Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

Species-specific gamete recognition initiates fusion-driving trimer formation by conserved fusogen HAP2

Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers.

An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency

Dengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization (NT50 < 0.1 μg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly prolonged the survival of interferon-α/β/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 that had lost their in vitro ADE activity showed enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits promising features for therapeutic application including a low NT50 value, potential for treatment of various kinds of mosquito-borne flavivirus infection, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

Potent neutralization of Rift Valley fever virus by human monoclonal antibodies through fusion inhibition

Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn–specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.

A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles.

An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency

Dengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization ability (NT50 < 0.1 µg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly improved the survival rate of interferon-α/β/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 molecules that had lost their in vitro ADE activity showed significantly enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits several promising features for therapeutic application including a low NT50 value, potential for pan-flavivirus infection treatment, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

Spike protein fusion loop controls SARS-CoV-2 fusogenicity and infectivity

Compared to the other human coronaviruses, SARS-CoV-2 has a higher reproductive number that is driving the COVID-19 pandemic. The high transmission of SARS-CoV-2 has been attributed to environmental, immunological, and molecular factors. The Spike protein is the foremost molecular factor responsible for virus fusion, entry and spread in the host, and thus holds clues for the rapid viral spread. The dense glycosylation of Spike, its high affinity of binding to the human ACE2 receptor, and the efficient priming by cleavage have already been proposed for driving efficient virus-host entry, but these do not explain its unusually high transmission rate. I have investigated the Spike from six β-coronaviruses, including the SARS-CoV-2, and find that their surface-exposed fusion peptides constituting the defined fusion loop are spatially organized contiguous to each other to work synergistically for triggering the virus-host membrane fusion process. The architecture of the Spike quaternary structure ensures the participation of the fusion peptides in the initiation of the host membrane contact for the virus fusion process. The SARS-CoV-2 fusion peptides have unique physicochemical properties, accrued in part from the presence of consecutive prolines that impart backbone rigidity which aids the virus fusogenicity. The specific contribution of these prolines shows significantly diminished fusogenicity in vitro and associated pathogenesis in vivo, inferred from comparative studies of their deletion-mutant in a fellow murine β-coronavirus MHV-A59. The priming of the Spike by its cleavage and subsequent fusogenic conformational transition steered by the fusion loop may be critical for the SARS-CoV-2 spread.Significance StatementThe three proximal fusion peptides constituting the fusion loop in Spike protein are the membranotropic segments most suitable for engaging the host membrane surface for its disruption. Spike’s unique quaternary structure architecture drives the fusion peptides to initiate the protein host membrane contact. The SARS-CoV-2 Spike trimer surface is relatively more hydrophobic among other human coronavirus Spikes, including the fusion peptides that are structurally more rigid owing to the presence of consecutive prolines, aromatic/hydrophobic clusters, a stretch of consecutive β-branched amino acids, and the hydrogen bonds. The synergy accrued from the location of the fusion peptides, their physicochemical features, and the fusogenic conformational transition appears to drive the virus fusion process and may explain the high spread of the SARS-CoV-2.

DMSA-camSPECT/US fusion imaging of children’s kidneys – Proof of feasibility

Aim SPECT using Tc-99m-dimercaptosuccinic acid (DMSA-camSPECT) and ultrasound (US) are useful diagnostic modalities in pediatric nephrology. Former studies proved SPECT/US fusion to be a problem-solving tool for thyroid and sentinel lymph node imaging. The purpose of this study was to demonstrate the feasibility of real-time DMSA-camSPECT/US fusion in children and to evaluate the technical implementation. Methods Subsequent to a clinical indicated DMSA-camSPECT showing a mostly normal result, 10 children received a SPECT/US fusion. The magnetic sensor-navigated equipment allows a real-time superposition of three-dimensional SPECT data to live US images according to the position of the transducer. Results 100 % of still images and 90 % of cine-loops were sufficient to depict the kidney; at least one sufficient camSPECT/US fusion loop could be accomplished for every kidney examined. Limitations are mainly caused by standard difficulties of conventional US. Conclusion Real-time DMSA-camSPECT/US is feasible in depiction of renal parenchyma. This newly implemented application broadens the scope of SPECT/US fusion imaging.