Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Structural diversity of the SARS-CoV-2 Omicron spike.

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

Diverse Murine Vaccinations Reveal Distinct Antibody Classes to Target Fusion Peptide and Variation in Peptide Length to Improve HIV Neutralization.

While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses - both X-ray and cryo-EM - revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability. IMPORTANCE The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.

Structures of Langya Virus Fusion Protein Ectodomain in Pre- and Postfusion Conformation.

Langya virus (LayV) is a paramyxovirus in the Henipavirus genus, closely related to the deadly Nipah (NiV) and Hendra (HeV) viruses, that was identified in August 2022 through disease surveillance following animal exposure in eastern China. Paramyxoviruses present two glycoproteins on their surface, known as attachment and fusion proteins, that mediate entry into cells and constitute the primary antigenic targets for immune response. Here, we determine cryo-electron microscopy (cryo-EM) structures of the uncleaved LayV fusion protein (F) ectodomain in pre- and postfusion conformations. The LayV-F protein exhibits pre- and postfusion architectures that, despite being highly conserved across paramyxoviruses, show differences in their surface properties, in particular at the apex of the prefusion trimer, that may contribute to antigenic variability. While dramatic conformational changes were visualized between the pre- and postfusion forms of the LayV-F protein, several domains remained invariant, held together by highly conserved disulfides. The LayV-F fusion peptide (FP) is buried within a highly conserved, hydrophobic interprotomer pocket in the prefusion state and is notably less flexible than the rest of the protein, highlighting its "spring-loaded" state and suggesting that the mechanism of pre-to-post transition must involve perturbations to the pocket and release of the fusion peptide. Together, these results offer a structural basis for how the Langya virus fusion protein compares to its Henipavirus relatives and propose a mechanism for the initial step of pre- to postfusion conversion that may apply more broadly to paramyxoviruses. IMPORTANCE The Henipavirus genus is quickly expanding into new animal hosts and geographic locations. This study compares the structure and antigenicity of the Langya virus fusion protein to other henipaviruses, which have important vaccine and therapeutic development implications. Furthermore, the study proposes a new mechanism to explain the early steps of the fusion initiation process that can be more broadly applied to the Paramyxoviridae family.

Study of the antivirus function mediated by STING in Micropterus salmoides.

Stimulator of interferon genes (STING) has been demonstrated as a critical mediator in the innate immune response to cytosolic DNA and RNA derived from different pathogens. While the role of Micropterus salmoides STING (MsSTING) in largemouth bass virus is still unknown. In this study, RT-qPCR assay and Western-blot assay showed that the expression levels of MsSTING and its downstream genes were up-regulated after LMBV infection. Pull down experiment proved that a small peptide called Fusion peptide (FP) that previously reported to target to marine and human STING as a selective inhibitor also interacted with MsSTING in vitro. Comparing with the RNA-seq of Largemouth bass infected with LMBV singly, 326 genes were significantly up-regulated and 379 genes were significantly down-regulated in the FP plus LMBV group in which Largemouth bass was treatment with FP before LMBV-challenged. KEGG analysis indicated that the differentially expressed genes (DEGs) were mainly related to signaling transduction, infectious disease viral, immune system and endocrine system. Besides, the survival rate of LMBV-infected largemouth bass was highly decreased following FP treatment. Taken together, our study showed that MsSTING played an important role in immune response against LMBV infection.

Current Status and Perspectives of Therapeutic Antibodies Targeting the Spike Protein S2 Subunit against SARS-CoV-2.

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has devastated public health and the global economy. New variants are continually emerging because of amino acid mutations within the SARS-CoV-2 spike protein. Existing neutralizing antibodies (nAbs) that target the receptor-binding domain (RBD) within the spike protein have been shown to have reduced neutralizing activity against these variants. In particular, the recently expanding omicron subvariants BQ 1.1 and XBB are resistant to nAbs approved for emergency use by the United States Food and Drug Administration. Therefore, it is essential to develop broad nAbs to combat emerging variants. In contrast to the massive accumulation of mutations within the RBD, the S2 subunit remains highly conserved among variants. Therefore, nAbs targeting the S2 region may provide effective cross-protection against novel SARS-CoV-2 variants. Here, we provide a detailed summary of nAbs targeting the S2 subunit: the fusion peptide, stem helix, and heptad repeats 1 and 2. In addition, we provide prospects to solve problems such as the weak neutralizing potency of nAbs targeting the S2 subunit.

Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody.

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti-HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

Killed whole-genome reduced-bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

Commonly Elicited Antibodies against the Base of the HIV-1 Env Trimer Guide the Population-Level Evolution of a Structure-Regulating Region in gp41.

The antibody response against the HIV-1 envelope glycoproteins (Envs) guides evolution of this protein within each host. Whether antibodies with similar target specificities are elicited in different individuals and affect the population-level evolution of Env is poorly understood. To address this question, we analyzed properties of emerging variants in the gp41 fusion peptide-proximal region (FPPR) that exhibit distinct evolutionary patterns in HIV-1 clade B. For positions 534, 536, and 539 in the FPPR, alanine was the major emerging variant. However, 534A and 536A show a constant frequency in the population between 1979 and 2016, whereas 539A is gradually increasing. To understand the basis for these differences, we introduced alanine substitutions in the FPPR of primary HIV-1 strains and examined their functional and antigenic properties. Evolutionary patterns could not be explained by fusion competence or structural stability of the emerging variants. Instead, 534A and 536A exhibited modest but significant increases in sensitivity to antibodies against the membrane-proximal external region (MPER) and gp120-gp41 interface. These Envs were also more sensitive to poorly neutralizing sera from HIV-1-infected individuals than the clade ancestral form or 539A variant. Competition binding assays confirmed for all sera tested the presence of antibodies against the base of the Env trimer that compete with monoclonal antibodies targeting the MPER and gp120-gp41 interface. Our findings suggest that weakly neutralizing antibodies against the trimer base are commonly elicited; they do not exert catastrophic population size reduction effects on emerging variants but, instead, determine their set point frequencies in the population and historical patterns of change. IMPORTANCE Infection by HIV-1 elicits formation of antibodies that target the viral Env proteins and can inactivate the virus. The specific targets of these antibodies vary among infected individuals. It is unclear whether some target specificities are shared among the antibody responses of different individuals. We observed that antibodies against the base of the Env protein are commonly elicited during infection. The selective pressure applied by such antibodies is weak. As a result, they do not completely eliminate the sensitive forms of the virus from the population, but maintain their frequency at a low level that has not increased since the beginning of the AIDS pandemic. Interestingly, the changes in Env do not occur at the sites targeted by the antibodies, but at a distinct region of Env, the fusion peptide-proximal region, which regulates their exposure.

Recombinant expression a novel fibronectin-collage fusion peptide modulating stem cell stemness via integrin ß3.

Constructing bionic extracellular matrix (ECM) is an attractive proposition for tissue engineering and clinical regeneration therapy involving the stemness of stem cells. Here, a novel recombinant protein fibronectin-collagen peptide (FCP) was designed to modulate the function of ECM expressed by Picha. pastoris strain X33. This FCP promotes cell migration and adhesion and maintains rBMSC stemness by binding integrin ß3. Its effects were blocked by both integrin ß3 siRNA and the integrin ß3 inhibitor Cilengitide. A template-independent ab initio prediction modeling approach is the best approach to construct a stable FCP protein model, which predicts the binding sites between FCP and integrin ß3. FCP may be used in the in vitro culture and clinical regeneration of stem cells that highly express integrin ß3, such as hematopoietic stem cells. The study provides information on the molecular structure of FCP and its bioactivity, which can be used to design new compounds. KEY POINTS: • Design a novel recombinant fibronectin-collagen peptide biomimetic ECM. • FCP promotes cell adhesion, migration, and proliferation. • Predicted and verified FCP structure and affinity with integrin ß3. • FCP binds integrin ß3 to maintain rBMSC stemness.

Antiviral Peptides as Anti-Influenza Agents.

Influenza viruses represent a leading cause of high morbidity and mortality worldwide. Approaches for fighting flu are seasonal vaccines and some antiviral drugs. The development of the seasonal flu vaccine requires a great deal of effort, as careful studies are needed to select the strains to be included in each year's vaccine. Antiviral drugs available against Influenza virus infections have certain limitations due to the increased resistance rate and negative side effects. The highly mutative nature of these viruses leads to the emergence of new antigenic variants, against which the urgent development of new approaches for antiviral therapy is needed. Among these approaches, one of the emerging new fields of "peptide-based therapies" against Influenza viruses is being explored and looks promising. This review describes the recent findings on the antiviral activity, mechanism of action and therapeutic capability of antiviral peptides that bind HA, NA, PB1, and M2 as a means of countering Influenza virus infection.

A TCR-like antibody against a proinsulin-containing fusion peptide ameliorates type 1 diabetes in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by destruction of insulin-producing ß cells. The response of autoreactive T cells to ß cell antigens plays a central role in the development of T1D. Recently, fusion peptides composed by insulin C-peptide fragments and other proteins were reported as ß cell target antigens for diabetogenic CD4+ T cells in non-obese diabetic (NOD) mice. In this study, we generated a T cell-receptor (TCR)-like monoclonal antibody (mAb) against a fusion peptide bound to major histocompatibility complex (MHC) class II component to elucidate the function of the fusion peptides in T1D. In addition, we developed a novel NFAT-GFP TCR reporter system to evaluate the TCR-like mAb. The NFAT-GFP reporter T cells expressing the diabetogenic TCR were specifically activated by the fusion peptide presented on the MHC class II molecules. By using the NFAT-GFP reporter T cells, we showed that the TCR-like mAb blocks the diabetogenic T cell response against the fusion peptide presented on the MHC class II molecules. Furthermore, the development of T1D was ameliorated when pre-diabetic NOD mice were treated with this mAb. These findings suggest that NFAT-GFP reporter T cells are useful to assess the function of specific TCR and the recognition of fusion peptides by T cells is crucial for the pathogenesis of T1D.

Development of a 3Mut-Apex-Stabilized Envelope Trimer That Expands HIV-1 Neutralization Breadth When Used To Boost Fusion Peptide-Directed Vaccine-Elicited Responses.

HIV-1 envelope (Env) trimers, stabilized in a prefusion-closed conformation, can elicit humoral responses capable of neutralizing HIV-1 strains closely matched in sequence to the immunizing strain. One strategy to increase elicited neutralization breadth involves vaccine priming of immune responses against a target site of vulnerability, followed by vaccine boosting of these responses with prefusion-closed Env trimers. This strategy has succeeded at the fusion peptide (FP) site of vulnerability in eliciting cross-clade neutralizing responses in standard vaccine-test animals. However, the breadth and potency of the elicited responses have been less than optimal. Here, we identify three mutations (3mut), Met302, Leu320, and Pro329, that stabilize the apex of the Env trimer in a prefusion-closed conformation and show antigenically, structurally, and immunogenically that combining 3mut with other approaches (e.g., repair and stabilize and glycine-helix breaking) yields well-behaved clade C-Env trimers capable of boosting the breadth of FP-directed responses. Crystal structures of these trimers confirmed prefusion-closed apexes stabilized by hydrophobic patches contributed by Met302 and Leu320, with Pro329 assuming canonically restricted dihedral angles. We substituted the N-terminal eight residues of FP (FP8, residues 512 to 519) of these trimers with the second most prevalent FP8 sequence (FP8v2, AVGLGAVF) and observed a 3mut-stabilized consensus clade C-Env trimer with FP8v2 to boost the breadth elicited in guinea pigs of FP-directed responses induced by immunogens containing the most prevalent FP8 sequence (FP8v1, AVGIGAVF). Overall, 3mut can stabilize the Env trimer apex, and the resultant apex-stabilized Env trimers can be used to expand the neutralization breadth elicited against the FP site of vulnerability.IMPORTANCE A major hurdle to the development of an effective HIV-1 vaccine is the elicitation of serum responses capable of neutralizing circulating strains of HIV, which are extraordinarily diverse in sequence and often highly neutralization resistant. Recently, we showed how sera with 20 to 30% neutralization breadth could, nevertheless, be elicited in standard vaccine test animals by priming with the most prevalent N-terminal 8 residues of the HIV-1 fusion peptide (FP8), followed by boosting with a stabilized BG505-envelope (Env) trimer. Here, we show that subsequent boosting with a 3mut-apex-stabilized consensus C-Env trimer, modified to have the second most prevalent FP8 sequence, elicits higher neutralization breadth than that induced by continued boosting with the stabilized BG505-Env trimer. With increased neutralizing breadth elicited by boosting with a heterologous trimer containing the second most prevalent FP8 sequence, the fusion peptide-directed immune-focusing approach moves a step closer toward realizing an effective HIV-1 vaccine regimen.

Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity.

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.

Polyethylene Glycol 40-Modified Peptide with High Therapeutic Efficacy in Simian-Human Immunodeficiency Virus-Acutely Infected Rhesus Monkeys.

Anti-human immunodeficiency virus type 1 (anti-HIV-1) fusion peptides have been studied for nearly 2 decades, but few candidates have found useful clinical applications. One factor underlying the failure of such agents to reach the clinic is their poor pharmacokinetic properties, and many efforts have been made to overcome this problem. In this study, we modified C34, a peptide inhibitor of HIV-1 fusion, at its conserved glycosylation site using polyethylene glycols (PEGs) of different molecular weights. PEG40-NC, a conjugate of C34 and branched PEG 40 kDa (PEG40), which has been previously shown to improve the pharmacokinetic profiles of proteins, showed a significantly extended half-life (t1/2; 10.39 h in rats), which compensated for decreased in vitro activity (50% effective concentration [EC50] of 18.51 nM). PEG40-NC also showed a mechanism of action similar to that of C34. PEG40-NC monotherapy in acutely simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys significantly suppressed viral load compared with a control treatment. Efficacy was linked to the extended half-life and lymphatic exposure conferred by attached PEG40. These results highlight the potential of further clinical investigations of PEG40-NC in combination with antiretroviral therapy or other anti-HIV agents.IMPORTANCE Poor pharmacokinetics have severely hindered the clinical use of anti-HIV peptides. Different small molecules, such as lipid, cholesterol, and small PEG, were designed to modify peptides to improve their pharmacokinetics. In this study, we incorporated large branched PEG to anti-HIV peptide and obtained a conjugate with extended half-life and improved in vivo efficacy. The strategy we developed in this study can also be applicable for the development of other peptide candidates.

HIV-1 anchor inhibitors and membrane fusion inhibitors target distinct but overlapping steps in virus entry.

HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) and represents an attractive target for therapeutic intervention. Two drugs that inhibit HIV entry are approved for clinical use: the membrane fusion-inhibitor T20 (Fuzeon, enfuvirtide) and the C-C chemokine receptor type 5 (CCR5) blocker maraviroc (Selzentry). Another class of entry inhibitors supposedly target the fusion peptide (FP) and are termed anchor inhibitors. These include the VIRIP peptide and VIRIP derivatives such as VIR165, VIR353, and VIR576. Here, we investigated the mechanism of inhibition by VIR165. We show that substitutions within the FP modulate sensitivity to VIR165, consistent with the FP being the drug target. Our results also revealed that VIR165 acts during an intermediate post-CD4-binding entry step that is overlapping but not identical to the step inhibited by fusion inhibitors such as T20. We found that some but not all resistance mutations to heptad repeat 2 (HR2)-targeting fusion inhibitors can provide cross-resistance to VIR165. In contrast, resistance mutations in the HR1-binding site for the fusion inhibitors did not cause cross-resistance to VIR165. However, Env with mutations located outside this binding site and thought to affect fusion kinetics, exhibited decreased sensitivity to VIR165. Although we found a strong correlation between Env stability and resistance to HR2-based fusion inhibitors, such correlation was not observed for Env stability and VIR165 resistance. We conclude that VIRIP analogs target the FP during an intermediate, post-CD4-binding entry step that overlaps with but is distinct from the step(s) inhibited by HR2-based fusion inhibitors.

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology.

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)-carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.

Membrane Composition Modulates Fusion by Altering Membrane Properties and Fusion Peptide Structure.

Membrane fusion, one of the most essential processes in the life of eukaryotes, occurs when two separate lipid bilayers merge into a continuous bilayer and internal contents of two separated membranes mingle. There is a certain class of proteins that assist the binding of the viral envelope to the target host cell and catalyzing fusion. All class I viral fusion proteins contain a highly conserved 20-25 amino-acid amphipathic peptide at the N-terminus, which is essential for fusion activity and is termed as the 'fusion peptide'. It has been shown that insertion of fusion peptides into the host membrane and the perturbation in the membrane generated thereby is crucial for membrane fusion. Significant efforts have been given in the last couple of decades to understand the lipid-dependence of structure and function of the fusion peptide in membranes to understand the role of lipid compositions in membrane fusion. In addition, the lipid compositions further change the membrane physical properties and alter the mechanism and extent of membrane fusion. Therefore, lipid compositions modulate membrane fusion by changing membrane physical properties and altering structure of the fusion peptide.

Reduced Susceptibility to VIRIP-Based HIV-1 Entry Inhibitors Has a High Genetic Barrier and Severe Fitness Costs.

VIRIP has been identified as natural HIV-1 inhibitor targeting the gp41 fusion peptide. An optimized analogue (VIR-576) was effective in a phase I/II clinical trial and initial studies showed that HIV-1 resistance to VIRIP-based inhibitors has a high genetic barrier. Partially resistant CXCR4 (X4)-tropic HIV-1 NL4-3 variants could be obtained, however, after more than 15 months of passaging in MT-4 cells in the presence of another derivative (VIR-353). Sequence analyses identified the accumulation of seven mutations across the HIV-1 envelope glycoprotein but outside the gp41 fusion peptide. The authors suggested that the three initial alterations conferred resistance, while subsequent changes restored viral fitness. Here, we introduced these mutations individually and in combination into X4- and CCR5 (R5)-tropic HIV-1 constructs and determined their impact on VIR-353 and VIR-576 susceptibility, viral infectivity, replication fitness, and fusogenicity. We found that essentially all seven mutations contribute to reduced susceptibility to VIRIP-based inhibitors. HIV-1 constructs containing ≥4 changes were substantially more resistant to both VIRIP-based inhibitors and the VRC34.01 antibody targeting the fusion peptide. However, they were also much less infectious and fusogenic than those harboring only the three initial alterations. Furthermore, the additional changes attenuated rather than rescued HIV-1 replication in primary human cells. Thus, the genetic barrier to HIV-1 resistance against VIRIP-based inhibitors is higher than previously suggested, and mutations reducing viral susceptibility come at a severe fitness cost that was not rescued during long-term cell culture passage.IMPORTANCE Many viral pathogens are critically dependent on fusion peptides (FPs) that are inserted into the cellular membrane for infection. Initially, it was thought that FPs cannot be targeted for therapy because they are hardly accessible. However, an optimized derivative (VIR-576) of an endogenous fragment of α1-antitrypsin, named VIRIP, targeting the gp41 FP reduced viral loads in HIV-1-infected individuals. Characterization of HIV-1 variants selected during long-term cell-culture passage in the presence of a VIRIP derivative suggested that just three mutations in the HIV-1 Env protein might be sufficient for VIRIP resistance and that four subsequent changes restored viral fitness. Here, we show that all seven mutations contribute to reduced viral susceptibility to VIRIP-based inhibitors and demonstrate that the additional changes strongly impair rather than rescue HIV-1 infectivity, fusogenicity, and replication fitness. High genetic barrier to resistance and severe fitness cost support further clinical development of this class of antiviral agents.

Computational Virology: Molecular Simulations of Virus Dynamics and Interactions.

Molecular modelling and simulations play a key role in computational virology, allowing us to study viruses and their components. This allows experimental structures and related information to be integrated into a single coherent model, enabling predictions about the behaviour of a viral system to go beyond what could be obtained experimentally. In this way, computational approaches provide a powerful complement to more traditional experimental and structural methods. In this chapter, we describe three main areas of computational virology to showcase the power of methods within this field. We begin by describing relatively small simulation systems and focusing on the behaviour of fusion peptides. Then, extending to longer timescales and larger systems, we discuss computational studies of viral capsid assembly and genome encapsidation. Finally, we describe recent developments which allow entire viral particles to be simulated.