Transmembrane conformation of the envelope protein of an alpha coronavirus, NL63.

The envelope (E) proteins of coronaviruses (CoVs) form cation-conducting channels that are associated with the pathogenicity of these viruses. To date, high-resolution structural information about these viroporins is limited to the SARS-CoV E protein. To broaden our structural knowledge of other members of this family of viroporins, we now investigate the conformation of the E protein of the human coronavirus (hCoV), NL63. Using two- and three-dimensional magic-angle-spinning NMR, we have measured 13 C and 15 N chemical shifts of the transmembrane domain of E (ETM), which yielded backbone (ϕ, ψ) torsion angles. We further measured the water accessibility of NL63 ETM at neutral pH versus acidic pH in the presence of Ca2+ ions. These data show that NL63 ETM adopts a regular α-helical conformation that is unaffected by pH and the N-terminal ectodomain. Interestingly, the water accessibility of NL63 ETM increases only modestly at acidic pH in the presence of Ca2+ compared to neutral pH, in contrast to SARS ETM, which becomes much more hydrated at acidic pH. This difference suggests a structural basis for the weaker channel conductance of α-CoV compared to ß-CoV E proteins. The weaker E channel activity may in turn contribute to the reduced virulence of hCoV-NL63 compared to SARS-CoV viruses.

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding.

Enveloped viruses carry one or multiple proteins with receptor-binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor-binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N- and C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties.

Investigating vulnerability of the conserved SARS-CoV-2 spike's heptad repeat 2 as target for fusion inhibitors using chimeric miniproteins.

Inhibition of SARS-CoV-2 membrane fusion is a highly desired target to combat COVID-19. The interaction between the spike's heptad repeat (HR) regions 1 (HR1) and 2 (HR2) is a crucial step during the fusion process and these highly conserved HR regions constitute attractive targets for fusion inhibitors. However, the relative importance of each subregion of the long HR1-HR2 interface for viral inhibition remains unclear. Here, we designed, produced, and characterized a series of chimeric miniproteins that mimic two different half subdomains of HR1. The proteins were designed as single polypeptide chains that spontaneously fold into antiparallel trimeric helical bundles aimed at structurally imitate the molecular surface of each HR1 half subregion. All the miniproteins folded stably as helical structures and could bind complementary HR2 peptides with moderate affinity. However, only the miniproteins mimicking the N-terminal HR1 half subdomain, but not those imitating C-terminal one, could inhibit cell infection by SARS-COV-2 real viruses in cell cultures. Most interestingly, the inhibitory activity of the miniproteins correlated with their structural stability, but not with their relative binding affinity for HR2 peptides. These results are highly relevant for designing more focused and active fusion inhibitors targeting the highly conserved HR2 region of the Spike.

The transmembrane and cytosolic domains of equine herpesvirus type 1 glycoprotein D determine Golgi retention by regulating vesicle formation.

Accumulating evidence underscores the pivotal role of envelope proteins in viral secondary envelopment. However, the intricate molecular mechanisms governing this phenomenon remain elusive. To shed light on these mechanisms, we investigated a Golgi-retained gD of EHV-1 (gDEHV-1), distinguishing it from its counterparts in Herpes Simplex Virus-1 (HSV-1) and Pseudorabies Virus (PRV). To unravel the specific sequences responsible for the Golgi retention phenotype, we employed a gene truncation and replacement strategy. The results suggested that Golgi retention signals in gDEHV-1 exhibiting a multi-domain character. The extracellular domain of gDEHV-1 was identified as an endoplasmic reticulum (ER)-resident domain, the transmembrane domain and cytoplasmic tail (TM-CT) of gDEHV-1 were integral in facilitating the protein's residence within the Golgi complex. Deletion or replacement of either of these dual domains consistently resulted in the mutant gDEHV-1 being retained in an ER-like structure. Moreover, (TM-CT)EHV-1 demonstrated a preference for binding to endomembranes, inducing the generation of a substantial number of vesicles, potentially originate from the Golgi complex or the ER-Golgi intermediate compartment. In conclusion, our findings provide insights into the intricate molecular mechanisms governing the Golgi retention of gDEHV-1, facilitating the comprehension of the processes underlying viral secondary envelopment.

Defining the impact of flavivirus envelope protein glycosylation site mutations on sensitivity to broadly neutralizing antibodies.

Antibodies targeting an envelope dimer epitope (EDE) cross-neutralize Zika virus (ZIKV) and dengue virus (DENV) and have thus inspired an epitope-focused vaccine design. There are two EDE antibody subclasses (EDE1, EDE2) distinguished by their dependence on viral envelope protein N-linked glycosylation at position N153 (DENV) or N154 (ZIKV) for binding. Here, we determined how envelope glycosylation site mutations affect neutralization by EDE and other broadly neutralizing antibodies. Consistent with structural studies, mutations abolishing the N153/N154 glycosylation site increased DENV and ZIKV sensitivity to neutralization by EDE1 antibodies. Surprisingly, despite their location at predicted contact sites, these mutations also increased sensitivity to EDE2 antibodies. Moreover, despite preserving the glycosylation site motif (N-X-S/T), substituting the threonine at ZIKV envelope residue 156 with a serine resulted in loss of glycan occupancy accompanied with increased neutralization sensitivity to EDE antibodies. For DENV, the presence of a serine instead of a threonine at envelope residue 155 retained glycan occupancy, but nonetheless increased sensitivity to EDE antibodies, in some cases to a similar extent as mutation at N153, which abolishes glycosylation. Envelope glycosylation site mutations also increased ZIKV and DENV sensitivity to other non-EDE broadly neutralizing antibodies, but had limited effects on ZIKV- or DENV-specific antibodies. Thus, envelope protein glycosylation is context-dependent and modulates the potency of broadly neutralizing antibodies in a manner not predicted by existing structures. Manipulating envelope protein glycosylation could be a novel strategy for engineering vaccine antigens to elicit antibodies that broadly neutralize ZIKV and DENV.IMPORTANCEAntibodies that potently cross-neutralize Zika (ZIKV) and dengue (DENV) viruses are attractive to induce via vaccination to protect against these co-circulating flaviviruses. Structural studies have shown that viral envelope protein glycosylation is important for binding by one class of these so-called broadly neutralizing antibodies, but less is known about its effect on neutralization. Here, we investigated how envelope protein glycosylation site mutations impact the potency of broadly neutralizing antibodies against ZIKV and DENV. We found that glycan occupancy was not always predicted by an intact N-X-S/T sequence motif. Moreover, envelope protein glycosylation site mutations alter the potency of broadly neutralizing antibodies in a manner unexpected from their predicted binding mechanism as determined by existing structures. We therefore highlight the complex role and determinants of envelope protein glycosylation that should be considered in the design of vaccine antigens to elicit broadly neutralizing antibodies.

Membrane Condensation and Curvature Induced by SARS-CoV-2 Envelope Protein.

The envelope (E) protein of SARS-CoV-2 participates in virion encapsulation and budding at the membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). The positively curved membrane topology required to fit an 80 nm viral particle is energetically unfavorable; therefore, viral proteins must facilitate ERGIC membrane curvature alteration. To study the possible role of the E protein in this mechanism, we examined the structural modification of the host lipid membrane by the SARS-CoV-2 E protein using synchrotron-based X-ray methods. Our reflectometry results on solid-supported planar bilayers show that E protein markedly condenses the surrounding lipid bilayer. For vesicles, this condensation effect differs between the two leaflets such that the membrane becomes asymmetric and increases its curvature. The formation of such a curved and condensed membrane is consistent with the requirements to stably encapsulate a viral core and supports a role for E protein in budding during SARS-CoV-2 virion assembly.

Evolutionary-Related High- and Low-Virulent Classical Swine Fever Virus Isolates Reveal Viral Determinants of Virulence.

Classical swine fever (CSF) has been eradicated from Western and Central Europe but remains endemic in parts of Central and South America, Asia, and the Caribbean. CSF virus (CSFV) has been endemic in Cuba since 1993, most likely following an escape of the highly virulent Margarita/1958 strain. In recent years, chronic and persistent infections with low-virulent CSFV have been observed. Amino acid substitutions located in immunodominant epitopes of the envelope glycoprotein E2 of the attenuated isolates were attributed to positive selection due to suboptimal vaccination and control. To obtain a complete picture of the mutations involved in attenuation, we applied forward and reverse genetics using the evolutionary-related low-virulent CSFV/Pinar del Rio (CSF1058)/2010 (PdR) and highly virulent Margarita/1958 isolates. Sequence comparison of the two viruses recovered from experimental infections in pigs revealed 40 amino acid differences. Interestingly, the amino acid substitutions clustered in E2 and the NS5A and NS5B proteins. A long poly-uridine sequence was identified previously in the 3' untranslated region (UTR) of PdR. We constructed functional cDNA clones of the PdR and Margarita strains and generated eight recombinant viruses by introducing single or multiple gene fragments from Margarita into the PdR backbone. All chimeric viruses had comparable replication characteristics in porcine monocyte-derived macrophages. Recombinant PdR viruses carrying either E2 or NS5A/NS5B of Margarita, with 36 or 5 uridines in the 3'UTR, remained low virulent in 3-month-old pigs. The combination of these elements recovered the high-virulent Margarita phenotype. These results show that CSFV evolution towards attenuated variants in the field involved mutations in both structural and non-structural proteins and the UTRs, which act synergistically to determine virulence.

Exploring Rigid and Flexible Scaffolds to Develop Potent Glucuronic Acid Glycodendrimers for Dengue Virus Inhibition.

Multivalent glycodendrimers are valuable tools for studying carbohydrate-protein interactions, and their scaffolds represent important components to increase specificity and affinity. Previous work by our group described the preparation of a tetravalent glucuronic acid rigid dendron that binds with good affinity to the dengue virus envelope protein (KD = 22 µM). Herein, the chemical synthesis and binding analysis of three new sets of rigid, semirigid, and flexible glucuronic acid-based dendrimers bearing different levels of multivalency and their interactions with the dengue virus envelope protein are described. The different oligoalkynyl scaffolds were coupled to glucuronic acid azides by a copper-catalyzed azide-alkyne cycloaddition reaction through optimized synthetic strategies to afford the desired glycodendrimers with good yields. Surface plasmon resonance studies have demonstrated that glycodendrimers 12b and 12c, with flexible scaffolds, give the best binding interactions with the dengue virus envelope protein (12b: KD = 0.487 µM and 12c: KD = 0.624 µM). Their binding constant values were 45 and 35 times higher than the one obtained in previous studies with a rigid tetravalent glucuronic acid dendron (KD = 22 µM), respectively. Molecular modeling studies were carried out in order to understand the difference in behavior observed for 12b and 12c. This work reports an efficient glycodendrimer chemical synthesis process that provides an appropriate scaffold that offers an easy and versatile strategy to find new active compounds against the dengue virus.

Multimerization of the heptad repeat regions of the SARS-CoV 2 spike protein.

The heptad repeat 1 and 2 (HR1, HR2) regions in the spike protein of SARS-CoV 2 play a key role in the fusogenic mechanism of the virus with the host cell. During the fusion process they are thought to rearrange into an interdomain multimer. Functional fragments of the heptad repeat 1 and 2 regions in the spike protein of SARS-CoV 2 were chemically synthesized, labeled with nitrofurazone (NBD) and their interactions investigated by fluorescence spectroscopy. Steady state emission, fluorescence quenching, anisotropy and lifetime measurements in combination with a fluorophore dilution scheme were used to dissect multimer formation of HR1 and HR2 in quantitative detail. In addition, the investigation of the multimers by homo-FRET (via anisotropy) and lifetime measurements reveals new insights into the mechanism of fluorophore-fluorophore interactions in biological samples.

Pathway analysis of host responses to dengue virus serotype 2 infection and inhibition of viral envelope protein by naringenin from Ganoderma lucidum.

Dengue fever cases are spiking over the last two decades. Incessant efforts are still being made to gain deeper insights on this arboviral disease and to identify bioactive antivirals. In this study, bioinformatics analysis was conducted to identify the differentially expressed genes (DEGs) in the expression profiling datasets of dengue virus serotype 2 (DENV2) patients. We found overexpressed genes in dengue patients that can interrupt cell cycle progression and phase transitions of mitosis inside the host to favour the viral replication process. These DEGs were associated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as cell cycle and DNA replication. A protein interaction network consisting of these significant pathways was also constructed using STRING. Futher, the traditional Chinese medicine (TCM) compounds from Ganoderma lucidum were screened to target DENV2 envelope protein, which was crucial for viral fusion activity. Docking, orbital energy, and toxicity prediction analysis revealed that naringenin was the best antiviral candidate. Following molecular dynamics simulations, the predicted binding energy of the protein-naringenin system using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach was slightly greater than the control system. It is recommended to perform in vitro inhibition of naringenin against DENV2 and use our findings to complement the experimental data obtained.

Non-overlapping epitopes on the gHgL-gp42 complex for the rational design of a triple-antibody cocktail against EBV infection.

Epstein-Barr virus (EBV) is closely associated with cancer, multiple sclerosis, and post-acute coronavirus disease 2019 (COVID-19) sequelae. There are currently no approved therapeutics or vaccines against EBV. It is noteworthy that combining multiple EBV glycoproteins can elicit potent neutralizing antibodies (nAbs) against viral infection, suggesting possible synergistic effects. Here, we characterize three nAbs (anti-gp42 5E3, anti-gHgL 6H2, and anti-gHgL 10E4) targeting different glycoproteins of the gHgL-gp42 complex. Two antibody cocktails synergistically neutralize infection in B cells (5E3+6H2+10E4) and epithelial cells (6H2+10E4) in vitro. Moreover, 5E3 alone and the 5E3+6H2+10E4 cocktail confer potent in vivo protection against lethal EBV challenge in humanized mice. The cryo-EM structure of a heptatomic gHgL-gp42 immune complex reveals non-overlapping epitopes of 5E3, 6H2, and 10E4 on the gHgL-gp42 complex. Structural and functional analyses highlight different neutralization mechanisms for each of the three nAbs. In summary, our results provide insight for the rational design of therapeutics or vaccines against EBV infection.

Widespread amyloid aggregates formation by Zika virus proteins and peptides.

Viral pathogenesis typically involves numerous molecular mechanisms. Protein aggregation is a relatively unknown characteristic of viruses, despite the fact that viral proteins have been shown to form terminally misfolded forms. Zika virus (ZIKV) is a neurotropic one with the potential to cause neurodegeneration. Its protein amyloid aggregation may link the neurodegenerative component to the pathogenicity associated with the viral infection. Therefore, we investigated protein aggregation in the ZIKV proteome as a putative pathogenic route and one of the alternate pathways. We discovered that it contains numerous anticipated aggregation-prone regions in this investigation. To validate our prediction, we used a combination of supporting experimental techniques routinely used for morphological characterization and study of amyloid aggregates. Several ZIKV proteins and peptides, including the full-length envelope protein, its domain III (EDIII) and fusion peptide, Pr N-terminal peptide, NS1 ß-roll peptide, membrane-embedded signal peptide 2K, and cytosolic region of NS4B protein, were shown to be highly aggregating in our study. Because our findings show that viral proteins can form amyloids in vitro, we need to do a thorough functional study of these anticipated APRs to understand better the role of amyloids in the pathophysiology of ZIKV infection.

Activity of domain III-specific antibodies in early convalescence: A case study.

The Dengue virus complex (DENV), formed by four serotypes, constitutes the most important arbovirus affecting humans. The structural domain III of their envelope protein (DIII) elicits strongly neutralizing serotype-specific antibodies. Contrasting results have been obtained regarding their role in the serum neutralizing activity of infected patients. We used a DENV immune serum from a secondary infection to examine the impact of characterizing the anti-DIII antibody response after affinity purification with recombinant DIII proteins to eliminate potential interferences from the interactions with human plasma proteins and other anti-DENV antibodies. Total anti-DENV IgG repertoire and anti-DIIIE antibodies were compared in functionality. In early convalescence, reactivity of anti-DIII antibodies is serotype specific and exhibits the strongest reactivity with infecting serotypes. Purification of anti-DIII antibodies emphasizes the reactivity profile as compared to total IgG fraction and serum. Serotype-specificity of the virus neutralization activity correlated with the apparent kD of the binding to recombinant DIIIs.

Computer-aided Affinity Enhancement of a Cross-reactive Antibody against Dengue Virus Envelope Domain III.

The dengue virus (DENV), composed of four distinct but serologically related Flaviviruses, causes the most important emerging viral disease, with nearly 400 million infections yearly. Currently, there are no approved therapies. Although DENV infection induces lifelong immunity against the same serotype, the antibodies raised contribute to severe disease in heterotypic infections. Therefore, understanding the mechanism of DENV neutralization by antibodies is crucial in the design of vaccines against all serotypes. This study reports a comparative structural and energetic analysis of the monoclonal antibody (mAb) 4E11 in complex with its target domain III of the envelope protein for all four DENV serotypes. We use extensive replica molecular dynamics simulations in conjunction with the binding free energy calculations. Further single point and double mutations were designed through computational site-directed mutagenesis and observed that the re-engineered antibody exhibits high affinity to binding and broadly neutralizing activity against serotypes. Our results showed improved binding affinity by the gain of enthalpy, which could be attributed to the stabilization of salt-bridge and hydrogen bond interactions at the antigen-antibody interface. The findings provide valuable results in understanding the structural dynamics and energetic contributions that will be helpful to the design of high-affinity antibodies against dengue infections.

A critical review on antiviral peptides derived from viral glycoproteins and host receptors to decoy herpes simplex virus.

The health of the human population has been continuously challenged by viral infections. Herpes simplex virus (HSV) is one of the common causes of illness and can lead to death in immunocompromised patients. Existing anti-HSV therapies are not completely successful in eliminating the infection due to anti-viral drug resistance, ineffectiveness against the latent virus and high toxicity over prolonged use. There is a need to update our knowledge of the current challenges faced in anti-HSV therapeutics and realize the necessity of developing alternative treatment approaches. Protein therapeutics are now being explored as a novel approach due to their high specificity and low toxicity. This review highlights the significance of HSV viral glycoproteins and host receptors in the pathogenesis of HSV infection. Proteins or peptides derived from HSV glycoproteins gC, gB, gD, gH and host cell receptors (HSPG, nectin and HVEM) that act as decoys to inhibit HSV attachment, entry, or fusion have been discussed. Few researchers have tried to improve the efficacy and stability of the identified peptides by modifying them using a peptidomimetic approach. With these efforts, we think developing an alternative treatment option for immunocompromised patients and drug-resistant organisms is not far off.

Zika-specific neutralizing antibodies targeting inter-dimer envelope epitopes.

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.

The Complex Proteolipidic Behavior of the SARS-CoV-2 Envelope Protein Channel: Weak Selectivity and Heterogeneous Oligomerization.

The envelope (E) protein is a small polypeptide that can form ion channels in coronaviruses. In SARS coronavirus 2 (SARS-CoV-2), the agent that caused the recent COVID-19 pandemic, and its predecessor SARS-CoV-1, E protein is found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), where virion budding takes place. Several reports claim that E protein promotes the formation of "cation-selective channels". However, whether this term represents specificity to certain ions (e.g., potassium or calcium) or the partial or total exclusion of anions is debatable. Herein, we discuss this claim based on the available data for SARS-CoV-1 and -2 E and on new experiments performed using the untagged full-length E protein from SARS-CoV-2 in planar lipid membranes of different types, including those that closely mimic the ERGIC membrane composition. We provide evidence that the selectivity of the E-induced channels is very mild and depends strongly on lipid environment. Thus, despite past and recent claims, we found no indication that the E protein forms cation-selective channels that prevent anion transport, and even less that E protein forms bona fide specific calcium channels. In fact, the E channel maintains its multi-ionic non-specific neutral character even in concentrated solutions of Ca2+ ions. Also, in contrast to previous studies, we found no evidence that SARS-CoV-2 E channel activation requires a particular voltage, high calcium concentrations or low pH, in agreement with available data from SARS-CoV-1 E. In addition, sedimentation velocity experiments suggest that the E channel population is mostly pentameric, but very dynamic and probably heterogeneous, consistent with the broad distribution of conductance values typically found in electrophysiological experiments. The latter has been explained by the presence of proteolipidic channel structures.

Why does the herpes simplex 1 virus-encoded UL49.5 protein fail to inhibit the TAP-dependent antigen presentation?

Herpes simplex virus 1 (HSV-1) is a well-studied herpesvirus that causes various human diseases. Like other herpesviruses, HSV-1 produces the transmembrane glycoprotein N (gN/UL49.5 protein), which has been extensively studied, but its function in HSV-1 remains largely unknown. The amino-acid sequences and lengths of UL49.5 proteins differ between herpesvirus species. It is, therefore, crucial to determine whether and to what extent the spatial structure of UL49.5 orthologs that are transporter associated with antigen processing (TAP) inhibitors (i.e., of bovine herpesvirus 1; BoHV-1) differ from that of non-TAP inhibitors (i.e., of HSV-1). Our study aimed to examine the 3D structure of the HSV-1-encoded UL49.5 protein in an advanced model of the endoplasmic reticulum (ER) membrane using circular dichroism, 2D nuclear magnetic resonance, and multiple-microsecond all-atom molecular dynamics simulations in an ER membrane mimetic environment. According to our findings, the N-terminus of the HSV-1-encoded UL49.5 adopts a highly flexible, unordered structure in the extracellular part due to the presence of a large number of proline and glycine residues. In contrast to the BoHV-1-encoded homolog, the transmembrane region of the HSV-1-encoded UL49.5 is formed by a single long transmembrane α-helix, rather than two helices oriented perpendicularly, while the cytoplasmic part of the protein (C-terminus) has a short unordered structure. Our findings provide valuable experimental structural information on the HSV-1-encoded UL49.5 protein and offer, based on the obtained structure, insight into its lack of biological activity in inhibiting the TAP-dependent antigen presentation pathway.

Phospholipid binding of the dengue virus envelope E protein segment containing the conserved His residue.

Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.

Immunoaffinity biosensors for the detection of SARS-CoV-1 using screened Fv-antibodies from an autodisplayed Fv-antibody library.

The detection of severe acute respiratory syndrome coronavirus (SARS-CoV-1) was demonstrated using screened Fv-antibodies for SPR biosensor and impedance spectrometry. The Fv-antibody library was first prepared on the outer membrane of E. coli using autodisplay technology and the Fv-variants (clones) with a specific affinity toward the SARS-CoV-1 spike protein (SP) were screened using magnetic beads immobilized with the SP. Upon screening the Fv-antibody library, two target Fv-variants (clones) with a specific binding affinity toward the SARS-CoV-1 SP were determined and the Fv-antibodies on two clones were named "Anti-SP1" (with CDR3 amino acid sequence: 1GRTTG5NDRPD11Y) and "Anti-SP2" (with CDR3 amino acid sequence: 1CLRQA5GTADD11V). The binding affinities of the two screened Fv-variants (clones) were analyzed using flow cytometry and the binding constants (KD) were estimated to be 80.5 ± 3.6 nM for Anti-SP1 and 45.6 ± 8.9 nM for Anti-SP2 (n = 3). In addition, the Fv-antibody including three CDR regions (CDR1, CDR2, and CDR3) and frame regions (FRs) between the CDR regions was expressed as a fusion protein (Mw. 40.6 kDa) with a green fluorescent protein (GFP) and the KD values of the expressed Fv-antibodies toward the SP estimated to be 15.3 ± 1.5 nM for Anti-SP1 (n = 3) and 16.3 ± 1.7 nM for Anti-SP2 (n = 3). Finally, the expressed Fv-antibodies screened against SARS-CoV-1 SP (Anti-SP1 and Anti-SP2) were applied for the detection of SARS-CoV-1. Consequently, the detection of SARS-CoV-1 was demonstrated to be feasible using the SPR biosensor and impedance spectrometry utilizing the immobilized Fv-antibodies against the SARS-CoV-1 SP.