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Bunyavirales constitute the largest order of enveloped RNA viruses, many members of which cause severe diseases in humans and domestic animals. In recent decades, innovative fluorescence‐based methods have paved the way to visualize and track single fluorescent bunyaviral particles in fixed and live cells. This technological breakthrough has enabled imaging of the early stages of infection and the quantification of every step in the bunyavirus cell entry process. Here, we describe the latest procedures for rendering bunyaviral particles fluorescent and discuss the advantages and disadvantages of each approach in light of the most recent advances in fluorescence detection and monitoring of bunyavirus entry. In this mini‐review, we also illustrate how fluorescent viral particles are a powerful tool for deciphering the cellular entry process of bunyaviruses, the vast majority of which have not yet been analyzed. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is a virus that causes acute respiratory infections in neonates and older adults. To infect host cells, the attachment glycoprotein (G) interacts with a cell surface receptor. This interaction determines the specific cell types that are susceptible to infection. RSV possesses a type I fusion protein F. Type I fusion proteins are metastable when rearrangement of the prefusion F occurs; the fusion peptide is exposed transforming the protein into postfusion form. The transition between the prefusion form and its postfusion form facilitates the viral envelope and the host cell membrane to fuse, enabling the virus to enter the host cell. Understanding the entry mechanism employed by RSV is crucial for developing effective antiviral therapies. In this review, we will discuss the various types of viral fusion proteins and explore the potential entry mechanisms utilized by RSV. A deeper understanding of these mechanisms will provide valuable insights for the development of novel approaches to treat RSV infections. [ABSTRACT FROM AUTHOR]

As COVID‐19 infections wreak havoc across the globe, attention has rightly been focused on the vital organ systems (lung, kidney and heart) that are vulnerable to viral attack and contribute to the acute pathology associated with this disease. However, we should not lose sight of the fact that COVID‐19 will attack any cell type in the body expressing ACE2 ‐ including human spermatozoa. These cells possess the entire repertoire of receptors (AT1R, AT2R, MAS) and ligand processing enzymes (ACE1 and ACE2) needed to support the angiotensin signalling cascade. The latter not only provides COVID‐19 with a foothold on the sperm surface but may also promote integration, given the additional presence of a range of proteases (TMPRSS2, TMPRSS11B, TMPRSS12, furin) capable of promoting viral fusion. This article reviews the roles played by these various cellular constituents in maintaining the vitality of human spermatozoa and their competence for fertilization. The reproductive consequences of a viral attack on these systems, in terms of fertility and the risk of sexual transmission, are currently unknown. However, we should be alive to the possibility that there may be reproductive consequences of COVID‐19 infection in young males that go beyond their capacity to survive a viral attack. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID‐19) is caused by SARS‐CoV‐2 and has spread across the globe. SARS‐CoV‐2 is a highly infectious virus with no vaccine or antiviral therapy available to control the pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS‐CoV‐2. SARS‐CoV‐2 is a new member of the betacoronavirus genus like other closely related viruses including SARS‐CoV and Middle East respiratory syndrome coronavirus (MERS‐CoV). Both SARS‐CoV and MERS‐CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon‐stimulated genes (ISGs), cholesterol 25‐hydroxylase (CH25H), is induced by SARS‐CoV‐2 infection in vitro and in COVID‐19‐infected patients. CH25H converts cholesterol to 25‐hydrocholesterol (25HC) and 25HC shows broad anti‐coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA‐WA1/2020 SARS‐CoV‐2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER‐localized acyl‐CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus–cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID‐19 and emerging viral diseases in the future. Synopsis: 25‐Hydroxycholesterol, produced by the interferon‐stimulated protein CH25H, inhibits the entry of SARS‐CoV‐2, SARS‐CoV, and MERS‐CoV. Mechanistically, 25HC activates the ER‐localized enzyme ACAT, whose activity depletes accessible cholesterol on the plasma membrane and restricts viral membrane fusion. An interferon‐stimulated gene, CH25H is induced in SARS‐CoV-2 infection in vitro and in vivo.25HC exhibits broad anti‐coronavirus activity by blocking membrane fusion.25HC activates ACAT to induce internalization of accessible cholesterol on the plasma membrane and restricts viral fusion.25HC inhibits USA‐WA1/2020 SARS‐CoV-2 infection in lung epithelial cells and virus entry in human lung organoids. [ABSTRACT FROM AUTHOR]

While it is now possible to identify and genetically fingerprint the causative agents of emerging viral diseases, often with extraordinary speed, suitable therapies cannot be developed with equivalent speed, because drug discovery requires information that goes beyond knowledge of the viral genome. Peptides, however, may represent a special opportunity. For all enveloped viruses, fusion between the viral and the target cell membrane is an obligatory step of the life cycle. Class I fusion proteins harbor regions with a repeating pattern of amino acids, the heptad repeats (HRs), that play a key role in fusion, and HR-derived peptides such as enfuvirtide, in clinical use for HIV, can block the process. Because of their characteristic sequence pattern, HRs are easily identified in the genome by means of computer programs, providing the sequence of candidate peptide inhibitors directly from genomic information. Moreover, a simple chemical modification, the attachment of a cholesterol group, can dramatically increase the antiviral potency of HR-derived inhibitors and simultaneously improve their pharmacokinetics. Further enhancement can be provided by dimerization of the cholesterol-conjugated peptide. The examples reported so far include inhibitors of retroviruses, paramyxoviruses, orthomyxoviruses, henipaviruses, coronaviruses, and filoviruses. For some of these viruses, in vivo efficacy has been demonstrated in suitable animal models. The combination of bioinformatic lead identification and potency/pharmacokinetics improvement provided by cholesterol conjugation may form the basis for a rapid response strategy, where development of an emergency cholesterol-conjugated therapeutic would immediately follow the availability of the genetic information of a new enveloped virus. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The fusion of Nipah with host cells is facilitated by two of their glycoproteins, the G and the F proteins. The binding of cellular ephrins to the G head domain causes the G stalk domain to interact differently with F, which activates F to mediate virus-host fusion. To gain insight into how the ephrin-binding signal transduces from the head to the stalk domain of G, we examine quantitatively the differences between the conformational ensembles of the G head domain in its ephrin-bound and unbound states. We consider the human ephrins B2 and B3, and a double mutant of B2, all of which trigger fusion. The ensembles are generated using molecular dynamics, and the differences between them are quantified using a new machine learning method. We find that the portion of the G head domain whose conformational density is altered equivalently by the three ephrins is large, and comprises ~25% of the residues in the G head domain. This subspace also includes the residues that are known to be important to F activation, which suggests that it contains at least one common signaling pathway. The spatial distribution of the residues constituting this subspace supports the model of signal transduction in which the signal transduces via the G head dimer interface. This study also adds to the growing list of examples where signaling does not depend solely on backbone deviations. In general, this study provides an approach to filter out conserved patterns in protein dynamics. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. [ABSTRACT FROM AUTHOR]

Vesicular stomatitis virus glycoprotein G (VSV-G) belongs to a new class of viral fusion proteins (Class III). The structure of VSV-G has been solved in two different conformations and fusion is known to be triggered by low pH. To investigate Class III fusion mechanisms, molecular dynamics simulations were performed on the VSV-G prefusion structure in two different protonation states: at physiological pH (pH 7) and low pH present in the endosome (pH 5). Domain IV containing the fusion loops, which need to interact with the target membrane, exhibits the highest mobility. Energetic analyses revealed weakened interaction between Domain IV and the protein core at pH 5, which can be attributed to two pairs of structurally neighboring conserved and differentially protonated residues in the Domain IV-core interface. Energetic calculations also demonstrated that the interaction between the subunits in the core of the trimeric VSV-G is strengthened at pH 5, mainly due to newly formed interactions between the C-terminal loop of Domain II and the N-terminus of the adjacent subunit. A pair of interacting residues in this interface that is affected by differential protonation was shown to be the main effectors of this phenomenon. The results of this study thus enhance the mechanistic understanding of the effects of protonation changes in VSV-G. Proteins 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Upon receptor-mediated activation, the gp41 hydrophobic, conserved fusion peptide inserts into the target membrane and promotes the kind of perturbations required for the progression of the HIV-cell fusion reaction. Using a synthetic combinatorial library we have identified all d-amino acid hexapeptide sequences that inhibited the fusion peptide capacity of perturbing model membranes. Two hexapeptides that effectively inhibited the fusion peptide in these systems were subsequently shown to inhibit cell–cell fusion promoted by gp41 expressed at cell surfaces. These observations might be of importance for understanding the mechanisms underlying fusion peptide activity and suggest new strategies for screening compounds that target these viral sequences. [Copyright &y& Elsevier]

Cell infection by alphaviruses is accompanied by membrane permeability changes. New predictive approaches, including the computation of interfacial affinity and corresponding hydrophobic moments, suggest a segmented amphipathic-at-interface domain in the stem region of Semliki Forest virus fusion protein E1. Expression of E1 sequences in Escherichia coli cells confirmed that the membrane proximal plus transmembrane (TM) domain unit permeabilizes cells as efficiently as the 6K viroporin. Both our predictive and experimental data support the involvement of the E1 stem-TM region in membrane insertion and permeabilization. We propose to combine Wimley–White hydrophobicity analysis with expression-coupled permeability assays in order to identify viral products implied in breaching cell membrane barriers during infection. [Copyright &y& Elsevier]

The fusogenic subdomain of the Ebola virus envelope glycoprotein is an internal sequence located ca. 20 residues downstream the N-terminus of the glycoprotein transmembrane subunit. Partitioning of the Ebola fusion peptide into membranes containing phosphatidylinositol in the absence of Ca2+ stabilizes an α-helical conformation, and gives rise to vesicle efflux but not vesicle fusion. In the presence of millimolar Ca2+ the membrane-bound peptide adopts an extended β-structure, and induces inter-vesicle mixing of lipids. The peptide conformational polymorphism may be related to the flexibility of the virus–cell intermembrane fusogenic complex. [Copyright &y& Elsevier]

The refolding of the prototypic fusogenic protein hemagglutinin (HA) at the pH of fusion is considered to be a concerted and irreversible discharge of a loaded spring, with no distinct intermediates between the initial and final conformations. Here, we show that HA refolding involves reversible conformations with a lifetime of minutes. After reneutralization, low pH-activated HA returns from the conformations wherein both the fusion peptide and the kinked loop of the IL&2 subunit are exposed, but the HA1 subunits have not yet dissociated, to a structure indistinguishable from the initial one in functional, biochemical and immunological characteristics. The rate of the transition from reversible conformations to irreversible refolding depends on the pH and on the presence of target membrane. Importantly, recovery of the initial conformation is blocked by the interactions between adjacent HA trimers. The existence of the identified reversible stage of refolding can be crucial for allowing multiple copies of HA to synchronize their release of conformational energy, as required for fusion. [ABSTRACT FROM AUTHOR]

Abstract: Through an integrated study of the reactivity of a monoclonal antibody, 803‐15.6, with synthetic peptides and native recombinant HIV‐1 envelope glycoprotein gp120, we have obtained structure–functional information on a region of rgp120 not yet elucidated by X‐ray crystallography. mAb 803‐15.6 binds with high affinity and broad cross‐clade specificity to the conserved C‐terminal region (amino acids 502–516) of HIV‐1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502–508. Using quantitative binding analyses, the affinity of mAb 803‐15.6 for native, monomeric recombinant gp120[sub HXB2] (rgp120) was found to be similar to that for the synthetic gp120 peptide (502–516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803‐15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502–504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803‐15.6 for rgp120 is not affected by binding of CD4 and vice‐versa. These results suggest either that the 502–504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120–gp41 oligomer. The detailed characterization of the 803‐15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process. [ABSTRACT FROM AUTHOR]