections. Our findings indicate that primary testing using a ZIKV NS1 IgG ELISA, the test of choice for large-scale ZIKV serosurvey studies, provided relatively high sensitivity. Moreover, the confirmation of positive ELISA results using the ZIKV NS1 BOB ELISA increased average specificity to 94.59% across serum samples. The combined use of two simple ELISAs for ZIKV serosurveys and the monitoring of ZIKV infection during pregnancy can elucidate the epidemiology, pathogenesis, and complications of ZIKV in DENV-endemic areas.

Non-structural protein 1 (Nsp1) is a virulence factor found in all beta coronaviruses (b-CoVs). Recent studies have shown that Nsp1 of SARS-CoV-2 virus interacts with the nuclear export receptor complex, which includes nuclear RNA export factor 1 (NXF1) and nuclear transport factor 2-like export factor 1 (NXT1). The NXF1-NXT1 complex plays a crucial role in the transport of host messenger RNA (mRNA). Nsp1 interferes with the proper binding of NXF1 to mRNA export adaptors and its docking to the nuclear pore complex. We propose that drugs targeting the binding surface between Nsp1 and NXF1-NXT1 may be a useful strategy to restore host antiviral gene expression. Exploring this strategy forms the main goals of this paper. Crystal structures of Nsp1 and the heterodimer of NXF1-NXT1 have been determined. We modeled the docking of Nsp1 to the NXF1-NXT1 complex, and discovered repurposed drugs that may interfere with this binding. To our knowledge, this is the first attempt at drug-repurposing of this complex. We used structural analysis to screen 1993 FDA-approved drugs for docking to the NXF1-NXT1 complex. The top hit was ganirelix, with a docking score of -14.49. Ganirelix competitively antagonizes the gonadotropin releasing hormone receptor (GNRHR) on pituitary gonadotrophs, and induces rapid, reversible suppression of gonadotropin secretion. The conformations of Nsp1 and GNRHR make it unlikely that they interact with each other. Additional drug leads were inferred from the structural analysis of this complex, which are discussed in the paper. These drugs offer several options for therapeutically blocking Nsp1 binding to NFX1-NXT1, which may normalize nuclear export in COVID-19 infection.

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses of public health relevance. Both viruses circulate in the same endemic settings and acute infections generally manifest similar symptoms. This highlights the importance of accurate diagnosis for clinical management and outbreak control. One of the commonly used acute diagnostic markers for flaviviruses is nonstructural protein 1 (NS1). However, false positives due to antigenic cross-reactivity have been reported between DENV and ZIKV infections when using DENV NS1 antigen (NS1 Ag) detection assays in acute cases. Therefore, we investigated the lowest detectable virus titres and cross-reactivity of three commercial dengue NS1 Ag rapid assays and two ELISAs for different flaviviruses. Our results showed that substantially high viral titres of ZIKV, Kunjin virus (KUNV) and yellow fever virus (YFV) are required to give false-positive results when using DENV NS1 rapid detection assays. Commercial DENV NS1 ELISAs did not react with ZIKV and YFV. In comparison, tested assays detected DENV at a significantly low virus titre. Given the relatively low viral loads reported in clinical samples, our findings suggest that commercially available dengue NS1 Ag detection assays are less likely to generate false-positive results among clinical samples in areas where multiple flaviviruses cocirculate., Competing Interests: The authors declare no conflict of interest.