SARS-CoV-2 Fusion Peptide Conjugated to a Tetravalent Dendrimer Selectively Inhibits Viral Infection.

Fusion is a key event for enveloped viruses, through which viral and cell membranes come into close contact. This event is mediated by viral fusion proteins, which are divided into three structural and functional classes. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein belongs to class I fusion proteins, characterized by a trimer of helical hairpins and an internal fusion peptide (FP), which is exposed once fusion occurs. Many efforts have been directed at finding antivirals capable of interfering with the fusion mechanism, mainly by designing peptides on the two heptad-repeat regions present in class I viral fusion proteins. Here, we aimed to evaluate the anti-SARS-CoV-2 activity of the FP sequence conjugated to a tetravalent dendrimer through a classical organic nucleophilic substitution reaction (SN2) using a synthetic bromoacetylated peptide mimicking the FP and a branched scaffold of poly-L-Lysine functionalized with cysteine residues. We found that the FP peptide conjugated to the dendrimer, unlike the monomeric FP sequence, has virucidal activity by impairing the attachment of SARS-CoV-2 to cells. Furthermore, we found that the peptide dendrimer does not have the same effects on other coronaviruses, demonstrating that it is selective against SARS-CoV-2.

Vaccination for Respiratory Syncytial Virus: A Narrative Review and Primer for Clinicians.

Respiratory syncytial virus (RSV) poses a significant burden on public health, causing lower respiratory tract infections in infants, young children, older adults, and immunocompromised individuals. Recent development and licensure of effective RSV vaccines provide a promising approach to lessening the associated morbidity and mortality of severe infections. This narrative review aims to empower clinicians with the necessary knowledge to make informed decisions regarding RSV vaccination, focusing on the prevention and control of RSV infections, especially among vulnerable populations. The paper explores the available RSV vaccines and existing evidence regarding their efficacy and safety in diverse populations. Synthesizing this information for clinicians can help the latter understand the benefits and considerations associated with RSV vaccination, contributing to improved patient care and public health outcomes.

Viral Membrane Fusion: A Dance Between Proteins and Lipids.

There are at least 21 families of enveloped viruses that infect mammals, and many contain members of high concern for global human health. All enveloped viruses have a dedicated fusion protein or fusion complex that enacts the critical genome-releasing membrane fusion event that is essential before viral replication within the host cell interior can begin. Because all enveloped viruses enter cells by fusion, it behooves us to know how viral fusion proteins function. Viral fusion proteins are also major targets of neutralizing antibodies, and hence they serve as key vaccine immunogens. Here we review current concepts about viral membrane fusion proteins focusing on how they are triggered, structural intermediates between pre- and postfusion forms, and their interplay with the lipid bilayers they engage. We also discuss cellular and therapeutic interventions that thwart virus-cell membrane fusion.

In Silico Physicochemical Characterization of Fusion Proteins from Emerging Amazonian Arboviruses.

Mayaro (MAYV), Saint Louis encephalitis (SLEV), and Oropouche (OROV) viruses are neglected members of the three main families of arboviruses with medical relevance that circulate in the Amazon region as etiological agents of outbreaks of febrile illnesses in humans. As enveloped viruses, MAYV, SLEV, and OROV largely depend on their class II fusion proteins (E1, E, and Gc, respectively) for entry into the host cell. Since many aspects of the structural biology of such proteins remain unclear, the present study aimed at physicochemically characterizing them by an in silico approach. The complete amino acid sequences of MAYV E1, SLEV E, and OROV Gc proteins derived by conceptual translation from annotated coding regions in the reference sequence genome of the respective viruses were obtained from the NCBI Protein database in the FASTA format and then submitted to the ClustalO, Protcalc, Pepstats, Predator, Proscan, PCprof, Phyre2, and 3Drefine web servers for the determination of sequence identities, the estimation of residual properties, the prediction of secondary structures, the identification of potential post-translational modifications, the recognition of antigenic propensities, and the modeling/refinement of three-dimensional structures. Sequence identities were 20.44%, 18.82%, and 13.70% between MAYV/SLEV, SLEV/OROV, and MAYV/OROV fusion proteins, respectively. As for the residual properties, MAYV E1 and SLEV E proteins showed a predominance of the non-polar profile (56% and 55% of the residues, respectively), whereas the OROV Gc protein showed a predominance of the polar profile (52% of the residues). Regarding predicted secondary structures, MAYV E1 and SLEV E proteins showed fewer alpha-helices (16.51% and 15.17%, respectively) than beta-sheets (21.79% and 25.15%, respectively), while the opposite was observed in the OROV Gc protein (20.39% alpha-helices and 12.14% beta-sheets). Regarding post-translational modifications, MAYV E1, SLEV E, and OROV Gc proteins showed greater relative potential for protein kinase C phosphorylation, N-myristoylation, and casein kinase II phosphorylation, respectively. Finally, antigenic propensities were higher in the N-terminus half than in the C-terminus half of these three proteins, whose three-dimensional structures revealed three distinctive domains. In conclusion, MAYV E1 and SLEV E proteins were found to share more physicochemical characteristics with each other than the OROV Gc protein, although they are all grouped under the same class of viral fusion proteins.

Molecular Modeling of Viral Type I Fusion Proteins: Inhibitors of Influenza Virus Hemagglutinin and the Spike Protein of Coronavirus.

The fusion of viral and cell membranes is one of the basic processes in the life cycles of viruses. A number of enveloped viruses confer fusion of the viral envelope and the cell membrane using surface viral fusion proteins. Their conformational rearrangements lead to the unification of lipid bilayers of cell membranes and viral envelopes and the formation of fusion pores through which the viral genome enters the cytoplasm of the cell. A deep understanding of all the stages of conformational transitions preceding the fusion of viral and cell membranes is necessary for the development of specific inhibitors of viral reproduction. This review systematizes knowledge about the results of molecular modeling aimed at finding and explaining the mechanisms of antiviral activity of entry inhibitors. The first section of this review describes types of viral fusion proteins and is followed by a comparison of the structural features of class I fusion proteins, namely influenza virus hemagglutinin and the S-protein of the human coronavirus.

Fusexins, HAP2/GCS1 and Evolution of Gamete Fusion.

Gamete fusion is the climax of fertilization in all sexually reproductive organisms, from unicellular fungi to humans. Similarly to other cell-cell fusion events, gamete fusion is mediated by specialized proteins, named fusogens, that overcome the energetic barriers during this process. In recent years, HAPLESS 2/GENERATIVE CELL-SPECIFIC 1 (HAP2/GCS1) was identified as the fusogen mediating sperm-egg fusion in flowering plants and protists, being both essential and sufficient for the membrane merger in some species. The identification of HAP2/GCS1 in invertebrates, opens the possibility that a similar fusogen may be used in vertebrate fertilization. HAP2/GCS1 proteins share a similar structure with two distinct families of exoplasmic fusogens: the somatic Fusion Family (FF) proteins discovered in nematodes, and class II viral glycoproteins (e.g., rubella and dengue viruses). Altogether, these fusogens form the Fusexin superfamily. While some attributes are shared among fusexins, for example the overall structure and the possibility of assembly into trimers, some other characteristics seem to be specific, such as the presence or not of hydrophobic loops or helices at the distal tip of the protein. Intriguingly, HAP2/GCS1 or other fusexins have neither been identified in vertebrates nor in fungi, raising the question of whether these genes were lost during evolution and were replaced by other fusion machinery or a significant divergence makes their identification difficult. Here, we discuss the biology of HAP2/GCS1, its involvement in gamete fusion and the structural, mechanistic and evolutionary relationships with other fusexins.

Proteomics Computational Analyses Suggest That the Envelope Glycoproteins of Segmented Jingmen Flavi-Like Viruses are Class II Viral Fusion Proteins (b-Penetrenes) with Mucin-Like Domains.

Jingmen viruses are newly described segmented flavi-like viruses that have a worldwide distribution in ticks and have been associated with febrile illnesses in humans. Computational analyses were used to predict that Jingmen flavi-like virus glycoproteins have structural features of class II viral fusion proteins, including an ectodomain consisting of beta-sheets and short alpha-helices, a fusion peptide with interfacial hydrophobicity and a three-domain architecture. Jingmen flavi-like virus glycoproteins have a sequence enriched in serine, threonine, and proline at the amino terminus, which is a feature of mucin-like domains. Several of the serines and threonines are predicted be modified by the addition of O-linked glycans. Some of the glycoproteins are predicted to have an additional mucin-like domain located prior to the transmembrane anchor, whereas others are predicted to have a stem consisting of two alpha-helices. The flavivirus envelope protein and Jingmen flavi-virus glycoproteins may have diverged from a common class II precursor glycoprotein with a mucin-like domain or domains acquired after divergence.

Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.

Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan "hole" naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

Structure and Dynamics of Membrane Proteins from Solid-State NMR.

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy elucidates membrane protein structures and dynamics in atomic detail to yield mechanistic insights. By interrogating membrane proteins in phospholipid bilayers that closely resemble biological membranes, SSNMR spectroscopists have revealed ion conduction mechanisms, substrate transport dynamics, and oligomeric interfaces of seven-transmembrane helix proteins. Research has also identified conformational plasticity underlying virus-cell membrane fusions by complex protein machineries, and ß-sheet folding and assembly by amyloidogenic proteins bound to lipid membranes. These studies collectively show that membrane proteins exhibit extensive structural plasticity to carry out their functions. Because of the inherent dependence of NMR frequencies on molecular orientations and the sensitivity of NMR frequencies to dynamical processes on timescales from nanoseconds to seconds, SSNMR spectroscopy is ideally suited to elucidate such structural plasticity, local and global conformational dynamics, protein-lipid and protein-ligand interactions, and protonation states of polar residues. New sensitivity-enhancement techniques, resolution enhancement by ultrahigh magnetic fields, and the advent of 3D and 4D correlation NMR techniques are increasingly aiding these mechanistically important structural studies.

Expression.purification and antigenicity of a recombinant respiratory syncyttal virus fusion gene encoded protein(amino acids 168-289)in the insect cells / 中华检验医学杂志

Objective To explore the antigenicity of the recombinant respiration syncytial virus (RSV)fusion protein (amino acids 168-289) encoded by 546-881 bases of the fusion gene expressed in insect baculoviruses expression system.Methods The fragment F' of fusion gene 546-881 bases was amplified from viral RNA( Long strain ) by the reverse transcription-polymerase chain reaction(RT-PCR).F' was inserted into transfer vector pBacPAK9 and the recombinant plasmid pBacRSV F' was constructed.Sf9 insect cells were then co-transfeeted with a mixture of recombinant plasmids pBacRSV F' and linearized BacPAK6 viral DNA( Bsu36 Ⅰ -digested).The recombinant baculoviruses BacPAK F' was constructed and was able to express the recombinant protein in Sf9 insect cells.The recombinant protein was purified by Ni2+ NTA chromatography and its antigenicity was identified by Western blot(WB) analysis.Specimens including the nasopharyngeal aspirates(NPAs) and sera were collected from 33 infants and young children with acute lower respiration tract infection. Indirect immunofluorecenee assay (IFA) and WB were used to detect the RSV antigen in NPAs and the anti-RSV antibody in sera respectively.Results The recombinant protein (molecular weight 13 000) was expressed in Sf9 insect cells.WB analysis demonstrated that the purified recombinant protein had a specific RSV antibody-binding activity. The recombinant protein could be recognized by positive serum infected by RSV.The positive rate was 27.3% and 33.3% respectively.There was no significant difference between them(X2 = 0.29 ,P ＞ 0.05).Conclusion The recombinant respiratory syncytial virus fusion gene (546-881 bp) encoding protein is expressed in Sf9 insect cells and it has strong antigenicity and could have clinical application value for detection of RSV infection.

Immunogenicity of a novel vaccine with VLP as vector for Wilm's tumor / 中华普通外科杂志

Objective To develop a vaccine for Wilm's tumor in order to lay a foundation for effective treatment and substitute for the radiotherapy and chemotherapy of this tumor. Methods DNA fragments encoding the WT1 antigens (including, HLA-A * 2402 and HLA-A * 2402x), and DNA fragments encoding the couple antigens HLA-A * 2402-HBc and HLA-A * 2402x-HBc were cloned and inserted into the eukaryotic expression vector pcDNA3.1(+) in sense orientation and baculovirus expression pFactbac respectively, then transfected into COS-7 cells and Sf 9 cell respectively. The expression of the target proteins in the cells were identified by CPE, SDS-PAGE and electron microscopy. Virus-like particles were observed under electron microscopy. Results The fusion gene of HLA-A * 2402 and HLA-A * 2402x and HBc express virus-like particle protein. Conclusions This preliminary result shows a hopeful future in developing an effective immunotherapy to Wilm's tumor.

Structure-activity relationships of anti-HIV-1 peptides with disulfide linkage between D- and L-cysteine at positions i and i+3, respectively, derived from HIV-1 gp41 C-peptide

The constrained alpha-helical structure of a C-peptide is useful for enhancing anti-HIV-1 activity. The i and i+3 positions in an alpha-helical structure are located close together, therefore D-Cys (dC) and L-Cys (C) were introduced at the positions, respectively, to make a dC-C disulfide bond in 28mer C-peptides. Accordingly, this study tested whether a dC-C disulfide bond would increase the alpha-helicity and anti-HIV-1 activity of peptides. A C-peptide can be divided into three domains, the N-terminal hydrophobic domain (HPD), middle interface domain (IFD), and C-terminal hydrogen domain (HGD), based on the binding property with an N-peptide. In general, the dC-C modifications in HPD enhanced the anti-HIV-1 activity, while those in IFD and HGD resulted in no or much less activity. The modified peptides with no activity clearly showed much less alpha-helicity than the native peptides, while those with higher activity showed an almost similar or slightly increased alpha-helicity. Therefore, the present results suggest that the introduction of a dC-C bridge in the N-terminal hydrophobic domain of a C-peptide may be useful for enhancing the anti-HIV-1 activity.

Monitoring antibody titers to recombinant Core-NS3 fusion polypeptide is useful for evaluating hepatitis C virus infection and responses to interferon-alpha therapy

To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment.