Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum.

Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.

Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age.

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA sequencing profiles of nasopharyngeal (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of the Th1 chemokines CXCL9/10/11 and their cognate receptor CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B cell-specific and NK cell-specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.

Human parainfluenza virus fusion complex glycoproteins imaged in action on authentic viral surfaces.

Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of the Respirovirus species of the Paramyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion.

Engineering Protease-Resistant Peptides to Inhibit Human Parainfluenza Viral Respiratory Infection.

The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with ß-amino acid residues to generate α/ß-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/ß-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.

Structure-Guided Improvement of a Dual HPIV3/RSV Fusion Inhibitor.

Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) are leading causes of lower respiratory tract infections. There are currently no vaccines or antiviral therapeutics to treat HPIV3 or RSV infections. We recently reported a peptide (VIQKI), derived from the C-terminal heptad repeat (HRC) domain of the HPIV3 fusion (F) glycoprotein that inhibits infection by both HPIV3 and RSV. The dual inhibitory activity of VIQKI is due to its unique ability to bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F, thereby preventing the native HRN-HRC interactions required for viral entry. Here we describe the structure-guided design of dual inhibitors of HPIV3 and RSV fusion with improved efficacy. We show that VIQKI derivatives possessing one (I456F) or two (I454F/I456F) phenylalanine substitutions near the N-terminus exhibit more stable assemblies with the RSV-HRN domain and enhanced antiviral efficacy against both HPIV3 and RSV infection. Cocrystal structures of the new Phe-substituted inhibitors coassembled with HPIV3 or RSV-HRN domains reveal that the I456F substitution makes intimate hydrophobic contact with the core trimers of both HPIV3 and RSV F.

Analysis of a Subacute Sclerosing Panencephalitis Genotype B3 Virus from the 2009-2010 South African Measles Epidemic Shows That Hyperfusogenic F Proteins Contribute to Measles Virus Infection in the Brain.

During a measles virus (MeV) epidemic in 2009 in South Africa, measles inclusion body encephalitis (MIBE) was identified in several HIV-infected patients. Years later, children are presenting with subacute sclerosing panencephalitis (SSPE). To investigate the features of established MeV neuronal infections, viral sequences were analyzed from brain tissue samples of a single SSPE case and compared with MIBE sequences previously obtained from patients infected during the same epidemic. Both the SSPE and the MIBE viruses had amino acid substitutions in the ectodomain of the F protein that confer enhanced fusion properties. Functional analysis of the fusion complexes confirmed that both MIBE and SSPE F protein mutations promoted fusion with less dependence on interaction by the viral receptor-binding protein with known MeV receptors. While the SSPE F required the presence of a homotypic attachment protein, MeV H, in order to fuse, MIBE F did not. Both F proteins had decreased thermal stability compared to that of the corresponding wild-type F protein. Finally, recombinant viruses expressing MIBE or SSPE fusion complexes spread in the absence of known MeV receptors, with MIBE F-bearing viruses causing large syncytia in these cells. Our results suggest that alterations to the MeV fusion complex that promote fusion and cell-to-cell spread in the absence of known MeV receptors is a key property for infection of the brain.IMPORTANCE Measles virus can invade the central nervous system (CNS) and cause severe neurological complications, such as MIBE and SSPE. However, mechanisms by which MeV enters the CNS and triggers the disease remain unclear. We analyzed viruses from brain tissue of individuals with MIBE or SSPE, infected during the same epidemic, after the onset of neurological disease. Our findings indicate that the emergence of hyperfusogenic MeV F proteins is associated with infection of the brain. We also demonstrate that hyperfusogenic F proteins permit MeV to enter cells and spread without the need to engage nectin-4 or CD150, known receptors for MeV that are not present on neural cells.

Measles Virus Bearing Measles Inclusion Body Encephalitis-Derived Fusion Protein Is Pathogenic after Infection via the Respiratory Route.

A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intrahost evolution of the wild-type (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of small-molecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for interhost spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37°C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro, neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV.IMPORTANCE Measles virus (MeV) infection can be severe in immunocompromised individuals and lead to complications, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE) occur even in the face of an intact immune response. While they are relatively rare complications of MeV infection, MIBE and SSPE are lethal. This work addresses the hypothesis that despite a dysregulated viral fusion complex, central nervous system (CNS)-adapted measles virus can spread outside the CNS within an infected host.

Dual Inhibition of Human Parainfluenza Type 3 and Respiratory Syncytial Virus Infectivity with a Single Agent.

Human parainfluenza virus 3 (HPIV3) and respiratory syncytial virus (RSV) cause lower respiratory infection in infants and young children. There are no vaccines for these pathogens, and existing treatments have limited or questionable efficacy. Infection by HPIV3 or RSV requires fusion of the viral and cell membranes, a process mediated by a trimeric fusion glycoprotein (F) displayed on the viral envelope. Once triggered, the pre-fusion form of F undergoes a series of conformational changes that first extend the molecule to allow for insertion of the hydrophobic fusion peptide into the target cell membrane and then refold the trimeric assembly into an energetically stable post-fusion state, a process that drives the merger of the viral and host cell membranes. Peptides derived from defined regions of HPIV3 F inhibit infection by HPIV3 by interfering with the structural transitions of the trimeric F assembly. Here we describe lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F that potently inhibit infection by both HPIV3 and RSV. The lead peptide inhibits RSV infection as effectively as does a peptide corresponding to the RSV HRC domain itself. We show that the inhibitors bind to the N-terminal heptad repeat (HRN) domains of both HPIV3 and RSV F with high affinity. Co-crystal structures of inhibitors bound to the HRN domains of HPIV3 or RSV F reveal remarkably different modes of binding in the N-terminal segment of the inhibitor.

Fusion Inhibitory Lipopeptides Engineered for Prophylaxis of Nipah Virus in Primates.

Background: The emerging zoonotic paramyxovirus Nipah virus (NiV) causes severe respiratory and neurological disease in humans, with high fatality rates. Nipah virus can be transmitted via person-to-person contact, posing a high risk for epidemic outbreaks. However, a broadly applicable approach for human NiV outbreaks in field settings is lacking. Methods: We engineered new antiviral lipopeptides and analyzed in vitro fusion inhibition to identify an optimal candidate for prophylaxis of NiV infection in the lower respiratory tract, and we assessed antiviral efficiency in 2 different animal models. Results: We show that lethal NiV infection can be prevented with lipopeptides delivered via the respiratory route in both hamsters and nonhuman primates. By targeting retention of peptides for NiV prophylaxis in the respiratory tract, we avoid its systemic delivery in individuals who need only prevention, and thus we increase the safety of treatment and enhance utility of the intervention. Conclusions: The experiments provide a proof of concept for the use of antifusion lipopeptides for prophylaxis of lethal NiV. These results advance the goal of rational development of potent lipopeptide inhibitors with desirable pharmacokinetic and biodistribution properties and a safe effective delivery method to target NiV and other pathogenic viruses.

Biophysical Properties and Antiviral Activities of Measles Fusion Protein Derived Peptide Conjugated with 25-Hydroxycholesterol.

Measles virus (MV) infection is re-emerging, despite the availability of an effective vaccine. The mechanism of MV entry into a target cell relies on coordinated action between the MV hemagglutinin (H) receptor binding protein and the fusion envelope glycoprotein (F) which mediates fusion between the viral and cell membranes. Peptides derived from the C-terminal heptad repeat (HRC) of F can interfere with this process, blocking MV infection. As previously described, biophysical properties of HRC-derived peptides modulate their antiviral potency. In this work, we characterized a MV peptide fusion inhibitor conjugated to 25-hydroxycholesterol (25HC), a cholesterol derivative with intrinsic antiviral activity, and evaluated its interaction with membrane model systems and human blood cells. The peptide (MV.

Antiviral Lipopeptide-Cell Membrane Interaction Is Influenced by PEG Linker Length.

A set of lipopeptides was recently reported for their broad-spectrum antiviral activity against viruses belonging to the Paramyxoviridae family, including human parainfluenza virus type 3 and Nipah virus. Among them, the peptide with a 24-unit PEG linker connecting it to a cholesterol moiety (VG-PEG24-Chol) was found to be the best membrane fusion inhibitory peptide. Here, we evaluated the interaction of the same set of peptides with biomembrane model systems and isolated human peripheral blood mononuclear cells (PBMC). VG-PEG24-Chol showed the highest insertion rate and it was among the peptides that induced a larger change on the surface pressure of cholesterol rich membranes. This peptide also displayed a high affinity towards PBMC membranes. These data provide new information about the dynamics of peptide-membrane interactions of a specific group of antiviral peptides, known for their potential as multipotent paramyxovirus antivirals.

Circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection.

UNLABELLED: Human parainfluenza viruses (HPIVs) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the hemagglutinin-neuraminidase receptor-binding protein (HN) and the fusion envelope glycoprotein (F), which together comprise the molecular fusion machinery; upon receptor engagement by HN, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo. IMPORTANCE: Acute respiratory infection is the leading cause of mortality in young children under 5 years of age, causing nearly 20% of childhood deaths worldwide each year. The paramyxoviruses, including human parainfluenza viruses (HPIVs), cause a large share of these illnesses. There are no vaccines or drugs for the HPIVs. Inhibiting entry of viruses into the human cell is a promising drug strategy that blocks the first step in infection. To develop antivirals that inhibit entry, it is critical to understand the first steps of infection. We found that clinical viruses isolated from patients have very different entry properties from those of the viruses generally studied in laboratories. The viral entry mechanism is less active and more sensitive to fusion inhibitory molecules. We propose that to interfere with viral infection, we test clinically circulating viruses in natural tissues, to develop antivirals against respiratory disease caused by HPIVs.

Improvement of HIV fusion inhibitor C34 efficacy by membrane anchoring and enhanced exposure.

OBJECTIVES: The aim of the present work was to evaluate the interaction of two new HIV fusion inhibitors {HIVP3 [C34-polyethylene glycol (PEG)4-cholesterol] and HIVP4 [(C34-PEG4)2-cholesterol]} with membrane model systems and human blood cells in order to clarify where and how the fusion inhibitors locate, allowing us to understand their mechanism of action at the molecular level, and which strategies may be followed to increase efficacy. METHODS: Lipid vesicles with defined compositions were used for peptide partition and localization studies, based on the intrinsic fluorescence of HIVP3 and HIVP4. Lipid monolayers were employed in surface pressure studies. Finally, human erythrocytes and peripheral blood mononuclear cells (PBMCs) isolated from blood samples were used in dipole potential assays. RESULTS: Membrane partition, dipole potential and surface pressure assays indicate that the new fusion inhibitors interact preferentially with cholesterol-rich liquid-ordered membranes, mimicking biological membrane microdomains known as lipid rafts. HIVP3 and HIVP4 are able to interact with human erythrocytes and PBMCs to a similar degree as a previously described simpler drug with monomeric C34 and lacking the PEG spacer, C34-cholesterol. However, the pocket-binding domain (PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol. CONCLUSIONS: The present data allow us to conclude that more efficient blocking of HIV entry results from the synergism between the membranotropic behaviour and the enhanced exposure of the PBD.

Measles virus fusion machinery activated by sialic acid binding globular domain.

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Fatal measles virus infection prevented by brain-penetrant fusion inhibitors.

Measles virus (MV) infection causes an acute childhood disease that can include infection of the central nervous system and can rarely progress to severe neurological disease for which there is no specific treatment. We generated potent antiviral peptide inhibitors of MV entry and spreading and MV-induced cell fusion. Dimers of MV-specific peptides derived from the C-terminal heptad repeat region of the MV fusion protein, conjugated to cholesterol, efficiently protect SLAM transgenic mice from fatal MV infection. Fusion inhibitors hold promise for the prophylaxis of MV infection in unvaccinated and immunocompromised people, as well as potential for the treatment of grave neurological complications of measles.

Identification of a region in the stalk domain of the nipah virus receptor binding protein that is critical for fusion activation.

Paramyxoviruses, including the emerging lethal human Nipah virus (NiV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral and target cell membranes. For paramyxoviruses, membrane fusion is the result of the concerted action of two viral envelope glycoproteins: a receptor binding protein and a fusion protein (F). The NiV receptor binding protein (G) attaches to ephrin B2 or B3 on host cells, whereas the corresponding hemagglutinin-neuraminidase (HN) attachment protein of NDV interacts with sialic acid moieties on target cells through two regions of its globular domain. Receptor-bound G or HN via its stalk domain triggers F to undergo the conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We show that chimeric proteins containing the NDV HN receptor binding regions and the NiV G stalk domain require a specific sequence at the connection between the head and the stalk to activate NiV F for fusion. Our findings are consistent with a general mechanism of paramyxovirus fusion activation in which the stalk domain of the receptor binding protein is responsible for F activation and a specific connecting region between the receptor binding globular head and the fusion-activating stalk domain is required for transmitting the fusion signal.

Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry.

Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread. IMPORTANCE: Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

How a paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody.

Paramyxoviruses including measles, Nipah, and parainfluenza viruses are public health threats with pandemic potential. Human parainfluenza virus type 3 (HPIV3) is a leading cause of illness in pediatric, older, and immunocompromised populations. There are no approved vaccines or therapeutics for HPIV3. Neutralizing monoclonal antibodies (mAbs) that target viral fusion are a potential strategy for mitigating paramyxovirus infection, however their utility may be curtailed by viral evolution that leads to resistance. Paramyxoviruses enter cells by fusing with the cell membrane in a process mediated by a complex consisting of a receptor binding protein (HN) and a fusion protein (F). Existing atomic resolution structures fail to reveal physiologically relevant interactions during viral entry. We present cryo-ET structures of pre-fusion HN-F complexes in situ on surfaces of virions that evolved resistance to an anti-HPIV3 F neutralizing mAb. Single mutations in F abolish mAb binding and neutralization. In these complexes, the HN protein that normally restrains F triggering has shifted to uncap the F apex. These complexes are more readily triggered to fuse. These structures shed light on the adaptability of the pre-fusion HN-F complex and mechanisms of paramyxoviral resistance to mAbs, and help define potential barriers to resistance for the design of mAbs.

A neutralizing antibody prevents postfusion transition of measles virus fusion protein.

Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.