When designing live-attenuated respiratory syncytial virus (RSV) vaccine candidates, attenuating mutations can be developed through biologic selection or reverse-genetic manipulation and may include point mutations, codon and gene deletions, and genome rearrangements. Attenuation typically involves the reduction in virus replication, due to direct effects on viral structural and replicative machinery or viral factors that antagonize host defense or cause disease. However, attenuation must balance reduced replication and immunogenic antigen expression. In the present study, we explored a new approach in order to discover attenuating mutations. Specifically, we used protein structure modeling and computational methods to identify amino acid substitutions in the RSV nonstructural protein 1 (NS1) predicted to cause various levels of structural perturbation. Twelve different mutations predicted to alter the NS1 protein structure were introduced into infectious virus and analyzed in cell culture for effects on viral mRNA and protein expression, interferon and cytokine expression, and caspase activation. We found the use of structure-based machine learning to predict amino acid substitutions that reduce the thermodynamic stability of NS1 resulted in various levels of loss of NS1 function, exemplified by effects including reduced multi-cycle viral replication in cells competent for type I interferon, reduced expression of viral mRNAs and proteins, and increased interferon and apoptosis responses.

The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets. Although SADS-CoV uses different strategies to evade the host\'s innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified. In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected. The results determined that nsp1 was a potent antagonist of IFN response. SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation. Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin-proteasome pathway. Furthermore, SADS-CoV nsp1 induced CREB-binding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling. In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK-STAT signaling pathway via the ubiquitin-proteasome pathway. This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK-STAT signaling pathway and the strategies of SADS-CoV in evading the host\'s innate immune system.

OBJECTIVE: Rotavirus is an enteric RNA virus that causes severe dehydrating gastroenteritis in infants and young children through infection of enterocytes in the small intestine. Timely clearance of the virus demands a robust innate immune response by cells associated with the small intestine, including the expression of interferon (IFN). Previous studies have shown that some rotavirus strains suppress the production of interferon, by inducing the degradation of mitochondrial antiviral signaling (MAVS) protein and interferon regulatory factor-3 (IRF3). In this study, we have used reverse genetics to generate recombinant rotaviruses expressing compromised forms of VP3 or NSP1, or both, to explore the function of these viral proteins in the degradation of MAVS and IRF3. Our results demonstrate that VP3 is responsible for MAVS depletion in rotavirus-infected cells, and through this activity, helps to suppress IFN production. Thus, VP3 functions to support the activity of rotavirus NSP1, the major interferon antagonist of the virus.

Flaviviruses include medically important mosquito-borne pathogens, such as Zika virus (ZIKV), Japanese encephalitis virus (JEV), dengue virus (DENV) and West Nile virus (WNV), that cause hundreds of millions of infections each year. Currently, there are no approved effect therapies against mosquito-borne flaviviruses. The flaviviruses encoded nonstructural protein 1 (NS1) is a secreted glycoprotein widely involved in viral replication, immune evasion, and directly causing tissue-specific damage during flaviviruses infection. Upon viral infection of host cell, NS1 can be found in multiple oligomeric forms and include a dimer on the cell surface, and a soluble secreted hexameric lipoparticle. In the recent decade, the detailed crystal structure of several flaviviruses NS1 have been determined and unraveled its broader and deeper functions. Consistent with the potential immune function revealed by its structure, NS1 is involved in the escaping of host signal immune pathway mediated by pattern recognition receptors (PRRs), including RIG-I-like receptors (RLRS) and Toll-like receptors (TLRs). Moreover, the flavivirus NS1 is efficiently secreted by infected cells and circulates in the blood of the host to directly induce specific tissues damage. The NS1 of ZIKV, JEV and WNV changes the permeability of brain microvascular endothelial cell to cause endothelial cell dysfunction and promote virus pathogenesis. DENV NS1 can induce systemic tissues damage in humans through multiple strategies. Mutations of several key amino acids in NS1 can reduce the neurovirulence of the flavivirus. In this article, we provide an overview of the latest research on this fascinating protein in these disparate areas.

The genus Flavivirus contains a wide variety of viruses that cause severe disease in humans, including dengue virus, yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus. Nonstructural protein 1 (NS1) is a glycoprotein that encodes a 352-amino-acid polypeptide and has a molecular weight of 46-55 kDa depending on its glycosylation status. NS1 is highly conserved among multiple flaviviruses and occurs in distinct forms, including a dimeric form within the endoplasmic reticulum, a cell-associated form on the plasma membrane, or a secreted hexameric form (sNS1) trafficked to the extracellular matrix. Intracellular dimeric NS1 interacts with other NSs to participate in viral replication and virion maturation, while extracellular sNS1 plays a critical role in immune evasion, flavivirus pathogenesis and interactions with natural vectors. In this review, we provide an overview of recent research progress on flavivirus NS1, including research on the structural details, the secretory pathways in mammalian and mosquito cells and the multiple functions in viral replication, immune evasion, pathogenesis and interaction with natural hosts, drawing together the previous data to determine the properties of this protein.

Since 2010, the duck Tembusu virus (DTMUV) has caused a severe outbreak of egg drop syndrome in laying ducks in China, which has resulted in substantial financial losses in the poultry industry. DTMUV nonstructural protein 1 (NS1), as the only secreted protein, could aid in the development of therapeutic antibodies and diagnostic techniques; however, there are few studies on the preparation and epitope identification of monoclonal antibodies (mAbs) against DTMUV NS1. In this study, by indirect enzyme-linked immunosorbent assay (ELISA), Western blotting, and indirect immunofluorescence assay, we screened 6 mAbs (8A4, 8E6, 10F12, 1H11, 3D5, 5C11) that could specifically recognize DTMUV NS1. For epitope mapping of mAbs, a series of GST-tagged truncated fusion proteins of DTMUV NS1 were constructed by prokaryotic expression. Finally, the 4 shortest linear epitopes were identified by indirect ELISA and Western blotting. The epitope 133FVIDGPK139 was recognized by 8A4, the epitope 243IPKTLGGP250 was recognized by 8E6, the epitope 267PWDEK271 was recognized by 10F12, and 156EDFGFGVL163 was recognized by 1H11, 3D5, and 5C11. By sequence alignment and cross-reaction tests, we found that 8A4 and 8E6 had high specificity for DTMUV NS1 compared with that of other mAbs, but 10F12, 1H11, 3D5, and 5C11 exhibited a clear degree of cross-reaction with dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) NS1. Finally, the predicted crystal structure analysis showed the approximate spatial positions of the 4 epitopes on the NS1 dimer. In summary, our study revealed 2 specific mAbs for DTMUV NS1 recognition and 4 multiflavivirus mAbs for DENV, JEV, WNV, and ZIKV NS1 recognition.

UNASSIGNED: Intestinal leakage commonly occurs in severe dengue infection with zonulin as a biomarker. The aim of this study was to determine the effects of NS1 on liver weight, zonulin expression and serum zonulin levels.
UNASSIGNED: This laboratory experiment used 18 ddY mice, which were randomly divided into control (C), PBS (T1), and PBS + NS1 (T2) groups. Mice in the T1 and T2 groups were intravenously injected with 500 μl PBS only and 50 μg NS1 respectively. Mice blood samples were collected before and after three-day treatment for measurement of zonulin level. The fresh liver was weighted directly and were then used for immunostaining.
UNASSIGNED: The C group had lower wet liver weight compared to the T groups (p=0.001). Increased expression of liver zonulin was found in the T2 group, significant different from the C (p=0.014) and T1 groups (p=0.020). After treatment, serum zonulin levels in the T1 group was higher than that of the T1 group before treatment (p=0.035) but not in control (p=0.753) and T2 groups (p=0.869).
UNASSIGNED: Administration of 50 μg NS 1 increases wet liver weight and zonulin expression in hepatocytes, but did not increase serum zonulin levels in ddY mice.

The effect of preformed protein coronas on immunoassays for Dengue nonstructural protein 1 (NS1) immunoassays was investigated. The composition of the protein corona that forms around nanoparticle-antibody conjugates in human serum was characterized, and selected proteins from the corona were used for preformed coronas (human serum albumin and apolipoprotein A1). Coronas were formed and characterized by dynamic light scattering (DLS), and the nanoparticle-conjugate was probed by optical absorption spectroscopy. Immunoassays were run, and performance was quantified by analyzing the strip intensity as a function of NS1 concentration. The preformed coronas influenced the limit of detection (LOD) of the assay and the affinity for the NS1 target (KD). The resulting KD and LODs for the NP-Ab-ApoA1 immunoprobes were 0.83 nM and 1.24 nM, respectively. For the NP-Ab -HSA coronas, the test line intensity was lower by 33% at a given NS1 concentration than for the NP-Ab immunoprobes, and KD was 0.14 nM, a slightly higher affinity. Due to the relatively large error of the negative control, a meaningful LOD for the NP-Ab with HSA coronas could not be determined.

Despite substantial morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika, and the currently available dengue vaccine is only recommended for dengue virus (DENV)-immune individuals. Thus, development of therapeutic and/or preventive drugs is urgently needed. DENV and Zika virus (ZIKV) nonstructural protein 1 (NS1) can directly trigger endothelial barrier dysfunction and induce inflammatory responses, contributing to vascular leak in vivo. Here we evaluated the efficacy of the (1-6,1-3)-β-D-glucan isolated from Agaricus subrufescens fruiting bodies (FR) and its sulfated derivative (FR-S) against DENV-2 and ZIKV infection and NS1-mediated pathogenesis. FR-S, but not FR, significantly inhibited DENV-2 and ZIKV replication in human monocytic cells (EC50 = 36.5 and 188.7 μg/mL, respectively) when added simultaneously with viral infection. No inhibitory effect was observed when FR or FR-S were added post-infection, suggesting inhibition of viral entry as a mechanism of action. In an in vitro model of endothelial permeability using human pulmonary microvascular endothelial cells (HPMECs), FR and FR-S (0.12 μg/mL) inhibited DENV-2 NS1- and ZIKV NS1-induced hyperpermeability by 50% and 100%, respectively, as measured by Trans-Endothelial Electrical Resistance. Treatment with 0.25 μg/mL of FR and FR-S inhibited DENV-2 NS1 binding to HPMECs. Further, FR-S significantly reduced intradermal hyperpermeability induced by DENV-2 NS1 in C57BL/6 mice and protected against DENV-induced morbidity and mortality in a murine model of dengue vascular leak syndrome. Thus, we demonstrate efficacy of FR-S against DENV and ZIKV infection and NS1-induced endothelial permeability in vitro and in vivo. These findings encourage further exploration of FR-S and other glycan candidates for flavivirus treatment alone or in combination with compounds with different mechanisms of action.

Molecular mechanisms of the non-structural protein 1 (NS1) in influenza A-induced pathological changes remain ambiguous. This study explored the pathogenesis of human infection by influenza A viruses (IAVs) through identifying human genes with codon usage bias (CUB) similar to NS1 gene of these viruses based on the relative synonymous codon usage (RSCU). CUB of the IAV subtypes H1N1, H3N2, H3N8, H5N1, H5N2, H5N8, H7N9 and H9N2 was analyzed and the correlation of RSCU values of NS1 sequences with those of the human genes was calculated. The CUB of NS1 was uneven and codons ending with A/U were preferred. The ENC-GC3 and neutrality plots suggested natural selection as the main determinant for CUB. The RCDI, CAI and SiD values showed that the viruses had a high degree of adaptability to human. A total of 2155 human genes showed significant RSCU-based correlation (p < 0.05 and r > 0.5) with NS1 coding sequences and was considered as human genes with CUB similar to NS1 gene of IAV subtypes. Differences and similarities in the subtype-specific human protein-protein interaction (PPI) networks and their functions were recorded among IAVs subtypes, indicating that NS1 of each IAV subtype has a specific pathogenic mechanism. Processes and pathways involved in influenza, transcription, immune response and cell cycle were enriched in human gene sets retrieved based on the CUB of NS1 gene of IAV subtypes. The present work may advance our understanding on the mechanism of NS1 in human infections of IAV subtypes and shed light on the therapeutic options.