Lactic acid bacterial surface display of scytovirin inhibitors for anti-ebolavirus infection.

Scytovirin (SVN) is a lectin from cyanobacteria which has a strong inhibitory activity against Ebola virus infection. We engineered scytovirin as the inhibitor for surface display of lactic acid bacteria to block Ebola virus infection. Two different bacterial strains (Lactobacillus casei and Lactococcus lactis) were successfully engineered for scytovirin expression on the bacterial surface. These bacteria were found to be effective at neutralizing pseudotyped Ebolavirus in a cell-based assay. This approach can be utilized for prophylactic prevention, as well as for treatment. Since lactic acid bacteria can colonize the human body, a long-term efficacy could be achieved. Furthermore, this approach is also simple and cost-effective and can be easily applied in the regions of Ebola outbreaks in the developing countries.

Determining Homogenization Parameters and Predicting 5182-Sc-Zr Alloy Properties by Artificial Neural Networks.

Artificial neural networks (ANNs) were established for the homogenization and recrystallization heat treatment processes of 5182-Sc-Zr alloy. Microhardness and conductivity testing were utilized to determine the precipitation state of Al3(ScxZr1-x) dispersoids during the homogenization treatment, while electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) were used to observe the microstructure evolution of the alloy. Tensile experiments were performed to test the mechanical properties of the alloy after recrystallization annealing. The two-stage homogenization parameters were determined by studying the changes in microhardness and electrical conductivity of 5182-Sc-Zr alloy after homogenization with the assistance of artificial neural networks: the first-stage homogenization at 275 °C for 20 h and the second-stage homogenization at 440 °C for 12 h. The dispersoids had entirely precipitated after homogenization, and the alloy segregation had improved. A high-accuracy prediction model, incorporating multiple influencing factors through artificial neural networks, was successfully established to predict the mechanical properties of the 5182-Sc-Zr alloy after annealing. Based on the atomic plane spacing in HRTEM, it was determined that the Al3(ScxZr1-x) dispersoids and the Al matrix maintained a good coherence relationship after annealing at 400 °C.

Displaying and delivering viral membrane antigens via WW domain-activated extracellular vesicles.

Membrane proteins expressed on the surface of enveloped viruses are conformational antigens readily recognized by B cells of the immune system. An effective vaccine would require the synthesis and delivery of these native conformational antigens in lipid membranes that preserve specific epitope structures. We have created an extracellular vesicle-based technology that allows viral membrane antigens to be selectively recruited onto the surface of WW domain-activated extracellular vesicles (WAEVs). Budding of WAEVs requires secretory carrier-associated membrane protein 3, which through its proline-proline-alanine-tyrosine motif interacts with WW domains to recruit fused viral membrane antigens onto WAEVs. Immunization with influenza and HIV viral membrane proteins displayed on WAEVs elicits production of virus-specific neutralizing antibodies and, in the case of influenza antigens, protects mice from the lethal viral infection. WAEVs thus represent a versatile platform for presenting and delivering membrane antigens as vaccines against influenza, HIV, and potentially many other viral pathogens.

Ligand-based design of peptide entry inhibitors targeting the endosomal receptor binding site of filoviruses.

Filoviruses enter cells through macropinocytosis and trafficking into the endosomes in which they bind to the receptor Niemann-Pick C1 protein (NPC1) for membrane fusion and entry into the cytoplasm. The endosomal receptor-binding is critical step for filovirus entry. Designing inhibitors to block receptor binding will prevent viral entry. Using available binding structural information from the co-crystal structures of the viral GP with the receptor NPC1 or with monoclonal antibodies, we have conducted structure-based design of peptide inhibitors to target the receptor binding site (RBS). The designed peptides were tested for their inhibition activity against pseudo-typed or replication-competent viruses in a cell-based assay. The results indicate that these peptides exhibited strong activities against both Ebola and Marburg virus infection. It is expected that these peptides can be further developed for therapeutic use to treat filovirus infection and combat the outbreaks.

Texture Evolution and Nanohardness in Cu-Nb Composite Wires.

Multifilamentary microcomposite copper-niobium (Cu-Nb) wires were fabricated by a series of accumulative drawing and bonding steps (ADB). The texture of the Cu matrix in these wires was studied using electron backscattered diffraction (EBSD) and transmission electron microscopy (TEM). Dynamic recrystallization during cold drawing caused a weakening of the <111> texture in the micron-scale Cu matrix at high values of true strain. A sharp <111> texture was observed in the nano-scale Cu matrix due to the suppression of dynamic recrystallization. The grain size was reduced by the higher level of dynamic recrystallization at high strains. The relation between the nanoindentation behavior of the different Cu matrix and the grain sizes, Cu-Nb interface, and texture was established.

Transcriptomic and photosynthetic responses to grafting of the Nod1 gene in nodulated and non-nodulated soybeans.

Legume plants form symbiotic relationships with rhizobia to convert N2 into ammonia, and the nodulation status can affect plant development including photosynthesis. However, the relationship between nitrogen fixation and photosynthesis during carbon and nitrogen metabolism remains unclear. This study was undertaken to unravel regulation of nodulation and photosynthesis using a spontaneous nonnodulated soybean mutant by grafting. The results of inheritance and gene mapping showed that the nonnodulated mutant was controlled by a recessive gene overlapped with the reported rj1 locus, and might be a new rj1 allele with 1 bp deletion in the fourth exon in comparison to the sequence of normal nodulation plants. According to grafting results, soybean nodulation is obviously determined by the roots, not the seedlings. Moreover, nitrogen content along with related metabolic enzyme activity, and photosynthetic capacity were enhanced by nonnodulated scions grafted with nodulated roots. Contrary results were obtained for nodulated scions grafted with nonnodulated roots. A total of 853 differentially expressed genes (DEGs) in the leaves and 1874 in the roots were identified by transcriptome analyses of the grafting treatments. We identified 285 differential gene ontology (GO) terms and 57 differential pathway terms identified in the leaves, while 856 differential GO terms and 207 differential pathway terms in the roots. Twenty DEGs interacting at translation level were selected, and the results of transcriptome analyses were verified by q-PCR. These findings indicated that the nodulation-related Nod allelic gene increases the nitrogen content of nonnodulated plants, which affects the enzymes involved in nitrogen metabolism, leading to changes in hormone levels and further regulation of photosynthesis and carbon metabolism.

Effects of Sc and Zr Addition on Microstructure and Mechanical Properties of AA5182.

The effects of 0.1 wt.% Sc and 0.1 wt.% Zr addition in AA5182 on microstructure and mechanical properties were investigated. Results show that Al3(ScxZr1-x) dispersoids formed in AA5182. Observation of ingots microstructures showed that the grain size of 5182-Sc-Zr alloy was 56% lower than that of based AA5182. Isothermal annealing between 230 °C and 500 °C for 2 h was performed to study the recrystallization, tensile properties and dispersoid coarsening. The recrystallization was inhibited by the dispersoids, and the alloy microstructure remained deformed after annealing. Al3(ScxZr1-x) in AA5182 was stable when annealing below 400 °C, while parts of dispersoids coarsened significantly when heating at 500 °C. The addition of Sc and Zr allowed YS of 5182 alloy to achieve 247.8 MPa, which is 100 MPa higher than the corresponding AA5182. The contributions of Orowan strengthening and grain boundary strengthening were obtained by calculation.

Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis.

Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

Identification of filovirus entry inhibitors targeting the endosomal receptor NPC1 binding site.

Filoviruses, mainly consisting of Ebola viruses (EBOV) and Marburg viruses (MARV), are enveloped negative-strand RNA viruses which can infect humans to cause severe hemorrhagic fevers and outbreaks with high mortality rates. The filovirus infection is mediated by the interaction of viral envelope glycoprotein (GP) and the human endosomal receptor Niemann-Pick C1 (NPC1). Blocking this interaction will prevent the infection. Therefore, we utilized an In silico screening approach to conduct virtual compound screening against the NPC1 receptor-binding site (RBS). Twenty-six top-hit compounds were purchased and evaluated by in vitro cell based inhibition assays against pseudotyped or replication-competent filoviruses. Two classes (A and U) of compounds were identified to have potent inhibitory activity against both Ebola and Marburg viruses. The IC50 values are in the lower level of micromolar concentrations. One compound (compd-A) was found to have a sub-micromolar IC50 value (0.86 µM) against pseudotyped Marburg virus. The cytotoxicity assay (MTT) indicates that compd-A has a moderate cytotoxicity level but the compd-U has much less toxicity and the CC50 value was about 100 µM. Structure-activity relationship (SAR) study has found some analogs of compd-A and -U have reduced the toxicity and enhanced the inhibitory activity. In conclusion, this work has identified several qualified lead-compounds for further drug development against filovirus infection.

Identifying Wild Versus Cultivated Gene-Alleles Conferring Seed Coat Color and Days to Flowering in Soybean.

Annual wild soybean (G. soja) is the ancestor of the cultivated soybean (G. max). To reveal the genetic changes from soja to max, an improved wild soybean chromosome segment substitution line (CSSL) population, SojaCSSLP5, composed of 177 CSSLs with 182 SSR markers (SSR-map), was developed based on SojaCSSLP1 generated from NN1138-2(max)×N24852(soja). The SojaCSSLP5 was genotyped further through whole-genome resequencing, resulting in a physical map with 1366 SNPLDBs (SNP linkage-disequilibrium blocks), which are composed of more markers/segments, shorter marker length and more recombination breakpoints than the SSR-map and caused 721 new wild substituted segments. Using the SNPLDB-map, two loci co-segregating with seed-coat color (SCC) and six loci for days to flowering (DTF) with 88.02% phenotypic contribution were identified. Integrated with parental RNA-seq and DNA-resequencing, two SCC and six DTF candidate genes, including three previously cloned (G, E2 and GmPRR3B) and five newly detected ones, were predicted and verified at nucleotide mutant level, and then demonstrated with the consistency between gene-alleles and their phenotypes in SojaCSSLP5. In total, six of the eight genes were identified with the parental allele-pairs coincided to those in 303 germplasm accessions, then were further demonstrated by the consistency between gene-alleles and germplasm phenotypes. Accordingly, the CSSL population integrated with parental DNA and RNA sequencing data was demonstrated to be an efficient platform in identifying candidate wild vs. cultivated gene-alleles.

Mandibulofacial Dysostosis Attributed to a Recessive Mutation of CYP26C1 in Hereford Cattle.

In spring 2020, six Hereford calves presented with congenital facial deformities attributed to a condition we termed mandibulofacial dysostosis (MD). Affected calves shared hallmark features of a variably shortened and/or asymmetric lower mandible and bilateral skin tags present 2-10 cm caudal to the commissure of the lips. Pedigree analysis revealed a single common ancestor shared by the sire and dam of each affected calf. Whole-genome sequencing (WGS) of 20 animals led to the discovery of a variant (Chr26 g. 14404993T>C) in Exon 3 of CYP26C1 associated with MD. This missense mutation (p.L188P), is located in an α helix of the protein, which the identified amino acid substitution is predicted to break. The implication of this mutation was further validated through genotyping 2 additional affected calves, 760 other Herefords, and by evaluation of available WGS data from over 2500 other individuals. Only the affected individuals were homozygous for the variant and all heterozygotes had at least one pedigree tie to the suspect founder. CYP26C1 plays a vital role in tissue-specific regulation of retinoic acid (RA) during embryonic development. Dysregulation of RA can result in teratogenesis by altering the endothelin-1 signaling pathway affecting the expression of Dlx genes, critical to mandibulofacial development. We postulate that this recessive missense mutation in CYP26C1 impacts the catalytic activity of the encoded enzyme, leading to excess RA resulting in the observed MD phenotype.

Viral myocarditis involves the generation of autoreactive T cells with multiple antigen specificities that localize in lymphoid and non-lymphoid organs in the mouse model of CVB3 infection.

Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971-990, and cardiac myosin heavy chain-α (Myhc) 334-352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21-40 dextramer+ T cells were inconsistently detected, the ß1-adrenergic receptor 181-200/211-230 or branched chain α-ketoacid dehydrogenase kinase 111-130 dextramer+ cells were absent. Interestingly, SERCA2a 971-990, Myhc 334-352 and ANT 21-40 dextramer+ cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971-990-reactive T cells generated in CVB3 infection could transfer disease to naïve mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.

Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Responses in Multiple Serotypes of Group B Coxsackievirus Infections.

Coxsackievirus group B (CVB) contains six serotypes that can affect various organs. Some of these organ-specific diseases such as myocarditis and pancreatitis can be caused by more than one serotype. Thus, development of immunological tools common to multiple serotypes is desired. This is especially critical for analyzing antigen-specific T cell responses at a single cell level. To this end, we made efforts to identify the immunogenic epitopes of CVB3 leading us to localize three T cell epitopes within the viral protein 1 (VP1) namely, VP1 681-700, VP1 721-740 and VP1 771-790. First, we confirmed their immunogenicity in the immunization settings. Second, we sought to verify the ability of VP1 epitopes to bind major histocompatibility complex (MHC) class II (IAk) molecules. Third, we created MHC class II (IAk) dextramers and tetramers and ascertained the T cell responses to be antigen-specific. Fourth, we analyzed the T cell responses in animals infected with CVB3 and noted the magnitude of antigen-specific T cell responses occurring in the order of VP1 721-740 and VP1 681-700 followed by VP1 771-790 as verified by proliferation assay and IAk tetramer staining. All epitopes induced interferon (IFN)-γ as a major cytokine. Finally, we investigated whether the VP1 tools generated for CVB3 can also be used to verify T cell responses in infections caused by other serotypes. To this end, we established the CVB4 infection model in A/J mice and found that the CVB4 infection led to the induction of IFN-γ-producing T cell responses primarily for VP1 721-740 and VP1 681-700. Thus, the VP1-specific tools, particularly IAk tetramers can be used to monitor anti-viral T cell responses in multiple CVB serotypes.

Identifying a wild allele conferring small seed size, high protein content and low oil content using chromosome segment substitution lines in soybean.

KEY MESSAGE: A wild soybean allele conferring 100-seed weight, protein content and oil content simultaneously was fine-mapped to a 329-kb region on Chromosome 15, in which Glyma.15g049200 was predicted a candidate gene. Annual wild soybean characterized with small 100-seed weight (100SW), high protein content (PRC), low oil content (OIC) may contain favourable alleles for broadening the genetic base of cultivated soybeans. To evaluate these alleles, a population composed of 195 chromosome segment substitution lines (SojaCSSLP4), with wild N24852 as donor and cultivated NN1138-2 as recurrent parent, was tested. In SojaCSSLP4, 10, 9 and 8 wild segments/QTL were detected for 100SW, PRC and OIC, respectively. Using a backcross-derived secondary population, one segment for the three traits (q100SW15, qPro15 and qOil15) and one for 100SW (q100SW18.2) were fine-mapped into a 329-kb region on chromosome 15 and a 286-kb region on chromosome 18, respectively. Integrated with the transcription data in SoyBase, 42 genes were predicted in the 329-kb region where Glyma.15g049200 showed significant expression differences at all seed development stages. Furthermore, the Glyma.15g049200 segments of the two parents were sequenced and compared, which showed two base insertions in CDS (coding sequence) in the wild N24852 comparing to the NN1138-2. Since only Glyma.15g049200 performed differential CDS between the two parents but related to the three traits, Glyma.15g049200 was predicted a pleiotropic candidate gene for 100SW, PRC and OIC. The functional annotation of Glyma.15g049200 indicated a bidirectional sucrose transporter belonging to MtN3/saliva family which might be the reason that this gene provides a same biochemical basis for 100SW, PRC and OIC, therefore, is responsible for the three traits. This result may facilitate isolation of the specific gene and provide prerequisite for understanding the other two pleiotropic QTL.

Blocking HIV-1 Infection by Chromosomal Integrative Expression of Human CD4 on the Surface of Lactobacillus acidophilus ATCC 4356.

Lactobacillus bacteria are potential delivery vehicles for biopharmaceutical molecules because they are well-recognized as safe microorganisms that naturally inhabit the human body. The goal of this study was to employ these lactobacilli to combat human immunodeficiency virus type 1 (HIV-1) infection and transmission. By using a chromosomal integration method, we engineered Lactobacillus acidophilus ATCC 4356 to display human CD4, the HIV-1 receptor, on the cell surface. Since human CD4 can bind to any infectious HIV-1 particles, the engineered lactobacilli can potentially capture HIV-1 of different subtypes and prevent infection. Our data demonstrate that the CD4-carrying bacteria are able to adsorb HIV-1 particles and reduce infection significantly in vitro and also block intrarectal HIV-1 infection in a humanized mouse model in preliminary tests in vivo Our results support the potential of this approach to decrease the efficiency of HIV-1 sexual transmission.IMPORTANCE In the absence of an effective vaccine, alternative approaches to block HIV-1 infection and transmission with commensal bacteria expressing antiviral proteins are being considered. This report provides a proof-of-concept by using Lactobacillus bacteria stably expressing the HIV-1 receptor CD4 to capture and neutralize HIV-1 in vitro and in a humanized mouse model. The stable expression of antiviral proteins, such as CD4, following genomic integration of the corresponding genes into this Lactobacillus strain may contribute to the prevention of HIV-1 sexual transmission.

Nucleolin Is a Functional Binding Protein for Salinomycin in Neuroblastoma Stem Cells.

The aim of this study is to illuminate a novel therapeutic approach by identifying a functional binding target of salinomycin, an emerging anticancer stem cell (CSC) agent, and to help dissect the underlying action mechanisms. By utilizing integrated strategies, we identify that nucleolin (NCL) is likely a salinomycin-binding target and a critical regulator involved in human neuroblastoma (NB) CSC activity. Salinomycin markedly suppresses NB CD34 expression and reduces CD34+ cell population in an NCL-dependent manner via disruption of the interaction of NCL with CD34 promoter. The elevated levels of NCL expression in NB tumors are associated with poor patient survival. Altogether, these results indicate that NCL is likely a novel functional salinomycin-binding target that exhibits the potential to be a prognostic marker for NB therapy.

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association.

HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity.IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

Contribution of the gp120 V3 loop to envelope glycoprotein trimer stability in primate immunodeficiency viruses.

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein (Env) becomes exposed after CD4 binding and contacts the coreceptor to mediate viral entry. Prior to CD4 engagement, a hydrophobic patch located at the tip of the V3 loop stabilizes the non-covalent association of gp120 with the Env trimer of HIV-1 subtype B strains. Here, we show that this conserved hydrophobic patch (amino acid residues 307, 309 and 317) contributes to gp120-trimer association in HIV-1 subtype C, HIV-2 and SIV. Changes that reduced the hydrophobicity of these V3 residues resulted in increased gp120 shedding and decreased Env-mediated cell-cell fusion and virus entry in the different primate immunodeficiency viruses tested. Thus, the hydrophobic patch is an evolutionarily conserved element in the tip of the gp120 V3 loop that plays an essential role in maintaining the stability of the pre-triggered Env trimer in diverse primate immunodeficiency viruses.

Characterization of twin-cysteine motif in the V2-loop region of gp120 in primate lentiviruses.

The twin-cysteine motif (TCM) in the V2 loop region of gp120, identified in our previous report on the simian immunodeficiency virus mac239 (SIVmac239), is a conserved evolutionary element in all primate lentiviruses except for HIV-1 which has lost the TCM during cross-species transmission. In this study, we have further explored the TCM in other SIV and HIV-2 strains. Our data shows that strains from different evolutionary lineages have different phenotypes when the twin-cysteines are removed. In the SIVsm/HIV-2 lineage, removal of the twin-cysteines decreases envelope trimer stability, but in the SIVagm lineage, a blockage of gp160 processing is observed. Molecular modeling has confirmed that the twin-cysteines do form a disulfide bond in the gp120 subunit, which interacts with the V1 loop to stabilize the envelope trimer. Therefore, we hypothesize that if the TCM is added back to HIV-1, it will enhance envelope stability for vaccine immunogen design.

Discovery of a non-nucleoside RNA polymerase inhibitor for blocking Zika virus replication through in silico screening.

Zika virus (ZIKV), an emerging arbovirus, has become a major human health concern globally due to its association with congenital abnormalities and neurological diseases. Licensed vaccines or antivirals against ZIKV are currently unavailable. Here, by employing a structure-based approach targeting the ZIKV RNA-dependent RNA polymerase (RdRp), we conducted in silico screening of a library of 100,000 small molecules and tested the top ten lead compounds for their ability to inhibit the virus replication in cell-based in vitro assays. One compound, 3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide (TPB), potently inhibited ZIKV replication at sub-micromolar concentrations. Molecular docking analysis suggests that TPB binds to the catalytic active site of the RdRp and therefore likely blocks the viral RNA synthesis by an allosteric effect. The IC50 and the CC50 of TPB in Vero cells were 94 nM and 19.4 µM, respectively, yielding a high selective index of 206. In in vivo studies using immunocompetent mice, TPB reduced ZIKV viremia significantly, indicating TPB as a potential drug candidate for ZIKV infections.