Search

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]

ABSTRACT Flavivirus particle maturation, a process essential for virus infectivity, has been mostly studied using dengue virus. In infected cells, immature icosahedral virions bud into the endoplasmic reticulum. Budding is induced by lateral contacts between heterodimers of transmembrane glycoproteins prM and E. During exocytosis through the trans-Golgi network (TGN), the acidic environment triggers a major particle reorganization in which E forms head-to-tail dimers and furin cleaves prM into globular pr and transmembrane M proteins. pr remains bound to E at acidic pH and blocks its fusogenic activity, but at neutral pH, its affinity for E drops and pr is shed from the particle in the extracellular environment, leaving a virion activated for fusion at low pH. We report here that recombinant yellow fever virus (YFV) pr retains high affinity for soluble E (sE) at neutral pH—significantly shifting the current paradigm. The X-ray structure of the YFV pr/sE complex shows essentially the same pattern of interactions reported for dengue virus, while the X-ray structure of YFV sE at neutral pH shows the same canonical head-to-tail sE dimer. pr binding to the sE dimer is precluded by the E “150-loop,” indicating it must adopt a different conformation in the E dimers on virions at acidic pH for pr binding. We had previously reported a similar local reorganization of the E 150-loop at acidic pH for the tick-borne encephalitis virus, with the important difference that pr stabilized a soluble sE head-to-tail dimer, which is not the case for YFV. IMPORTANCE All enveloped viruses enter cells by fusing their envelope with a target cell membrane while avoiding premature fusion with membranes of the producer cell—the latter being particularly important for viruses that bud at internal membranes. Flaviviruses bud in the endoplasmic reticulum, are transported through the TGN to reach the external milieu, and enter other cells via receptor-mediated endocytosis. The trigger for membrane fusion is the acidic environment of early endosomes, which has a similar pH to the TGN of the producer cell. The viral particles therefore become activated to react to mildly acidic pH only after their release into the neutral pH extracellular environment. Our study shows that for yellow fever virus (YFV), the mechanism of activation involves actively knocking out the fusion brake (protein pr) through a localized conformational change of the envelope protein upon exposure to the neutral pH external environment. Our study has important implications for understanding the molecular mechanism of flavivirus fusion activation in general and points to an alternative way of interfering with this process as an antiviral treatment.

The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation, and functional activity. In addition, protons (H1 or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. Although the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the H1 concentration ([H1]) of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen together with advances in genomic, proteomic, and electrophysiological techniques have led to the identification and characterization of unique pumps, channels, and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications for cellular function in health and disease are the subject of this review. [ABSTRACT FROM AUTHOR]

Membrane fusion plays a lead role in the transport of vesicles, neurotransmission, mitochondrial dynamics, and viral infection. There are fusion proteins that catalyze and regulate the fusion. Interestingly, various types of fusion proteins are present in nature and they possess diverse mechanisms of action. We have highlighted the importance of the functional domains of intracellular heterotypic fusion, homotypic endoplasmic reticulum (ER), homotypic mitochondrial, and type-I viral fusion. During intracellular heterotypic fusion, the SNAREs and four-helix bundle formation are prevalent. Type-I viral fusion is controlled by the membrane destabilizing properties of fusion peptide and six-helix bundle formation. The ER/mitochondrial homotypic fusion is controlled by GTPase activity and the membrane destabilization properties of the amphipathic helix(s). Although the mechanism of action of these fusion proteins is diverse, they have some similarities. In all cases, the lipid composition of the membrane greatly affects membrane fusion. Next, examples of lipidation of the fusion proteins were discussed. We suggest that the fatty acyl hydrophobic tail not only acts as an anchor but may also modulate the energetics of membrane fusion intermediates. Lipidation is also important to design more effective peptide-based fusion inhibitors. Together, we have shown that membrane lipid composition and lipidation are important to modulate membrane fusion. [ABSTRACT FROM AUTHOR]

Serine incorporator 5 (SER5) is a protein that upon incorporation into virions inhibits HIV-1 infectivity by interfering with the ability of the Env glycoprotein to promote viral fusion. The mechanisms by which SER5 antagonizes HIV-1 fusion are not well understood. A recent study of SER5's structure revealed a lipid-binding pocket, suggesting the ability to sequester lipids. This finding, along with the well-documented modulation of HIV-1 infectivity by viral lipids, especially cholesterol, prompted our examination of SER5′s effect on the general lipid order of the HIV-1 membrane. Pseudoviruses bearing the SER5-sensitive HXB2-Env and containing SER5 or SER2, a control protein that lacks antiviral activity, were analyzed using two distinct lipid-order probes. We show that SER5 incorporation does not noticeably affect the lipid order of pseudoviruses. Although viral cholesterol extraction reduces HIV-1 infectivity, SER5+ viruses are less sensitive to cholesterol extraction than the control samples. In contrast, the virus' sensitivity to cholesterol oxidation was not affected by SER5 incorporation. The hydrolytic release of sphingomyelin-sequestered cholesterol had a minimal impact on the apparent resistance to cholesterol extraction. Based on these results, we propose that a subpopulation of more stable Env glycoproteins responsible for the residual infectivity of SER5+ viruses is less sensitive to the cholesterol content of the viral membrane. [ABSTRACT FROM AUTHOR]

Cell fusion (fusogenesis) occurs in natural and pathological conditions in prokaryotes and eukaryotes. Cells of monocyte–macrophage lineage are highly fusogenic. They create syncytial multinucleated giant cells (MGCs) such as osteoclasts (OCs), MGCs associated with the areas of infection/inflammation, and foreign body-induced giant cells (FBGCs). The fusion of monocytes/macrophages with tumor cells may promote cancer metastasis. We describe types and examples of monocyte–macrophage lineage cell fusion and the role of actin-based structures in cell fusion. [ABSTRACT FROM AUTHOR]

ABSTRACT Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a serious threat to global public health, underscoring the urgency of developing effective therapies. Therapeutics and, more specifically, direct-acting antiviral development are still very much in their infancy. Here, we report that two hepatitis C virus (HCV) fusion inhibitors identified in our previous study, dichlorcyclizine and fluoxazolevir, broadly block human coronavirus entry into various cell types. Both compounds were effective against various human-pathogenic CoVs in multiple assays based on vesicular stomatitis virus (VSV) pseudotyped with the spike protein and spike-mediated syncytium formation. The antiviral effects were confirmed in SARS-CoV-2 infection systems. These compounds were equally effective against recently emerged variants, including the delta variant. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket near the fusion peptide of S protein, consistent with their potential mechanism of action as fusion inhibitors. In summary, these fusion inhibitors have broad-spectrum antiviral activities and may be promising leads for treatment of SARS-CoV-2, its variants, and other pathogenic CoVs. IMPORTANCE SARS-CoV-2 is an enveloped virus that requires membrane fusion for entry into host cells. Since the fusion process is relatively conserved among enveloped viruses, we tested our HCV fusion inhibitors, dichlorcyclizine and fluoxazolevir, against SARS-CoV-2. We performed in vitro assays and demonstrated their effective antiviral activity against SARS-CoV-2 and its variants. Cross-linking experiments and structural modeling suggest that the compounds bind to a hydrophobic pocket in spike protein to exert their inhibitory effect on the fusion step. These data suggest that both dichlorcyclizine and fluoxazolevir are promising candidates for further development as treatment for SARS-CoV-2.

Pestiviruses, within the family Flaviviridae , are economically important viruses of livestock. In recent years, new pestiviruses have been reported in domestic animals and non-cloven-hoofed animals. Among them, atypical porcine pestivirus (APPV) and Norway rat pestivirus (NRPV) have relatively little sequence conservation in their surface glycoprotein E2. Despite E2 being the main target for neutralizing antibodies and necessary for cell attachment and viral fusion, the mechanism of viral entry remains elusive. To gain further insights into the pestivirus E2 mechanism of action and to assess its diversity within the genus, we report X-ray structures of the pestivirus E2 proteins from APPV and NRPV. Despite the highly divergent structures, both are able to dimerize through their C-terminal domain and contain a solvent-exposed β-hairpin reported to be involved in host receptor binding. Functional analysis of this β-hairpin in the context of BVDV revealed its ability to rescue viral infectivity. [Display omitted] • The X-ray crystal structures of APPV and NRPV E2 glycoproteins were determined • APPV and NRPV E2 form a dimer in solution Aitkenhead et al. report the X-ray crystal structures of pestivirus E2 surface glycoproteins from atypical porcine pestivirus and Norway rat pestivirus. These glycoproteins serve as primary targets for neutralizing antibodies and play crucial roles in cell attachment and viral fusion. [ABSTRACT FROM AUTHOR]

Serine incorporator 5 (SER5) is a protein that upon incorporation into virions inhibits HIV-1 infectivity by interfering with the ability of the Env glycoprotein to promote viral fusion. The mechanisms by which SER5 antagonizes HIV-1 fusion are not well understood. A recent study of SER5’s structure revealed a lipid-binding pocket, suggesting the ability to sequester lipids. This finding, along with the well-documented modulation of HIV-1 infectivity by viral lipids, especially cholesterol, prompted our examination of SER5′s effect on the general lipid order of the HIV-1 membrane. Pseudoviruses bearing the SER5-sensitive HXB2-Env and containing SER5 or SER2, a control protein that lacks antiviral activity, were analyzed using two distinct lipid-order probes. We show that SER5 incorporation does not noticeably affect the lipid order of pseudoviruses. Although viral cholesterol extraction reduces HIV-1 infectivity, SER5+ viruses are less sensitive to cholesterol extraction than the control samples. In contrast, the virus’ sensitivity to cholesterol oxidation was not affected by SER5 incorporation. The hydrolytic release of sphingomyelin-sequestered cholesterol had a minimal impact on the apparent resistance to cholesterol extraction. Based on these results, we propose that a subpopulation of more stable Env glycoproteins responsible for the residual infectivity of SER5+ viruses is less sensitive to the cholesterol content of the viral membrane.

The intricate lattice of Gn and Gc glycoprotein spike complexes on the hantavirus envelope facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Through study of a neutralizing monoclonal antibody termed mAb P-4G2, which neutralizes the zoonotic pathogen Puumala virus (PUUV), we provide a molecular-level basis for antibody-mediated targeting of the hantaviral glycoprotein lattice. Crystallographic analysis demonstrates that P-4G2 binds to a multi-domain site on PUUV Gc and may preclude fusogenic rearrangements of the glycoprotein that are required for host-cell entry. Furthermore, cryo-electron microscopy of PUUV-like particles in the presence of P-4G2 reveals a lattice-independent configuration of the Gc, demonstrating that P-4G2 perturbs the (Gn-Gc)4 lattice. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

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]

ABSTRACT African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) causing a lethal hemorrhagic disease that currently threatens the global pig industry. Despite its relevance in the infectious cycle, very little is known about the internalization of ASFV in the host cell. Here, we report the characterization of ASFV protein pE199L, a cysteine-rich structural polypeptide with similarity to proteins A16, G9, and J5 of the entry fusion complex (EFC) of poxviruses. Using biochemical and immunomicroscopic approaches, we found that, like the corresponding poxviral proteins, pE199L localizes to the inner viral envelope and behaves as an integral transmembrane polypeptide with cytosolic intramolecular disulfide bonds. Using an ASFV recombinant that inducibly expresses the E199L gene, we found that protein pE199L is not required for virus assembly and egress or for virus-cell binding and endocytosis but is required for membrane fusion and core penetration. Interestingly, similar results have been previously reported for ASFV protein pE248R, an inner membrane virion component related to the poxviral L1 and F9 EFC proteins. Taken together, these findings indicate that ASFV entry relies on a form of fusion machinery comprising proteins pE248R and pE199L that displays some similarities to the unconventional fusion apparatus of poxviruses. Also, these results provide novel targets for the development of strategies that block the first stages of ASFV replication. IMPORTANCE African swine fever virus (ASFV) causes a highly lethal swine disease that is currently present in many countries of Eastern Europe, the Russian Federation, and Southeast Asia, severely affecting the pig industry. Despite extensive research, effective vaccines or antiviral strategies are still lacking and relevant gaps in knowledge of the fundamental biology of the viral infection cycle exist. In this study, we identified pE199L, a protein of the inner viral membrane that is required for virus entry. More specifically, pE199L is necessary for the fusion event that leads to the penetration of the genome-containing core in the host cell. Our results significantly increase our knowledge of the process of internalization of African swine fever virus, which may instruct future research on antiviral strategies.

Fusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by EM. To capture fusion events, the process must be curtailed by trapping Env-receptor binding at an intermediate stage. We have used fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2–4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate of HIV-1 Env and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.

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]

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes frequent outbreaks of severe neurologic and respiratory disease in humans with high case fatality rates. The 2 glycoproteins displayed on the surface of the virus, NiV-G and NiV-F, mediate host-cell attachment and membrane fusion, respectively, and are targets of the host antibody response. Here, we provide a molecular basis for neutralization of NiV through antibody-mediated targeting of NiV-F. Structural characterization of a neutralizing antibody (nAb) in complex with trimeric prefusion NiV-F reveals an epitope at the membrane-distal domain III (DIII) of the molecule, a region that undergoes substantial refolding during host-cell entry. The epitope of this monoclonal antibody (mAb66) is primarily protein-specific and we observe that glycosylation at the periphery of the interface likely does not inhibit mAb66 binding to NiV-F. Further characterization reveals that a Hendra virus-F-specific nAb (mAb36) and many antibodies in an antihenipavirus-F polyclonal antibody mixture (pAb835) also target this region of the molecule. Integrated with previously reported paramyxovirus F-nAb structures, these data support a model whereby the membrane-distal region of the F protein is targeted by the antibody-mediated immune response across henipaviruses. Notably, our domain-specific sequence analysis reveals no evidence of selective pressure at this region of the molecule, suggestive that functional constraints prevent immune-driven sequence variation. Combined, our data reveal the membrane-distal region of NiV-F as a site of vulnerability on the NiV surface. [ABSTRACT FROM AUTHOR]

To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool.

ABSTRACT Paramyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategy in vitro. Conformational changes in the F protein leading to adoption of the postfusion form of the protein—prior to receptor engagement of HN at the host cell membrane—render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevant ex vivo system, we have the potential to inform the development of therapeutics to block viral infection. IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategy in vitro. We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.

Influenza infection requires viral escape from early endosomes into the cytosol, which is enabled by an acid-induced irreversible conformational transformation in the viral protein hemagglutinin. Despite the direct relationship between this conformational change and infectivity, label-free methods for characterizing this and other protein conformational changes in biological mixtures are limited. While the chemical reactivity of the protein backbone and side-chain residues is a proxy for protein conformation, coupling this reactivity to quantitative mass spectrometry is a challenge in complex environments. Herein, we evaluate whether electrophilic amidination coupled with pseudo-parallel reaction monitoring is an effective label-free approach to detect the fusion-associated conformational transformation in recombinant hemagglutinin (rHA). We identified rHA peptides that are differentially amidinated between the pre- and post-fusion states, and validated that this difference relies upon the fusion-associated conformational switch. We further demonstrate that we can distinguish the fusion profile in a matrix of digested cellular lysate. This fusion assay can be used to evaluate fusion competence for modified HA. [ABSTRACT FROM AUTHOR]

A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intrahost evolution of the wild-type (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of smallmolecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for interhost spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37°C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro, neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV. [ABSTRACT FROM AUTHOR]

During a measles virus (MeV) epidemic in 2009 in South Africa, measles inclusion body encephalitis (MIBE) was identified in several HIV-infected patients. Years later, children are presenting with subacute sclerosing panencephalitis (SSPE). To investigate the features of established MeV neuronal infections, viral sequences were analyzed from brain tissue samples of a single SSPE case and compared with MIBE sequences previously obtained from patients infected during the same epidemic. Both the SSPE and the MIBE viruses had amino acid substitutions in the ectodomain of the F protein that confer enhanced fusion properties. Functional analysis of the fusion complexes confirmed that both MIBE and SSPE F protein mutations promoted fusion with less dependence on interaction by the viral receptor-binding protein with known MeV receptors. While the SSPE F required the presence of a homotypic attachment protein, MeV H, in order to fuse, MIBE F did not. Both F proteins had decreased thermal stability compared to that of the corresponding wild-type F protein. Finally, recombinant viruses expressing MIBE or SSPE fusion complexes spread in the absence of known MeV receptors, with MIBE F-bearing viruses causing large syncytia in these cells. Our results suggest that alterations to the MeV fusion complex that promote fusion and cell-to-cell spread in the absence of known MeV receptors is a key property for infection of the brain. [ABSTRACT FROM AUTHOR]

Viral glycoprotein-mediated membrane fusion is an essential step for productive infection of host cells by enveloped viruses; however, due to its rarity and challenges in detection, little is known about the details of fusion events at the single particle level. Here, we have developed dual-color foamy viruses (FVs) composed of eGFP-tagged prototype FV (PFV) Gag and mCherry-tagged Env of either PFV or macaque simian FV (SFVmac) origin that have been optimized for detection of the fusion process. Using our recently developed tracking imaging correlation (TrIC) analysis, we were able to detect the fusion process for both PFV and SFVmac Env containing virions. PFV Env-mediated fusion was observed both at the plasma membrane as well as from endosomes, whereas SFVmac Env-mediated fusion was only observed from endosomes. PFV Env-mediated fusion was observed to happen more often and more rapidly than as for SFVmac Env. Strikingly, using the TrIC method, we detected a novel intermediate state where the envelope and capsids are still tethered but separated by up to 400 nm before final separation of Env and Gag occurred.

The ongoing pandemic of coronavirus disease-2019 (COVID-19) is being caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The disease continues to present significant challenges to the health care systems around the world. This is primarily because of the lack of vaccines to protect against the infection and the lack of highly effective therapeutics to prevent and/or treat the illness. Nevertheless, researchers have swiftly responded to the pandemic by advancing old and new potential therapeutics into clinical trials. In this review, we summarize potential anti-COVID-19 therapeutics that block the early stage of the viral life cycle. The review presents the structures, mechanisms, and reported results of clinical trials of potential therapeutics that have been listed in clinicaltrials.gov. Given the fact that some of these therapeutics are multi-acting molecules, other relevant mechanisms will also be described. The reviewed therapeutics include small molecules and macromolecules of sulfated polysaccharides, polypeptides, and monoclonal antibodies. The potential therapeutics target viral and/or host proteins or processes that facilitate the early stage of the viral infection. Frequent targets are the viral spike protein, the host angiotensin converting enzyme 2, the host transmembrane protease serine 2, and clathrin-mediated endocytosis process. Overall, the review aims at presenting update-to-date details, so as to enhance awareness of potential therapeutics, and thus, to catalyze their appropriate use in combating the pandemic.

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.

Background Serum hemagglutination inhibition (HAI) and microneutralization (MN) antibodies are often used as a correlate of protection for influenza. However, these manual assays are labor-intensive and difficult to standardize due to variability in biologic reagents used and subjective interpretation of the results. Methods Sera with known HAI and MN titers were used to assess a novel test based on the inhibition of fluorescence ‘dequenching’. Whole influenza virions (A/California/07/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2) and B/Brisbane/60/2008) labelled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) were exposed to serial dilutions of serum and mixed with turkey red blood cells followed by acidification of the media (pH 5.0–5.5). The H1N1 and B/Brisbane strains were high hemagglutinating while the H3N2 strain had low hemagglutinating activity. In some experiments, labelled virions were subjected to repetitive freeze-thaw cycles prior to use in the assay. Results In the absence of detectable HAI/MN antibodies, there were consistent and substantial increases from baseline DiD fluorescence upon acidification. Sera with known high titer HAI/MN antibodies reduced or completely prevented DiD dequenching at low dilutions with progressive increases in fluorescence at higher dilutions, which permitted a reproducible assignment of an antibody ‘titer’ based on baseline and acidified DiD fluorescence values. The ‘titers’ measured by the DiD dequenching assay were highly correlated with HAI/MN results for the H1N1 and B strains (Spearman’s correlation coefficients (r s ) 0.874 to 0.946, p < 10 −7 to 10 −35 ). Correlations with HAI/MN titres for the low-hemagglutinating H3N2 strain tested were lower but remained statistically significant (r s 0.547–0.551, p < 0.004). Freeze-thawing of the DiD pre-stained virus stocks had no significant impact on the results of the assay. Conclusions The DiD dequenching assay may be a labour-saving and more objective alternative to the classic serologies. This novel assay could theoretically be standardized across laboratories using pre-stained virions and has the potential to be fully automated. [ABSTRACT FROM AUTHOR]

Influenza hemagglutinin (HA) is a viral membrane protein responsible for the initial steps of the entry of influenza virus into the host cell. It mediates binding of the virus particle to the host-cell membrane and catalyzes fusion of the viral membrane with that of the host. HA is therefore a major target in the development of antiviral strategies. The fusion of two membranes involves high activation barriers and proceeds through several intermediate states. Here, we provide a biophysical description of the membrane fusion process, relating its kinetic and thermodynamic properties to the large conformational changes taking place in HA and placing these in the context of multiple HA proteins working together to mediate fusion. Furthermore, we highlight the role of novel single-particle experiments and computational approaches in understanding the fusion process and their complementarity with other biophysical approaches. [ABSTRACT FROM AUTHOR]

Fusion of cellular membranes during normal biological processes, including proliferation, or synaptic transmission, is mediated and controlled by sophisticated protein machinery ensuring the preservation of the vital barrier function of the membrane throughout the process. Fusion of virus particles with host cell membranes is more sparingly arranged and often mediated by a single fusion protein, and the virus can afford to be less discriminative towards the possible different outcomes of fusion attempts. Formation of leaky intermediates was recently observed in some fusion processes, and an alternative trajectory of the process involving formation of -shaped structures was suggested. In this study, we apply the methods of elasticity theory and Lagrangian formalism augmented by phenomenological and molecular geometry constraints and boundary conditions to investigate the traits of this trajectory and the drivers behind the choice of one of the possible scenarios depending on the properties of the system. The alternative pathway proved to be a dead end, and, depending on the parameters of the participating membranes and fusion proteins, the system can either reversibly enter the corresponding "leaky" configuration or be trapped in it. A parametric study in the biologically relevant range of variables emphasized the fusion protein properties crucial for the choice of the fusion scenario. [ABSTRACT FROM AUTHOR]

Emerging infections of mosquito-borne Zika virus (ZIKV) pose an increasing threat to human health, as documented over the recent years in South Pacific islands and the Americas in recent years. To better understand molecular mechanisms underlying the increase in human cases with severe pathologies, we recently demonstrated the functional roles of structural proteins capsid (C), pre-membrane (prM), and envelop (E) of ZIKV epidemic strains with the initiation of viral infection in human cells. Specifically, we found that the C-prM region contributes to permissiveness of human host cells to ZIKV infection and ZIKV-induced cytopathic effects, whereas the E protein is associated with viral attachment and early infection. In the present study, we further characterize ZIKV E proteins by investigating the roles of residues isoleucine 152 (Ile152), threonine 156 (Thr156), and histidine 158 (His158) (i.e., the E-152/156/158 residues), which surround a unique N-glycosylation site (E-154), in permissiveness of human host cells to epidemic ZIKV infection. For comparison purpose, we generated mutant molecular clones of epidemic BeH819015 (BR15) and historical MR766-NIID (MR766) strains that carry each other’s E-152/156/158 residues, respectively. We observed that the BR15 mutant containing the E-152/156/158 residues from MR766 was less infectious in A549-Dual™ cells than parental virus. In contrast, the MR766 mutant containing E-152/156/158 residues from BR15 displayed increased infectivity. The observed differences in infectivity were, however, not correlated with changes in viral binding onto host-cells or cellular responses to viral infection. Instead, the E-152/156/158 residues from BR15 were associated with an increased efficiency of viral membrane fusion inside infected cells due to conformational changes of E protein that enhance exposure of the fusion loop. Our data highlight an important contribution of E-152/156/158 residues to the early steps of ZIKV infection in human cells.

Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot (Scophthalmus maximus) NK-lysine short peptide (Nkl71⁻100) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this work several biophysical methods, such as vesicle aggregation, leakage and fluorescence anisotropy, are employed to investigate the interaction of Nkl71⁻100 with different glycerophospholipid vesicles. At acidic pH, Nkl71⁻100 preferably interacts with phosphatidylserine (PS), disrupts PS membranes, and allows the content leakage from vesicles. Furthermore, Nkl71⁻100 exerts strong antiviral activity against spring viremia of carp virus (SVCV) by inhibiting not only the binding of viral particles to host cells, but also the fusion of virus and cell membranes, which requires a low pH context. Such antiviral activity seems to be related to the important role that PS plays in these steps of the replication cycle of SVCV, a feature that is shared by other families of virus-comprising members with health and veterinary relevance. Consequently, Nkl71⁻100 is shown as a promising broad-spectrum antiviral candidate.

Lassa virus (LASV) is an Old World arenavirus, endemic to West Africa, capable of causing hemorrhagic fever. Currently, there are no approved vaccines or effective antivirals for LASV. However, thorough understanding of the LASV glycoprotein and entry into host cells could accelerate therapeutic design. LASV entry is a two-step process involving the viral glycoprotein (GP). First, the GP subunit 1 (GP1) binds to the cell surface receptor and the viral particle is engulfed into an endosome. Next, the drop in pH triggers GP rearrangements, which ultimately leads to the GP subunit 2 (GP2) forming a six-helix-bundle (6HB). The process of GP2 forming 6HB fuses the lysosomal membrane with the LASV envelope, allowing the LASV genome to enter the host cell. The aim of this study was to identify residues in GP2 that are crucial for LASV entry. To achieve this, we performed alanine scanning mutagenesis on GP2 residues. We tested these mutant GPs for efficient GP1-GP2 cleavage, cell-to-cell membrane fusion, and transduction into cells expressing α-dystroglycan and secondary LASV receptors. In total, we identified seven GP2 mutants that were cleaved efficiently but were unable to effectively transduce cells: GP-L280A, GP-L285A/I286A, GP-I323A, GP-L394A, GP-I403A, GP-L415A, and GP-R422A. Therefore, the data suggest these residues are critical for GP2 function in LASV entry.

Summary To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool. [ABSTRACT FROM AUTHOR]

Orthomyxoviruses are an important family of RNA viruses, which include the various influenza viruses. Despite global efforts to eradicate orthomyxoviral pathogens, these infections remain pervasive. One such orthomyxovirus, infectious salmon anemia virus (ISAV), spreads easily throughout farmed and wild salmonids, constituting a significant economic burden. ISAV entry requires the interplay of the virion-attached hemagglutinin-esterase and fusion glycoproteins. Preventing infections will rely on improved understanding of ISAV entry. Here, we present the crystal structures of ISAV hemagglutinin-esterase unbound and complexed with receptor. Several distinctive features observed in ISAV HE are not seen in any other viral glycoprotein. The structures reveal a unique mode of receptor binding that is dependent on the oligomeric assembly of hemagglutinin-esterase. Importantly, ISAV hemagglutinin-esterase receptor engagement does not initiate conformational rearrangements, suggesting a distinct viral entry mechanism. This work improves our understanding of ISAV pathogenesis and expands our knowledge on the overall diversity of viral glycoprotein-mediated entry mechanisms. Finally, it provides an atomic-resolution model of the primary neutralizing antigen critical for vaccine development. [ABSTRACT FROM AUTHOR]

Fusion of cellular membranes during normal biological processes, including proliferation, or synaptic transmission, is mediated and controlled by sophisticated protein machinery ensuring the preservation of the vital barrier function of the membrane throughout the process. Fusion of virus particles with host cell membranes is more sparingly arranged and often mediated by a single fusion protein, and the virus can afford to be less discriminative towards the possible different outcomes of fusion attempts. Formation of leaky intermediates was recently observed in some fusion processes, and an alternative trajectory of the process involving formation of π-shaped structures was suggested. In this study, we apply the methods of elasticity theory and Lagrangian formalism augmented by phenomenological and molecular geometry constraints and boundary conditions to investigate the traits of this trajectory and the drivers behind the choice of one of the possible scenarios depending on the properties of the system. The alternative pathway proved to be a dead end, and, depending on the parameters of the participating membranes and fusion proteins, the system can either reversibly enter the corresponding “leaky” configuration or be trapped in it. A parametric study in the biologically relevant range of variables emphasized the fusion protein properties crucial for the choice of the fusion scenario.

Avian influenza virus (AIV) causes acute infectious diseases in poultry, critically impacting food supply. Highly pathogenic avian influenza viruses (HPAIVs), in particular, cause morbidity and mortality, resulting in significant economic losses in the poultry industry. To prevent the spread of HPAIVs, detection at early stages is critical to implement effective countermeasures such as quarantine and isolation. Through a viral fusion mechanism, cell-mimetic nanoparticles (CMPs), developed in the current study, can rapidly detect HPAIV and low pathogenic AIV (LPAIV). The CMPs comprise polymeric nanoparticles, which are constructed using sialic acid and fluorescence resonance energy transfer (FRET) dye pairs that expose the FRET off signal in response to LPAIV and HPAIV, after activation by enzymatic cleavage in the endosomal environment. The CMPs detect a wide variety of LPAIVs and HPAIVs in biological environments. Additionally, the cross-reactivity of CMPs is determined by testing their function with different viral species. Therefore, these findings demonstrate the significant potential of the proposed strategy for mimicking viral infection in vitro and using them as a highly effective diagnostic assay to rapidly detect LPAIV and HPAIV, preventing economic losses associated with viral outbreaks. [ABSTRACT FROM AUTHOR]

Rabies virus (RABV) causes lethal encephalitis and is responsible for approximately 60,000 deaths per year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates host-cell entry. RABV-G's pre-fusion trimeric conformation displays epitopes bound by protective neutralizing antibodies that can be induced by vaccination or passively administered for post-exposure prophylaxis. We report a 2.8-Å structure of a RABV-G trimer in the pre-fusion conformation, in complex with two neutralizing and protective monoclonal antibodies, 17C7 and 1112-1, that recognize distinct epitopes. One of these antibodies is a licensed prophylactic (17C7, Rabishield), which we show locks the protein in pre-fusion conformation. Targeted mutations can similarly stabilize RABV-G in the pre-fusion conformation, a key step toward structure-guided vaccine design. These data reveal the higher-order architecture of a key therapeutic target and the structural basis of neutralization by antibodies binding two key antigenic sites, and this will facilitate the development of improved vaccines and prophylactic antibodies. [Display omitted] • 2.8-Å cryo-EM structure of trimeric pre-fusion rabies virus glycoprotein • Structure reveals trimerization interface and binding of two neutralizing antibodies • Conformational transition can be inhibited by both antibodies and targeted mutation Ng, Fedosyuk et al. present the structure of a complex of pre-fusion trimeric rabies glycoprotein (RABV-G) with two neutralizing antibodies, one of which (17C7) is used clinically for post-exposure prophylaxis. They show that 17C7 inhibits pre- to post-fusion conformational transition, and they identify amino acid substitutions with a similar effect. [ABSTRACT FROM AUTHOR]

Background: HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Results: Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr—monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. Conclusions: The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Influenza virus, the causative agent of flu, enters the host cell by endocytosis. The low pH encountered inside endosomes triggers conformational changes in the viral glycoprotein hemagglutinin (HA), that mediate fusion of the viral and cellular membranes. This releases the viral genome into the cytoplasm of the infected cell, establishing the onset of the replication cycle. To investigate the structural basis of HA-mediated membrane fusion, a number of techniques have been employed. These include X-ray crystallography, which has provided atomic models of the HA ectodomain in its initial (pre-fusion) state and of part of HA in its final (post-fusion) state. However, this left an information deficit concerning many other aspects of the fusion process. Electron microscopy (EM) approaches are helping to fill this void. For example, influenza virions at neutral pH have been imaged by cryo-EM and cryo-electron tomography (cryo-ET); thin section EM has shown that influenza viruses enter the cell by endocytosis; the large-scale structural changes in HA when virions are exposed to low pH (pre-fusion to post-fusion states) have been visualized by negative staining and cryo-EM; acidification also induces structural changes in the M1 matrix layer and its separation from the viral envelope; intermediate HA conformations between its pre- and post-fusion states have been detected by cryo-ET supplemented with subtomogram averaging; and fusion of influenza virions with liposomes has been visualized by cryo-ET. In this review, we survey EM-based contributions towards the characterization of influenza virus-mediated membrane fusion and anticipate the potential for future developments. [ABSTRACT FROM AUTHOR]

HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ~-23 kBT, but folding of one hairpin successively accelerated the folding rate of the next one by ~20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a forcedependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention. [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]

Abstract: Chitosan and chitosan derived compounds are marine byproducts which have been shown to exhibit bioactivities including antibacterial, antioxidant, antidiabetic and anti-HIV. Proteins are among the most potent and selective molecules offering an endless potential of different structure and sequence preferences. The tripeptides which consist of tryptophan (W), methionine (M) and glutamine (Q) have been conjugated with chitosan oligomers. Among them QMW-COS and WMQ-COS protected C8166 cells from cell-lytic effects of HIV-1RF strain. Furthermore, these two compounds inhibited HIV-induced syncytia formation. We confirmed the decrease in viral-load in cell culture using p24 ELISA assay. To determine the specific mode of action of QMW-COS and WMQ-COS to inhibit HIV, we checked the inhibitory action of these compounds on viral reverse transcriptase and protease enzymes. However, we could not detect any inhibitory activity of conjugated compounds on recombinant reverse transcriptase and protease enzyme in vitro. When we co-cultured HIV-infected and uninfected C8166 cells, these two compounds actively inhibited the syncytia formation upon co-culture assay. Time-dependent addition of compounds and finally gp120-CD4 ELISA assay revealed that QMW-COS and WMQ-COS are bioactive compounds inhibiting HIV-induced cytopathic effects via exerting their effects on HIV entry stage. [Copyright &y& Elsevier]

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]

Abstract: C8, a short peptide characterized by three regularly spaced Trp residues, belongs to the membrane-proximal external functional domains of the feline immunodeficiency virus coat protein gp36. It elicits antiviral activity as a result of blocking cell entry and exhibits membranotropic and fusogenic activities. Membrane-proximal external functional domains of virus coat proteins are potential targets in the development of new anti-HIV drugs that overcome the limitations of the current anti-retroviral therapy. In the present work, we studied the conformation of C8 and its interaction with micellar surfaces using circular dichroism, nuclear magnetic resonance and fluorescence spectroscopy. The experimental data were integrated by molecular dynamics simulations in a micelle–water system. Our data provide insight into the environmental conditions related to the presence of the fusogenic peptide C8 on zwitterionic or negatively charged membranes. The membrane charge modulates the conformational features of C8. A zwitterionic membrane surface induces C8 to assume canonical secondary structures, with hydrophobic interactions between the Trp residues and the phospholipid chains of the micelles. A negatively charged membrane surface favors disordered C8 conformations and unspecific superficial interactions, resulting in membrane destabilization. [Copyright &y& Elsevier]

Human immunodeficiency virus type 1 is the cause of a modern global pandemic associated with progressive acquired immune deficiency. The infection is characterized by the loss of the primary target of viral infection, the CD4+ T cell. The measurement of plasma viremia in patients can predict the rate of CD4+ cell decline; however, it is not clear whether this cell-free plasma virus represents the engine that drives viral spread. Active viral replication is mainly observed within lymphoid tissues that are hotbeds of cell-cell interactions that initiate and organize immune responses. It is well established that cell-cell interactions enhance viral spread in vitro. Dendritic cell-T cell interactions, which lie at the heart of adaptive immune responses, enhance viral infection in vitro. Interactions between infected and uninfected CD4+ T cells are a dominant route of viral spread in vitro and are likely to play a central role in viral dissemination in vivo. Future studies will test existing paradigms of HIV-1 dissemination to determine whether virus-transmitting contacts between infected and uninfected T cells called virological synapses are the dominant mode of viral spread in vivo. Here, we review the status of our understanding of this mode of infection with a focus on T cell-T cell interactions and examine how it may explain resistance to neutralizing antibodies and or the generation of genetic diversity of HIV. [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: Lipid membranes play a key role in the viral life cycle. Enveloped viruses particularly require a sequence of fusion and fission events between the viral envelope and the target membranes for entry into the cell and egress from it. These processes are controlled by one or more viral glycoproteins that undergo conformational changes favoring the necessary micro- and mesoscopic lipid re-arrangements. Multiple regions from these glycoproteins are thought to interact with the membranes, according to a concerted mechanism, in order to generate the distortion necessary for fusion. In this work, we perform an EPR study on the role played by the membrane composition in tuning the interaction between lipid bilayers and two peptides, gH626–644 and gB632–650, that are highly fusogenic fragments of the gH and gB glycoproteins of herpes simplex virus. Our results show that both peptides interact with lipid bilayers, perturbing the local lipid packing. gH626–644 localizes close to the hydrophilic bilayer surface, while gB632–650 penetrates deeply into the membrane. Chain perturbation by the peptides increases in the presence of charged phospholipids. Finally, cholesterol does not alter the ability of gB632–650 to penetrate deeply in the membrane, whereas it limits penetration of the gH626–644 peptide to the more external layer. The different modes of interaction result in a higher fusogenic ability of gB632–650 towards cholesterol-enriched membranes, as demonstrated by lipid mixing assays. These results suggest that the mechanism of action of the gH and gB glycoproteins is modulated by the properties and composition of the phospholipid bilayer. [Copyright &y& Elsevier]

Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we presentthe high resolution crystal structure of TMC3531 21, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation. [ABSTRACT FROM AUTHOR]

Abstract: We present a universal mimetic approach of the prehairpin intermediate of gp41, which represents the active drug target for fusion inhibitors of HIV (human immunodeficiency virus) and SIV (simian immunodeficiency virus) based on membrane anchored lipopeptides. For this purpose, we have in situ coupled terminal cysteine-modified peptides originating from the NHR of SIV and HIV to a maleimide-functionalized DOPC bilayer and monitored the interactions with potential antagonists of the trimer-of-hairpin conformation C34 and T20 peptides by means of atomic force microscopy and ellipsometry. FT-IR analysis in conjugation with CD-spectroscopy of hydrated N36-lipopeptides, incorporated in multilamellar bilayer stacks was employed to investigate peptide conformation prior to antagonist binding. In contrast to solution studies substantial secondary structure formation of S-N36 after in situ coupling to the bilayer was found. We could show that S-N36-lipopeptide-aggregates in bilayers were selectively able to bind T20 or the corresponding C-peptides (C34) and similar results could be achieved by using H-N36 lipopeptides. It was found that T20 binding to coiled coil S-N36 lipopeptide assemblies was fully reversible at elevated temperatures, while T20 binds irreversibly to H-N36 bundles. [Copyright &y& Elsevier]

The recent development of functional models to analyze the early steps of the hepatitis C virus (HCV) life cycle has highlighted that HCV entry is a slow and complex multistep process involving the presence of several entry factors. Initial host cell attachment may involve glycosaminoglycans and the low-density lipoprotein receptor, after which the particle appears to interact sequentially with three entry factors: the scavenger receptor class B type I, the tetraspanin CD81 and the tight-junction protein claudin-1. Several serum components may also modulate HCV entry, while the recently discovered CD81 partner EWI-2wint can block the interaction of the viral particle with CD81, potentially preventing infection in the cell types in which it is expressed. After binding to the host cell, the HCV particle is internalized by clathrin-mediated endocytosis, with fusion likely occuring in early endosomes. This review summarizes our current knowledge on HCV entry. [ABSTRACT FROM AUTHOR]