Viral infection requires stable binding of viral fusion proteins to host membranes, which contain hundreds of lipid species. The mechanisms by which fusion proteins utilize specific host lipids to drive virus-host membrane fusion remains elusive. We conducted molecular simulations of classes I, II, and III fusion proteins interacting with membranes of diverse lipid compositions. Free energy calculations reveal that class I fusion proteins generally exhibit stronger membrane binding compared to classes II and III - a trend consistent across 74 fusion proteins from 13 viral families as suggested by sequence analysis. Class II fusion proteins utilize a lipid binding pocket formed by fusion protein monomers, stabilizing the initial binding of monomers to the host membrane prior to assembling into fusogenic trimers. In contrast, class III fusion proteins form a lipid binding pocket at the monomer-monomer interface through a unique fusion loop crossover. The distinct lipid binding modes correlate with the differing maturation pathways of classes II and III proteins. Binding affinity was predominantly controlled by cholesterol and gangliosides as well as via local enrichment of polyunsaturated lipids, thereby locally enhancing membrane disorder. Our study reveals energetics and atomic details underlying lipid recognition and reorganization by different viral fusion protein classes, offering insights into their specialized membrane fusion pathways.

Epstein-Barr Virus (EBV) as the first characterized tumorigenic virus in humans causes heavy disease burdens. An effective vaccine is urgently needed to block EBV infection. Glycoprotein B (gB) is the essential fusogen for EBV infection of all susceptible cell types. We previously demonstrated that neutralizing antibody 3A3 targeting gB effectively blocked EBV infection in a humanized mouse model, indicating that the epitope recognized by 3A3 is the potential immunogen candidate. Hence, we rationally designed a chimeric virus-like particle (cVLP) vaccine based on the hepatitis B core antigen (HBc149) to display gB peptide, αB, recognized by 3A3 (149-αB cVLP). The engineered 149-αB cVLP vaccine self-assembled into spherical particles presenting multiple copies of αB peptide. The 149-αB cVLP vaccine induced much higher antibody titers against αB peptides than gB protein immunization. Importantly, sera antibodies elicited by the 149-αB cVLP vaccine more efficiently blocked EBV infection and membrane fusion of epithelial cells and B cells. Sera from 149-αB cVLP vaccine-immunized rabbits conferred 100% protection against EBV infection in a humanized mouse model. We demonstrated that the 149-αB cVLP vaccine induced potent antigen-specific protective immune responses and shed light on the research of peptide-based vaccines against EBV infection.

The structural integrity of viral envelopes is a critical determinant of infectivity for enveloped viruses, directly influencing vector stability, functional accuracy of surface-displayed epitopes, and preservation of native conformational states required for membrane protein studies. However, conventional purification methods often disrupt envelope integrity and cause envelope proteins to lose their activity. Here, we systematically compared discontinuous, continuous, and optimized continuous sucrose density gradient centrifugation protocols for purifying Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Through cryo-EM, we demonstrated that our optimized continuous sucrose gradient protocol significantly increased the proportion of AcMNPV budded virions with intact envelopes from 36% to 81%, while preserving the metastable prefusion conformation of the fusion protein GP64. This advancement should prove useful for structural studies of viral envelope proteins and may enhance applications in gene therapy and vaccine development utilizing enveloped viruses.

The tick-borne apicomplexan parasite Babesia bovis causes bovine babesiosis which leads to enormous food and economic losses around the world. The existing resources to manage this disease are limited and have pitfalls, therefore, introduction of new strategies is urgently needed. B. bovis reproduces sexually in the midgut of its tick vector. HAP2, a well conserved ancient protein, plays a crucial role in the gamete fusion of this parasite and is a strong candidate for developing transmission-blocking vaccines. We previously demonstrated that immunization of cattle with full size B. bovis HAP2 blocks transmission of the parasite by Rhipicephalus microplus. Understanding the conserved structural features and antigenicity of HAP2 protein and its domains will facilitate developing effective methods to control pathogen transmission. In this study, we analyzed and compared AlphaFold2-predicted 3D structure of B. bovis HAP2 with the well-characterized crystal structures of HAP2 of Chlamydomonas reinhardtii and Arabidopsis thaliana. The comparisons and structural analysis resulted in the definition of three domains' sequences, fusion loops, and disulfide bonds in the B. bovis HAP2. In addition, recombinant versions of each three predicted HAP2 domains were recognized by antibodies from HAP2 immunized and transmission-protected cattle, confirming their antigenicity. Remarkably, domain II was highly recognized compared to the other two domains. This study introduces new directions in designing novel functional assays and improved vaccine design through targeting the HAP2 protein.

SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM has revealed stable prefusion and postfusion conformations of the spike, the transient intermediate states during the fusion process have remained poorly understood. Here, we designed a near-native viral fusion system that recapitulates SARS-CoV-2 entry and used cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that is dependent on protease cleavage and inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2' cleavage concurrently transition to postfusion conformations encircling the hemifusion and pre-fusion pores in a distinct conical arrangement. Subtomogram averaging revealed that the WS6 S2 antibody binds to the spike's stem-helix, crosslinks and clusters prefusion spikes and inhibits refolding of fusion intermediates. These findings elucidate the complete process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.

In 2021, at the height of the COVID-19 pandemic, coronavirus research spiked, with over 83,000 original research articles related to the word "coronavirus" added to the online resource PubMed. Just 2 years later, in 2023, only 30,900 original research articles related to the word "coronavirus" were added. While, irrefutably, the funding of coronavirus research drastically decreased, a possible explanation for the decrease in interest in coronavirus research is that projects on SARS-CoV-2, the causative agent of COVID-19, halted due to the challenge of establishing a good cellular or animal model system. Most laboratories do not have the capabilities to culture SARS-CoV-2 'in house' as this requires a Biosafety Level (BSL) 3 laboratory. Until recently, BSL 2 laboratory research on endemic coronaviruses was arduous due to the low cytopathic effect in isolated cell culture infection models and the lack of means to quantify viral loads. The purpose of this review article is to compare the human coronaviruses and provide an assessment of the latest techniques that use the endemic coronaviruses-HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1-as lower-biosafety-risk models for the more pathogenic coronaviruses-SARS-CoV-2, SARS-CoV, and MERS-CoV.

Flaviviruses include important human pathogens such as Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as some emerging viruses that affect millions of people worldwide. They fuse their membrane with the late endosomal one in a pH-dependent way and therefore the merging of the membranes is one of the main goals for obtaining new antivirals. The envelope E protein, a membrane fusion protein, is accountable for fusion and encompasses different domains involved in the fusion mechanism, including the fusion peptide segment. In this work we have used molecular dynamics to study the interaction of the distal end of domain II of the DENV envelope E protein with a membrane like the late endosomal membrane in order to observe the initiation of membrane fusion carried out by a number of trimers of the DENV envelope E protein interacting with a complex biomembrane and demonstrate its feasibility. Our results demonstrate the likelihood of membrane disorganization and pore formation by trimer complex organization, the amino acids responsible for such condition and the secondary structure arrangements needed for such fundamental process. At the same time, we define new targets of the envelope E protein sequence which could permit designing potent antiviral bioactive molecules.

Nipah virus (NiV) is a zoonotic paramyxovirus that has recently emerged as a crucial public health issue. It can elicit severe encephalitis and respiratory diseases in animals and humans, leading to fatal outcomes, exhibiting a wide range of host species tropism, and directly transmitting from animals to humans or through an intermediate host. Human-to-human transmission associated with recurrent NiV outbreaks is a potential global health threat. Currently, the lack of effective therapeutics or licensed vaccines for NiV necessitates the primary utilization of supportive care. In this review, we summarize current knowledge of the various aspects of the NiV, including therapeutics, vaccines, and its biological characteristics, epidemiology, pathogenesis, and clinical features. The objective is to provide valuable information from scientific and clinical research and facilitate the formulation of strategies for preventing and controlling the NiV.

Methicillin-Resistant Staphylococcus aureus forming into biofilms can trigger chronic inflammation and disrupt skin wound healing processes. Prolonged and excessive use of antibiotics can expedite the development of resistance, primarily because of their limited ability to penetrate microbial membranes and biofilms, especially antibiotics with intracellular drug targets. Herein, we devise a strategy in which virus-inspired nanoparticles control the release of antibiotics through rapid penetration into both bacterial cells and biofilms, thereby combating antimicrobial-resistant infections and promoting skin wound healing. Lipid-based nanoparticles based on stearamine and cholesterol were designed to mimic viral highly ordered nanostructures. To mimic the arginine-rich fragments in viral protein transduction domains, the primary amines on the surface of the lipid-based nanoparticles were exchanged by guanidine segments. Levofloxacin, an antibiotic that inhibits DNA replication, was chosen as the model drug to be incorporated into nanoparticles. Hyaluronic acid was coated on the surface of nanoparticles acting as a capping agent to achieve bacterial-specific degradation and guanidine explosion in the bacterial microenvironment. Our virus-inspired nanoparticles displayed long-acting antibacterial effects and powerful biofilm elimination to overcome antimicrobial-resistant infections and promote skin wound healing. This work demonstrates the ability of virus-inspired nanoparticles to achieve a dual penetration of microbial cell membranes and biofilm structures to address antimicrobial-resistant infections and trigger skin wound healing.

Guanylate-binding protein (GBP) 5 is an interferon-inducible cellular factor with broad anti-viral activity. Recently, GBP5 has been shown to antagonize the glycoproteins of a number of enveloped viruses, in part by disrupting the host enzyme furin. Here we show that GBP5 strongly impairs the infectivity of virus particles bearing not only viral glycoproteins that depend on furin cleavage for infectivity-the envelope (Env) glycoproteins of HIV-1 and murine leukemia virus and the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-but also viral glycoproteins that do not depend on furin cleavage: vesicular stomatitis virus glycoprotein and SARS-CoV S. We observe that GBP5 disrupts proper N-linked protein glycosylation and reduces the incorporation of viral glycoproteins into virus particles. The glycosylation of the cellular protein CD4 is also altered by GBP5 expression. Flow cytometry analysis shows that GBP5 expression reduces the cell-surface levels of HIV-1 Env and the S glycoproteins of SARS-CoV and SARS-CoV-2. Our data demonstrate that, under the experimental conditions used, inhibition of furin-mediated glycoprotein cleavage is not the primary anti-viral mechanism of action of GBP5. Rather, the antagonism appears to be related to impaired trafficking of glycoproteins to the plasma membrane. These results provide novel insights into the broad antagonism of viral glycoprotein function by the cellular host innate immune response.

The surface of enveloped viruses contains viral envelope glycoproteins, an important structural component facilitating virus attachment and entry while also acting as targets for the host adaptive immune system. In this study, we show that expression of GBP5 in virus-producer cells alters the glycosylation, cell-surface expression, and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into the broad impact of the host cell anti-viral factor GBP5 on protein glycosylation and trafficking.

Viral glycoproteins drive membrane fusion in enveloped viruses and determine host range, tissue tropism and pathogenesis1. Despite their importance, there is a fragmentary understanding of glycoproteins within the Flaviviridae2, a large virus family that include pathogens such as hepatitis C, dengue and Zika viruses, and numerous other human, animal and emergent viruses. For many flaviviruses the glycoproteins have not yet been identified, for others, such as the hepaciviruses, the molecular mechanisms of membrane fusion remain uncharacterized3. Here we combine phylogenetic analyses with protein structure prediction to survey glycoproteins across the entire Flaviviridae. We find class II fusion systems, homologous to the Orthoflavivirus E glycoprotein in most species, including highly divergent jingmenviruses and large genome flaviviruses. However, the E1E2 glycoproteins of the hepaciviruses, pegiviruses and pestiviruses are structurally distinct, may represent a novel class of fusion mechanism, and are strictly associated with infection of vertebrate hosts. By mapping glycoprotein distribution onto the underlying phylogeny, we reveal a complex evolutionary history marked by the capture of bacterial genes and potentially inter-genus recombination. These insights, made possible through protein structure prediction, refine our understanding of viral fusion mechanisms and reveal the events that have shaped the diverse virology and ecology of the Flaviviridae.

The COVID-19 pandemic highlighted the need for antivirals against emerging coronaviruses (CoV). Inhibiting spike (S) glycoprotein-mediated viral entry is a promising strategy. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses. We employed a three-step process to screen nearly 200,000 small molecules. First, we identified hits that inhibit pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Counter-screening against pseudoviruses with the vesicular stomatitis virus glycoprotein (VSV-G), yielded sixty-five SARS-CoV-2 S-specific inhibitors. These were further tested against pseudoviruses bearing the MERS-CoV S glycoprotein, which uses a different receptor. Out of these, five compounds, which included the known broad-spectrum inhibitor Nafamostat, were subjected to further validation and tested against pseudoviruses bearing the S glycoprotein of the Alpha, Delta, and Omicron variants as well as bona fide SARS-CoV-2. This rigorous approach revealed an unreported inhibitor and its derivative as potential broad-spectrum antivirals.

Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332, but Asn at this position is not absolutely conserved or required for HIV-1 entry based on the prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes, with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity with high levels of cell surface expression and slightly higher gp120 shedding than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs were in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states.

Glycan attached to amino acid asparagine at position 332 of HIV-1 envelope glycoproteins is a main target of a subset of broadly neutralizing antibodies that block HIV-1 infection. Here, we defined the contribution of different amino acids at this position to Env antigenicity, stability on ice, and conformational states.

Neutralizing antibodies (NAbs) are naturally produced by our immune system to combat viral infections. Clinically, neutralizing antibodies with potent efficacy and high specificity have been extensively used to prevent and treat a wide variety of viral infections, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Immunodeficiency Virus (HIV), Dengue Virus (DENV) and Hepatitis B Virus (HBV). An overwhelmingly large subset of clinically effective NAbs operates by targeting viral envelope proteins to inhibit viral entry into the host cell. Binding of viral envelope protein to the host receptor is a critical rate limiting step triggering a cascade of downstream events, including endocytosis, membrane fusion and pore formation to allow viral entry. In recent years, improved structural knowledge on these processes have allowed researchers to also leverage NAbs as an indispensable tool in guiding discovery of novel antiviral entry inhibitors, providing drug candidates with high efficacy and pan-genus specificity. This review will summarize the latest progresses on the applications of NAbs as effective entry inhibitors and as important tools to develop antiviral therapeutics by high-throughput drug screenings, rational design of peptidic entry inhibitor mimicking NAbs and in silico computational modeling approaches.

Banna virus (BAV) is the prototype Seadornavirus, a class of reoviruses for which there has been little structural study. Here, we report atomic cryo-EM structures of three states of BAV virions-surrounded by 120 spikes (full virions), 60 spikes (partial virions), or no spikes (cores). BAV cores are double-layered particles similar to the cores of other non-turreted reoviruses, except for an additional protein component in the outer capsid shell, VP10. VP10 was identified to be a cementing protein that plays a pivotal role in the assembly of BAV virions by directly interacting with VP2 (inner capsid), VP8 (outer capsid), and VP4 (spike). Viral spikes (VP4/VP9 heterohexamers) are situated on top of VP10 molecules in full or partial virions. Asymmetrical electrostatic interactions between VP10 monomers and VP4 trimers are disrupted by high pH treatment, which is thus a simple way to produce BAV cores. Low pH treatment of BAV virions removes only the flexible receptor binding protein VP9 and triggers significant conformational changes in the membrane penetration protein VP4. BAV virions adopt distinct spatial organization of their surface proteins compared with other well-studied reoviruses, suggesting that BAV may have a unique mechanism of penetration of cellular endomembranes.

Rift Valley fever is a zoonotic viral disease transmitted by mosquitoes, impacting both humans and livestock. Currently, there are no approved vaccines or antiviral treatments for humans. This study aimed to evaluate the in vitro efficacy of chemical compounds targeting the Gc fusion mechanism. These compounds were identified through virtual screening of millions of commercially available small molecules using a structure-based artificial intelligence bioactivity predictor. In our experiments, a pretreatment with small molecule compounds revealed that 3 out of 94 selected compounds effectively inhibited the replication of the Rift Valley fever virus MP-12 strain in Vero cells. As anticipated, these compounds did not impede viral RNA replication when administered three hours after infection. However, significant inhibition of viral RNA replication occurred upon viral entry when cells were pretreated with these small molecules. Furthermore, these compounds exhibited significant inhibition against Arumowot virus, another phlebovirus, while showing no antiviral effects on tick-borne bandaviruses. Our study validates AI-based virtual high throughput screening as a rational approach for identifying effective antiviral candidates for Rift Valley fever virus and other bunyaviruses.

Viruses are intracellular parasites that hijack the cellular machinery for their own replication. Therefore, an obligatory step in the virus life cycle is the delivery of the viral genome inside the cell. Enveloped viruses (i.e., viruses with a lipid envelope) use a two-step procedure to release their genetic material into the cell: (1) they first bind to specific surface receptors of the target cell membrane and then (2) they fuse the viral and cell membranes. This last step may occur at the cell surface or after internalization of the virus particle by endocytosis or by some other route (e.g., macropinocytosis). Remarkably, the virus-cell membrane fusion process goes essentially along the same intermediate steps than other membrane fusions that occur, for instance, in vesicular fusion at the nerve synapsis or cell-cell fusion in yeast mating. Specialized viral proteins, fusogens, promote virus-cell membrane fusion. The viral fusogens experience drastic structural rearrangements during fusion, releasing the energy required to overcome the repulsive forces that prevent spontaneous fusion of the two membranes. This chapter provides an overview of the different types of viral fusogens and their mode of action, as they are currently known. Furthermore, it outlines novel strategies for vaccine development related to stabilized viral fusogens.

While the phenomenon of excipient mediated membrane fusion has been studied widely, the inherent role of interfacial hydration involved in the process has mostly remained unaddressed. Here we report the experimental validation of the fact that PEG-induced membrane fusion is associated with the dehydration of the membrane(s). We explore the explicit hydration behavior at three different lipids (DOPC, POPC and DPPC) membranes with different aliphatic tails as they undergo fusogenic transition in the presence of PEG of average molecular weight of 4000 using THz-FTIR spectroscopy in the frequency window of 1.5-13.5 THz. Dynamic light scattering and electron microscopic measurements confirm the formation of different intermediate steps of the liposomes during the fusion process: bilayer aggregation, destabilization and finally lipid fusion. We observe that membrane hydration follows a systematic trend with the lipid specificity as the fusion process sets in.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332 but Asn at this position is not absolutely conserved or required for HIV-1 entry based on prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity even though cell surface expression levels are lower than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and to evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs was in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states.

Host cell entry of vaccinia virus (a poxvirus) proceeds through multiple steps that involve many viral proteins to mediate cell infection. Upon binding to cells, vaccinia virus membrane fuses with host membranes via a viral entry fusion protein complex comprising 11 proteins: A16, A21, A28, F9, G3, G9, H2, J5, L1, L5 and O3. Despite vaccinia virus having two infectious forms, mature and enveloped, that have different membrane layers, both forms require an identical viral entry fusion complex for membrane fusion. Components of the poxvirus entry fusion complex that have been structurally assessed to date share no known homology with all other type I, II and III viral fusion proteins, and the large number of fusion protein components renders it a unique system to investigate poxvirus-mediated membrane fusion. Here, we determined the NMR structure of a truncated version of vaccinia A28 protein. We also expressed a soluble H2 protein and showed that A28 interacts with H2 protein at a 1:1 ratio in vitro. Furthermore, we performed extensive in vitro alanine mutagenesis to identify A28 protein residues that are critical for H2 binding, entry fusion complex formation, and virus-mediated membrane fusion. Finally, we used molecular dynamic simulations to model full-length A28-H2 subcomplex in membranes. In summary, we characterized vaccinia virus A28 protein and determined residues important in its interaction with H2 protein and membrane components. We also provide a structural model of the A28-H2 protein interaction to illustrate how it forms a 1:1 subcomplex on a modeled membrane.

Alphaviruses threaten public health continuously, and Getah virus (GETV) is a re-emerging alphavirus that can potentially infect humans. Approved antiviral drugs and vaccines against alphaviruses are few available, but several host antiviral factors have been reported. Here, we used GETV as a model of alphaviruses to screen for additional host factors. Tetrachlorodibenzo-p-dioxin-inducible poly(ADP ribose) polymerase was identified to inhibit GETV replication by inducing ubiquitination of the glycoprotein E2, causing its degradation by recruiting the E3 ubiquitin ligase membrane-associated RING-CH8 (MARCH8). Using GETV as a model virus, focusing on the relationship between viral structural proteins and host factors to screen antiviral host factors provides new insights for antiviral studies on alphaviruses.

Hantaviruses are a significant and emerging global public health threat, impacting more than 200,000 individuals worldwide each year. The single-stranded RNA viruses belong to the Hantaviridae family and are responsible for causing two acute febrile diseases in humans: Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Currently, there are no licensed treatments or vaccines available globally for HTNV infection. Various candidate drugs have shown efficacy in increasing survival rates during the early stages of HTNV infection. Some of these drugs include lactoferrin, ribavirin, ETAR, favipiravir and vandetanib. Immunotherapy utilizing neutralizing antibodies (NAbs) generated from Hantavirus convalescent patients show efficacy against HTNV. Monoclonal antibodies such as MIB22 and JL16 have demonstrated effectiveness in protecting against HTNV infection. The development of vaccines and antivirals, used independently and/or in combination, is critical for elucidating hantaviral infections and the impact on public health. RNA interference (RNAi) arised as an emerging antiviral therapy, is a highly specific degrades RNA, with post-transcriptional mechanism using eukaryotic cells platform. That has demonstrated efficacy against a wide range of viruses, both in vitro and in vivo. Recent antiviral methods involve using small interfering RNA (siRNA) and other, immune-based therapies to target specific gene segments (S, M, or L) of the Hantavirus. This therapeutic approach enhances viral RNA clearance through the RNA interference process in Vero E6 cells or human lung microvascular endothelial cells. However, the use of siRNAs faces challenges due to their low biological stability and limited in vivo targeting ability. Despite their successful inhibition of Hantavirus replication in host cells, their antiviral efficacy may be hindered. In the current review, we focus on advances in therapeutic strategies, as antiviral medications, immune-based therapies and vaccine candidates aimed at enhancing the body's ability to control the progression of Hantavirus infections, with the potential to reduce the risk of severe disease.

SARS-CoV-2 infection induces non-physiological syncytia when its spike fusogenic protein on the surface of the host cells interacts with the ACE2 receptor on adjacent cells. Spike-induced syncytia are beneficial for virus replication, transmission, and immune evasion, and contribute to the progression of COVID-19. In this review, we highlight the properties of viral fusion proteins, mainly the SARS-CoV-2 spike, and the involvement of the host factors in the fusion process. We also highlight the possible use of anti-fusogenic factors as an antiviral for the development of therapeutics against newly emerging SARS-CoV-2 variants and how the fusogenic property of the spike could be exploited for biomedical applications.

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.

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.

Chikungunya virus (CHIKV) is an infectious agent spread by mosquitos, that has engendered endemic or epidemic outbreaks of Chikungunya fever (CHIKF) in Africa, South-East Asia, America, and a few European countries. Like most tropical infections, CHIKV is frequently misdiagnosed, underreported, and underestimated; it primarily affects areas with limited resources, like developing nations. Due to its high transmission rate and lack of a preventive vaccine or effective treatments, this virus poses a serious threat to humanity. After a 32-year hiatus, CHIKV reemerged as the most significant epidemic ever reported, in India in 2006. Since then, CHIKV-related research was begun in India, and up to now, more than 800 peer-reviewed research papers have been published by Indian researchers and medical practitioners. This review gives an overview of the outbreak history and CHIKV-related research in India, to favor novel high-quality research works intending to promote effective treatment and preventive strategies, including vaccine development, against CHIKV infection.

In many applications such as diagnostics and therapy development, small peptide fragments consisting of only a few amino acids are often attractive alternatives to bulky proteins. This is due to factors such as the ease of scalable chemical synthesis and numerous methods for their discovery. One drawback of using peptides is that their activity can often be negatively impacted by the lack of a rigid, 3D stabilizing structure provided by the rest of the protein. In many cases, this can be alleviated by different methods of rational templating onto nanomaterials, which provides additional possibilities to use concepts of multivalence or rational nano-engineering to enhance or even create new types of function or structure. In recent years, nanostructures made from the self-assembly of DNA strands have been used as scaffolds to create functional arrangements of peptides, often leading to greatly enhanced biological activity or new material properties. This review will give an overview of nano-templating approaches based on the combination of DNA nanotechnology and peptides. This will include both bioengineering strategies to control interactions with cells or other biological systems, as well as examples where the combination of DNA and peptides has been leveraged for the rational design of new functional materials.

The fusion of biological membranes is ubiquitous in natural processes like exo- and endocytosis, intracellular trafficking and viral entry. Membrane fusion is also utilized in artificial biomimetic fusion systems, e.g. for drug delivery. Both the natural and the biomimetic fusion systems rely on a wide range of (artificial) proteins mediating the fusion process. Although the exact mechanisms of these proteins differ, clear analogies in their general behavior can be observed in bringing the membranes in close proximity and mediating the fusion reaction. In our study, we use molecular dynamics simulations with coarse grained models, mimicking the general behavior of fusion proteins (spikes), to systematically examine the effects of specific characteristics of these proteins on the fusion process. The protein characteristics considered are (i) the type of membrane embedding, i.e., either transmembrane or not, (ii) the rigidity, and (iii) the transmembrane domain (TMD) length. The results show essential differences in fusion pathway between monotopic and transmembrane spikes, in which transmembrane spikes seem to inhibit the formation of hemifusion diaphragms, leading to a faster fusion development. Furthermore, we observed that an increased rigidity and a decreased TMD length both proved to contribute to a faster fusion development. Finally, we show that a single spike may suffice to successfully induce a fusion reaction, provided that the spike is sufficiently rigid and attractive. Not only does this shed light on biological fusion of membranes, it also provides clear design rules for artificial membrane fusion systems.

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.

Flaviviruses are enveloped viruses causing high public concerns. Their maturation spans several cellular compartments having different pH. Thus, complex control mechanisms are in place to avoid premature maturation. Here we report the dynamical behavior at neutral and acidic pH of the precursor of the membrane fusion protein E of tick-borne encephalitis, showing the different stabilizations of the E dimer and the role played by the small fusion-assisting protomer (pr). The comprehension, at atomic resolution, of the fine regulation of viral maturation will be fundamental to the development of efficient strategies against emerging viral threats.

As obligate pathogens, viruses have developed diverse mechanisms to deliver their genome across host cell membranes to sites of virus replication. While enveloped viruses utilize viral fusion proteins to accomplish fusion of their envelope with the cellular membrane, non-enveloped viruses rely on machinery that causes local membrane ruptures and creates an opening through which the capsid or viral genome is released. Both membrane fusion and membrane penetration take place at the plasma membrane or in intracellular compartments, often involving the engagement of the cellular machinery and antagonism of host restriction factors. Enveloped and non-enveloped viruses have evolved intricate mechanisms to enable virus uncoating and modulation of membrane fusion in a spatiotemporally controlled manner. This chapter summarizes and discusses the current state of understanding of the mechanisms of viral membrane fusion and penetration. The focus is on the role of lipids, viral scaffold uncoating, viral membrane fusion inhibitors, and host restriction factors as physicochemical modulators. In addition, recent advances in visualizing and detecting viral membrane fusion and penetration using cryo-electron microscopy methods are presented.

Infections caused by enveloped viruses require fusion with cellular membranes for viral genome entry. Viral entry occurs following an interaction of viral and cellular membranes allowing the formation of fusion pores, by which the virus accesses the cytoplasm. Here, we focus on interferon-induced transmembrane protein 3 (IFITM3) and its antiviral activity. IFITM3 is predicted to block or stall viral fusion at an intermediate state, causing viral propagation to fail. After introducing IFITM3, we describe the generalized lipid membrane fusion pathway and how it can be stalled, particularly with respect to IFITM3, and current questions regarding IFITM3's topology, with specific emphasis on IFITM3's amphipathic α-helix (AAH) ₅₉V-₆₈M, which is necessary for the antiviral activity. We report new hydrophobicity and hydrophobic moment calculations for this peptide and a variety of active site peptides from known membrane-remodeling proteins. Finally, we discuss the effects of posttranslational modifications and localization, how IFITM3's AAH may block viral fusion, and possible ramifications of membrane composition.

In sexually reproducing organisms, the genetic information is transmitted from one generation to the next via the merger of male and female gametes. Gamete fusion is a two-step process involving membrane recognition and apposition through ligand-receptor interactions and lipid mixing mediated by fusion proteins. HAP2 (also known as GCS1) is a bona fide gamete fusogen in flowering plants and protists. In vertebrates, a multitude of surface proteins have been demonstrated to be pivotal for sperm-egg fusion, yet none of them exhibit typical fusogenic features. In this Cell Science at a Glance article and the accompanying poster, we summarize recent advances in the mechanistic understanding of gamete fusion in eukaryotes, with a particular focus on mammalian species.

Sexual reproduction involves the fusion of two gametes of opposite sex. Although the sperm-expressed fusogen HAPLESS 2 (HAP2) or GENERATIVE CELL SPECIFIC 1 (GCS1) plays a vital role in this process in many eukaryotic organisms and an understanding of its regulation is emerging in unicellular systems [J. Zhang et al., Nat. Commun. 12, 4380 (2021); J. F. Pinello et al. Dev. Cell 56, 3380-3392.e9 (2021)], neither HAP2/GCS1 interactors nor mechanisms for delivery and activation at the fusion site are known in multicellular plants. Here, we show that Arabidopsis thaliana HAP2/GCS1 interacts with two sperm DUF679 membrane proteins (DMP8 and DMP9), which are required for the EGG CELL 1 (EC1)-induced translocation of HAP2/GCS1 from internal storage vesicle to the sperm plasma membrane to ensure successful fertilization. Our studies in Arabidopsis and tobacco provide evidence for a conserved function of DMP8/9-like proteins as HAP2/GCS1 partner in seed plants. Our data suggest that seed plants evolved a DMP8/9-dependent fusogen translocation process to achieve timely acquisition of sperm fusion competence in response to egg cell-derived signals, revealing a previously unknown critical step for successful fertilization.

Mammarenaviruses include many significant worldwide-widespread human pathogens, among them Lassa virus (LASV), having a dramatic morbidity and mortality rate. They are a potential high-risk menace to the worldwide public health since there are no treatments and there is a high possibility of animal-to-human and human-to-human viral transmission. These viruses enter into the cells by endocytosis fusing its membrane envelope with the late endosomal membrane thanks to the glycoprotein GP2, a membrane fusion protein of class I. This protein contains different domains, among them the N-terminal fusion peptide (NFP), the internal fusion loop (IFL), the membrane proximal external region (MPER) and the transmembrane domain (TMD). All these domains are implicated in the membrane fusion process. In this work, we have used an all-atom molecular dynamics study to know the binding of these protein domains with a complex membrane mimicking the late endosome one. We show that the NFP/IFL domain is capable of spontaneously inserting into the membrane without a significant change of secondary structure, the MPER domain locates at the bilayer interface with an orientation parallel to the membrane surface and tends to interact with other MPER domains, and the TMD domain tilts inside the bilayer. Moreover, they predominantly interact with negatively charged phospholipids. Overall, these membrane-interacting domains would characterise a target that would make possible to find effective antiviral molecules against LASV in particular and Mammarenaviruses in general.

The current view of nucleic acid-mediated innate immunity is that binding of intracellular sensors to nucleic acids is sufficient for their activation. Here, we report that endocytosis of virus or foreign DNA initiates a priming signal for the DNA sensor cyclic GMP-AMP synthase (cGAS)-mediated innate immune response. Mechanistically, viral infection or foreign DNA transfection triggers recruitment of the spleen tyrosine kinase (SYK) and cGAS to the endosomal vacuolar H⁺ pump (V-ATPase), where SYK is activated and then phosphorylates human cGAS^Y214/215 (mouse cGas^Y200/201) to prime its activation. Upon binding to DNA, the primed cGAS initiates robust cGAMP production and mediator of IRF3 activation/stimulator of interferon genes-dependent innate immune response. Consistently, blocking the V-ATPase-SYK axis impairs DNA virus- and transfected DNA-induced cGAMP production and expression of antiviral genes. Our findings reveal that V-ATPase-SYK-mediated tyrosine phosphorylation of cGAS following endocytosis of virus or other cargos serves as a priming signal for cGAS activation and innate immune response.

During sexual reproduction in the unicellular green alga Chlamydomonas, gametes undergo the conserved cellular events that define fertilization across the tree of life. After initial ciliary adhesion, plus and minus gametes attach to each other at plasma membrane sites specialized for fusion, their bilayers merge, and cell coalescence into a quadri-ciliated cell signals for nuclear fusion. Recent findings show that these conserved cellular events are driven by 3 conserved protein families, FUS1/GEX2, HAP2/GCS1, and KAR5/GEX1. New results also show that species-specific recognition in Chlamydomonas activates the ancestral, viral-like fusogen HAP2 to drive fusion; that the conserved nuclear envelope fusion protein KAR5/GEX1 is also essential for nuclear fusion in Arabidopsis; and that heterodimerization of BELL-KNOX proteins signals for nuclear fusion in Chlamydomonas through early diverging land plants. This review outlines how Chlamydomonas's Janus-like position in evolution along with the ease of working with its gametes have revealed broadly conserved mechanisms.

Liposomes represent important drug carrier vehicles in biological systems. A fusogenic liposomal system composed of equimolar mixtures of the cationic lipid DOTAP and the phospholipid DOPE showed high fusion and delivery efficiencies with cells and lipid vesicles. However, aspects of the thermodynamics involving the interaction of these fusogenic liposomes and biomimetic systems remain unclear. Here, we investigate the fusion of this system with large unilamellar vesicles (LUVs) composed of the zwitterionic lipid POPC and increasing fractions of the anionic lipid POPG and up to 30 mol % cholesterol. The focus here is to concomitantly follow changes in size, zeta-potential, and enthalpy binding upon membrane interaction and fusion. Isothermal titration calorimetry (ITC) data showed that membrane fusion in our system is an exothermic process in the absence of cholesterol, suggesting that electrostatic attraction is the driving force for fusion. An endothermic component appeared and eventually dominated the titration at 30 mol % cholesterol, which we propose is caused by membrane fluidification when cholesterol is diluted upon fusion. The inflection points of the ITC data occurred around 0.5-0.7 POPG/DOTAP for all systems, the same stoichiometry for which zeta-potential and dynamic light scattering measurements showed an increase in size coupled with charge neutralization of the system, which is consistent with the fact that fusion in our system is charge-mediated. Microscopy observations of the final mixtures revealed the presence of giant vesicles, which is a clear indication of fusion, coexisting with intermediate-sized objects that could be the result of both fusion and/or aggregation. The results show that the fusion efficiency of the DOTAP:DOPE fusogenic system is modulated by the charge and membrane packing of the acceptor membrane and explain why the system fuses very efficiently with cells.

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV's complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein's post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

COVID-19 is a new complex multisystem disease caused by the novel coronavirus SARS-CoV-2. In slightly over 2 years, it infected nearly 500 million and killed 6 million human beings worldwide, causing an unprecedented coronavirus pandemic. Currently, the international scientific community is engaged in elucidating the molecular mechanisms of the pathophysiology of SARS-CoV-2 infection as a basis of scientific developments for the future control of COVID-19. Global exome and genome analysis efforts work to define the human genetics of protective immunity to SARS-CoV-2 infection. Here, we review the current knowledge regarding the SARS-CoV-2 infection, the implications of COVID-19 to Public Health and discuss genotype to phenotype association approaches that could be exploited through the selection of candidate genes to identify the genetic determinants of severe COVID-19.

E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery.

The flavivirus envelope glycoproteins prM and E drive the assembly of icosahedral, spiky immature particles that bud across the membrane of the endoplasmic reticulum. Maturation into infectious virions in the trans-Golgi network involves an acid-pH-driven rearrangement into smooth particles made of (prM/E)2 dimers exposing a furin site for prM cleavage into "pr" and "M". Here we show that the prM "pr" moiety derives from an HSP40 cellular chaperonin. Furthermore, the X-ray structure of the tick-borne encephalitis virus (pr/E)2 dimer at acidic pH reveals the E 150-loop as a hinged-lid that opens at low pH to expose a positively-charged pr-binding pocket at the E dimer interface, inducing (prM/E)2 dimer formation to generate smooth particles in the Golgi. Furin cleavage is followed by lid-closure upon deprotonation in the neutral-pH extracellular environment, expelling pr while the 150-loop takes the relay in fusion loop protection, thus revealing the elusive flavivirus mechanism of fusion activation.