Integrated cryoEM structure of a spumaretrovirus reveals cross-kingdom evolutionary relationships and the molecular basis for assembly and virus entry.

Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.

Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection.

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

Convergent evolution and targeting of diverse E2 epitopes by human broadly neutralizing antibodies are associated with HCV clearance.

The early appearance of broadly neutralizing antibodies (bNAbs) in serum is associated with spontaneous hepatitis C virus (HCV) clearance, but to date, the majority of bNAbs have been isolated from chronically infected donors. Most of these bNAbs use the VH1-69 gene segment and target the envelope glycoprotein E2 front layer. Here, we performed longitudinal B cell receptor (BCR) repertoire analysis on an elite neutralizer who spontaneously cleared multiple HCV infections. We isolated 10,680 E2-reactive B cells, performed BCR sequencing, characterized monoclonal B cell cultures, and isolated bNAbs. In contrast to what has been seen in chronically infected donors, the bNAbs used a variety of VH genes and targeted at least three distinct E2 antigenic sites, including sites previously thought to be non-neutralizing. Diverse front-layer-reactive bNAb lineages evolved convergently, acquiring breadth-enhancing somatic mutations. These findings demonstrate that HCV clearance-associated bNAbs are genetically diverse and bind distinct antigenic sites that should be the target of vaccine-induced bNAbs.

Neutralizing antibodies evolve to exploit vulnerable sites in the HCV envelope glycoprotein E2 and mediate spontaneous clearance of infection.

Individuals who clear primary hepatitis C virus (HCV) infections clear subsequent reinfections more than 80% of the time, but the mechanisms are poorly defined. Here, we used HCV variants and plasma from individuals with repeated clearance to characterize longitudinal changes in envelope glycoprotein E2 sequences, function, and neutralizing antibody (NAb) resistance. Clearance of infection was associated with early selection of viruses with NAb resistance substitutions that also reduced E2 binding to CD81, the primary HCV receptor. Later, peri-clearance plasma samples regained neutralizing capacity against these variants. We identified a subset of broadly NAbs (bNAbs) for which these loss-of-fitness substitutions conferred resistance to unmutated bNAb ancestors but increased sensitivity to mature bNAbs. These data demonstrate a mechanism by which neutralizing antibodies contribute to repeated immune-mediated HCV clearance, identifying specific bNAbs that exploit fundamental vulnerabilities in E2. The induction of bNAbs with these specificities should be a goal of HCV vaccine development.

Deep mutational scanning reveals functional constraints and antibody-escape potential of Lassa virus glycoprotein complex.

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.

A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection.

Infections with dengue virus (DENV) and Zika virus (ZIKV) can induce cross-reactive antibody responses. Two immunodominant epitopes-one to precursor membrane protein and one to the fusion loop epitope on envelope (E) protein-are recognized by cross-reactive antibodies1-3 that are not only poorly neutralizing, but can also promote increased viral replication and disease severity via Fcγ receptor-mediated infection of myeloid cells-a process termed antibody-dependent enhancement (ADE)1,4,5. ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly neutralizing cross-reactive antibodies may prime an individual for ADE on natural infection. In this report, we describe the design and production of covalently stabilized ZIKV E dimers, which lack precursor membrane protein and do not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection.

Human Adaptation of Ebola Virus during the West African Outbreak.

The 2013-2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages.

Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic.

The magnitude of the 2013-2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013-2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.

Interaction of chikungunya virus glycoproteins with macrophage factors controls virion production.

Despite their role as innate sentinels, macrophages can serve as cellular reservoirs of chikungunya virus (CHIKV), a highly-pathogenic arthropod-borne alphavirus that has caused large outbreaks among human populations. Here, with the use of viral chimeras and evolutionary selection analysis, we define CHIKV glycoproteins E1 and E2 as critical for virion production in THP-1 derived human macrophages. Through proteomic analysis and functional validation, we further identify signal peptidase complex subunit 3 (SPCS3) and eukaryotic translation initiation factor 3 subunit K (eIF3k) as E1-binding host proteins with anti-CHIKV activities. We find that E1 residue V220, which has undergone positive selection, is indispensable for CHIKV production in macrophages, as its mutation attenuates E1 interaction with the host restriction factors SPCS3 and eIF3k. Finally, we show that the antiviral activity of eIF3k is translation-independent, and that CHIKV infection promotes eIF3k translocation from the nucleus to the cytoplasm, where it associates with SPCS3. These functions of CHIKV glycoproteins late in the viral life cycle provide a new example of an intracellular evolutionary arms race with host restriction factors, as well as potential targets for therapeutic intervention.

The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein.

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

The Pentamer glycoprotein complex inhibits viral Immediate Early transcription during Human Cytomegalovirus infections.

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

N123I mutation in the ALV-J receptor-binding domain region enhances viral replication ability by increasing the binding affinity with chNHE1.

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

RNA structures within Venezuelan equine encephalitis virus E1 alter macrophage replication fitness and contribute to viral emergence.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne +ssRNA virus belonging to the Togaviridae. VEEV is found throughout Central and South America and is responsible for periodic epidemic/epizootic outbreaks of febrile and encephalitic disease in equines and humans. Endemic/enzootic VEEV is transmitted between Culex mosquitoes and sylvatic rodents, whereas epidemic/epizootic VEEV is transmitted between mosquitoes and equids, which serve as amplification hosts during outbreaks. Epizootic VEEV emergence has been shown to arise from mutation of enzootic VEEV strains. Specifically, epizootic VEEV has been shown to acquire amino acid mutations in the E2 viral glycoprotein that facilitate viral entry and equine amplification. However, the abundance of synonymous mutations which accumulate across the epizootic VEEV genome suggests that other viral determinants such as RNA secondary structure may also play a role in VEEV emergence. In this study we identify novel RNA structures in the E1 gene which specifically alter replication fitness of epizootic VEEV in macrophages but not other cell types. We show that SNPs are conserved within epizootic lineages and that RNA structures are conserved across different lineages. We also identified several novel RNA-binding proteins that are necessary for altered macrophage replication. These results suggest that emergence of VEEV in nature requires multiple mutations across the viral genome, some of which alter cell-type specific replication fitness in an RNA structure-dependent manner.

Rapid adaptive evolution of avian leukosis virus subgroup J in response to biotechnologically induced host resistance.

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

Spatial connectivity matches direction selectivity in visual cortex.

The selectivity of neuronal responses arises from the architecture of excitatory and inhibitory connections. In the primary visual cortex, the selectivity of a neuron in layer 2/3 for stimulus orientation and direction is thought to arise from intracortical inputs that are similarly selective1-8. However, the excitatory inputs of a neuron can have diverse stimulus preferences1-4,6,7,9, and inhibitory inputs can be promiscuous10 and unselective11. Here we show that the excitatory and inhibitory intracortical connections to a layer 2/3 neuron accord with its selectivity by obeying precise spatial patterns. We used rabies tracing1,12 to label and functionally image the excitatory and inhibitory inputs to individual pyramidal neurons of layer 2/3 of the mouse visual cortex. Presynaptic excitatory neurons spanned layers 2/3 and 4 and were distributed coaxial to the preferred orientation of the postsynaptic neuron, favouring the region opposite to its preferred direction. By contrast, presynaptic inhibitory neurons resided within layer 2/3 and favoured locations near the postsynaptic neuron and ahead of its preferred direction. The direction selectivity of a postsynaptic neuron was unrelated to the selectivity of presynaptic neurons, but correlated with the spatial displacement between excitatory and inhibitory presynaptic ensembles. Similar asymmetric connectivity establishes direction selectivity in the retina13-17. This suggests that this circuit motif might be canonical in sensory processing.

Virological assessment of hospitalized patients with COVID-2019.

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.

Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.

Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo'.

On 21 February 2020, a resident of the municipality of Vo', a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection1. This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo' at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1-3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8-1.8%). Notably, 42.5% (95% CI: 31.5-54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9-9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections (P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon-Mann-Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures.

Polyvalent immunization elicits a synergistic broadly neutralizing immune response to hypervariable region 1 variants of hepatitis C virus.

A hepatitis C virus (HCV) vaccine is urgently needed. Vaccine development has been hindered by HCV's genetic diversity, particularly within the immunodominant hypervariable region 1 (HVR1). Here, we developed a strategy to elicit broadly neutralizing antibodies to HVR1, which had previously been considered infeasible. We first applied a unique information theory-based measure of genetic distance to evaluate phenotypic relatedness between HVR1 variants. These distances were used to model the structure of HVR1's sequence space, which was found to have five major clusters. Variants from each cluster were used to immunize mice individually, and as a pentavalent mixture. Sera obtained following immunization neutralized every variant in a diverse HCVpp panel (n = 10), including those resistant to monovalent immunization, and at higher mean titers (1/ID50 = 435) than a glycoprotein E2 (1/ID50 = 205) vaccine. This synergistic immune response offers a unique approach to overcoming antigenic variability and may be applicable to other highly mutable viruses.

Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.