TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remained genetically stable during the first 3 months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early-occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. IMPORTANCE The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions. The Alpha and Delta variants, which spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic compared to the original strain. Interestingly, Alpha and Delta both carried the mutations P681H/R in a cleavage site that made the spike more cleaved and more efficient at mediating viral fusion. We show here that variants with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of the D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite for the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.

The Mechanism and Consequences of SARS-CoV-2 Spike-Mediated Fusion and Syncytia Formation.

Syncytia are formed when individual cells fuse. SARS-CoV-2 induces syncytia when the viral spike (S) protein on the surface of an infected cell interacts with receptors on neighboring cells. Syncytia may potentially contribute to pathology by facilitating viral dissemination, cytopathicity, immune evasion, and inflammatory response. SARS-CoV-2 variants of concern possess several mutations within the S protein that enhance receptor interaction, fusogenicity and antibody binding. In this review, we discuss the molecular determinants of S mediated fusion and the antiviral innate immunity components that counteract syncytia formation. Several interferon-stimulated genes, including IFITMs and LY6E act as barriers to S protein-mediated fusion by altering the composition or biophysical properties of the target membrane. We also summarize the effect that the mutations associated with the variants of concern have on S protein fusogenicity. Altogether, this review contextualizes the current understanding of Spike fusogenicity and the role of syncytia during SARS-CoV-2 infection and pathology.

SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation.

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization.

The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India1-5. Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries6. The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2-also termed the Delta variant-is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12 months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein.

Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 and B.1.351 variants were first identified in the United Kingdom and South Africa, respectively, and have since spread to many countries. These variants harboring diverse mutations in the gene encoding the spike protein raise important concerns about their immune evasion potential. Here, we isolated infectious B.1.1.7 and B.1.351 strains from acutely infected individuals. We examined sensitivity of the two variants to SARS-CoV-2 antibodies present in sera and nasal swabs from individuals infected with previously circulating strains or who were recently vaccinated, in comparison with a D614G reference virus. We utilized a new rapid neutralization assay, based on reporter cells that become positive for GFP after overnight infection. Sera from 58 convalescent individuals collected up to 9 months after symptoms, similarly neutralized B.1.1.7 and D614G. In contrast, after 9 months, convalescent sera had a mean sixfold reduction in neutralizing titers, and 40% of the samples lacked any activity against B.1.351. Sera from 19 individuals vaccinated twice with Pfizer Cominarty, longitudinally tested up to 6 weeks after vaccination, were similarly potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Neutralizing titers increased after the second vaccine dose, but remained 14-fold lower against B.1.351. In contrast, sera from convalescent or vaccinated individuals similarly bound the three spike proteins in a flow cytometry-based serological assay. Neutralizing antibodies were rarely detected in nasal swabs from vaccinees. Thus, faster-spreading SARS-CoV-2 variants acquired a partial resistance to neutralizing antibodies generated by natural infection or vaccination, which was most frequently detected in individuals with low antibody levels. Our results indicate that B1.351, but not B.1.1.7, may increase the risk of infection in immunized individuals.

Syncytia formation by SARS-CoV-2-infected cells.

Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2-infected cells express the Spike protein (S) at their surface and fuse with ACE2-positive neighboring cells. Expression of S without any other viral proteins triggers syncytia formation. Interferon-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that SARS-CoV-2 pathological effects are modulated by cellular proteins that either inhibit or facilitate syncytia formation.

A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations.

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.

Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication.

The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

Contemporary Zika Virus Isolates Induce More dsRNA and Produce More Negative-Strand Intermediate in Human Astrocytoma Cells.

The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. Here we demonstrate that two contemporary (2015) ZIKV isolates from Puerto Rico and Brazil may have increased replicative fitness in human astrocytoma cells. Over a single infectious cycle, the Brazilian isolate replicates to higher titers and induces more severe cytopathic effects in human astrocytoma cells than the historical African reference strain or an early Asian lineage isolate. In addition, both contemporary isolates induce significantly more double-stranded RNA in infected astrocytoma cells, despite similar numbers of infected cells across isolates. Moreover, when we quantified positive- and negative-strand viral RNA, we found that the Asian lineage isolates displayed substantially more negative-strand replicative intermediates than the African lineage isolate in human astrocytoma cells. However, over multiple rounds of infection, the contemporary ZIKV isolates appear to be impaired in cell spread, infecting a lower proportion of cells at a low MOI despite replicating to similar or higher titers. Taken together, our data suggests that contemporary ZIKV isolates may have evolved mechanisms that allow them to replicate with increased efficiency in certain cell types, thereby highlighting the importance of cell-intrinsic factors in studies of viral replicative fitness.

Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice.

Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies.

Zika Virus: Emergence, Phylogenetics, Challenges, and Opportunities.

Zika virus (ZIKV) is an emerging arthropod-borne pathogen that has recently gained notoriety due to its rapid and ongoing geographic expansion and its novel association with neurological complications. Reports of ZIKV-associated Guillain-Barré syndrome as well as fetal microcephaly place emphasis on the need to develop preventative measures and therapeutics to combat ZIKV infection. Thus, it is imperative that models to study ZIKV replication and pathogenesis and the immune response are developed in conjunction with integrated vector control strategies to mount an efficient response to the pandemic. This paper summarizes the current state of knowledge on ZIKV, including the clinical features, phylogenetic analyses, pathogenesis, and the immune response to infection. Potential challenges in developing diagnostic tools, treatment, and prevention strategies are also discussed.

Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans.

Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states ("closed" vs. "open") of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of "closed" syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1-depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the "open" syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between "closed" syntaxin-1A and Munc18-1, whereas the same mutation in the "open" syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.