ABSTRACT
A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic--Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.
ABSTRACT
SARS-CoV-2 adaptation to its human host is evidenced by the emergence of new viral lineages with distinct genotypic and phenotypic characteristics, termed variants of concern (VOCs). Particular VOCs have become sequentially dominant globally (Alpha, Delta, Omicron) with each evolving independently from the ancestral Wuhan strain. Omicron is notable for its large number of spike mutations1 found to promote immune escape and re-infection2. Most recently, Omicron BA.4 and BA.5 subvariants have emerged with increasing levels of adaptive immune escape threatening vaccine effectiveness and increasing hospitalisations1,3-12. Here, we demonstrate that the most recent Omicron variants have enhanced capacity to antagonise or evade human innate immune defenses. We find Omicron BA.4 and BA.5 replication is associated with reduced activation of epithelial innate immune responses versus earlier BA.1 and BA.2 subvariants. We also find enhanced expression of innate immune antagonist proteins Orf6 and N, similar to Alpha, suggesting common pathways of human adaptation and linking VOC dominance to improved innate immune evasion. We conclude that Omicron BA.4 and BA.5 have combined evolution of antibody escape with enhanced antagonism of human innate immunity to improve transmission and possibly reduce immune protection from severe disease.
ABSTRACT
Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron, the fifth VOC to be described, harbours 30 amino acid mutations in spike including 15 in the receptor-binding domain. Here, we demonstrate substantial evasion of neutralisation by Omicron in vitro using sera from vaccinated individuals. Importantly, these data are mirrored by a substantial reduction in real-world vaccine effectiveness that is partially restored by booster vaccination. We also demonstrate that Omicron does not induce cell syncytia and favours a TMPRSS2-independent endosomal entry pathway. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant.
ABSTRACT
The SARS-CoV-2 Omicron/BA.1 lineage emerged in late 2021 and rapidly displaced the Delta variant before being overtaken itself globally by, the Omicron/BA.2 lineage in early 2022. Here, we describe how Omicron BA.1 and BA.2 show a lower severity phenotype in a hamster model of pathogenicity which maps specifically to the spike gene. We further show that Omicron is attenuated in a lung cell line but replicates more rapidly, albeit to lower peak titres, in human primary nasal cells. This replication phenotype also maps to the spike gene. Omicron spike (including the emerging Omicron lineage BA.4) shows attenuated fusogenicity and a preference for cell entry via the endosomal route. We map the altered Omicron spike entry route and partially map the lower fusogenicity to the S2 domain, particularly the substitution N969K. Finally, we show that pseudovirus with Omicron spike, engineered in the S2 domain to confer a more Delta-like cell entry route retains the antigenic properties of Omicron. This shows a distinct separation between the genetic determinants of these two key Omicron phenotypes, raising the concerning possibility that future variants with large antigenic distance from currently circulating and vaccine strains will not necessarily display the lower intrinsic severity seen during Omicron infection.
ABSTRACT
Variants of concern (VOCs) of severe acute respiratory syndrome coronavirus type-2 (SARS-CoV-2) threaten the global response to the COVID-19 pandemic. The alpha (B.1.1.7) variant appeared in the UK became dominant in Europe and North America in early 2021. The Spike glycoprotein of alpha has acquired a number mutations including the P681H mutation in the polybasic cleavage site that has been suggested to enhance Spike cleavage. Here, we show that the alpha Spike protein confers a level of resistance to the effects of interferon-{beta} (IFN{beta}) in lung epithelial cells. This correlates with resistance to restriction mediated by interferon-induced transmembrane protein-2 (IFITM2) and a pronounced infection enhancement by IFITM3. Furthermore, the P681H mutation is necessary for comparative resistance to IFN{beta} in a molecularly cloned SARS-CoV-2 encoding alpha Spike. Overall, we suggest that in addition to adaptive immune escape, mutations associated with VOCs also confer replication advantage through adaptation to resist innate immunity.
ABSTRACT
SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.
ABSTRACT
Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spikes polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.
ABSTRACT
Vaccines are proving to be highly effective in controlling hospitalisation and deaths associated with SARS-CoV-2 infection but the emergence of viral variants with novel antigenic profiles threatens to diminish their efficacy. Assessment of the ability of sera from vaccine recipients to neutralise SARS-CoV-2 variants will inform the success of strategies for minimising COVID19 cases and the design of effective antigenic formulations. Here, we examine the sensitivity of variants of concern (VOCs) representative of the B.1.617.1 and B.1.617.2 (first associated with infections in India) and B.1.351 (first associated with infection in South Africa) lineages of SARS-CoV-2 to neutralisation by sera from individuals vaccinated with the BNT162b2 (Pfizer/BioNTech) and ChAdOx1 (Oxford/AstraZeneca) vaccines. Across all vaccinated individuals, the spike glycoproteins from B.1.617.1 and B.1.617.2 conferred reductions in neutralisation of 4.31 and 5.11-fold respectively. The reduction seen with the B.1.617.2 lineage approached that conferred by the glycoprotein from B.1.351 (South African) variant (6.29-fold reduction) that is known to be associated with reduced vaccine efficacy. Neutralising antibody titres elicited by vaccination with two doses of BNT162b2 were significantly higher than those elicited by vaccination with two doses of ChAdOx1. Fold decreases in the magnitude of neutralisation titre following two doses of BNT162b2, conferred reductions in titre of 7.77, 11.30 and 9.56-fold respectively to B.1.617.1, B.1.617.2 and B.1.351 pseudoviruses, the reduction in neutralisation of the delta variant B.1.617.2 surpassing that of B.1.351. Fold changes in those vaccinated with two doses of ChAdOx1 were 0.69, 4.01 and 1.48 respectively. The accumulation of mutations in these VOCs, and others, demonstrate the quantifiable risk of antigenic drift and subsequent reduction in vaccine efficacy. Accordingly, booster vaccines based on updated variants are likely to be required over time to prevent productive infection. This study also suggests that two dose regimes of vaccine are required for maximal BNT162b2 and ChAdOx1-induced immunity.
ABSTRACT
Cell autonomous antiviral defenses can inhibit the replication of viruses and reduce transmission and disease severity. To better understand the antiviral response to SARS-CoV-2, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that while some people can express a prenylated OAS1 variant, that is membrane-associated and blocks SARS-CoV-2 infection, other people express a cytosolic, nonprenylated OAS1 variant which does not detect SARS-CoV-2 (determined by the splice-acceptor SNP Rs10774671). Alleles encoding nonprenylated OAS1 predominate except in people of African descent. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response. Remarkably, approximately 55 million years ago, retrotransposition ablated the OAS1 prenylation signal in horseshoe bats (the presumed source of SARS-CoV-2). Thus, SARS-CoV-2 never had to adapt to evade this defense. As prenylated OAS1 is widespread in animals, the billions of people that lack a prenylated OAS1 could make humans particularly vulnerable to the spillover of coronaviruses from horseshoe bats.
ABSTRACT
SARS-CoV-2 infection is generally mild or asymptomatic in children but the biological basis for this is unclear. We studied the profile of antibody and cellular immunity in children aged 3-11 years in comparison with adults. Antibody responses against spike and receptor binding domain (RBD) were high in children and seroconversion boosted antibody responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Seroneutralisation assays against alpha, beta and delta SARS-CoV-2 variants demonstrated comparable neutralising activity between children and adults. T cell responses against spike were >2-fold higher in children compared to adults and displayed a TH1 cytokine profile. SARS-CoV-2 spike-specific T cells were also detected in many seronegative children, revealing pre-existing responses that were cross-reactive with seasonal Alpha and Beta-coronaviruses. Importantly, all children retained high antibody titres and cellular responses at 6 months after infection whilst relative antibody waning was seen in adults. Spike-specific responses in children also remained broadly stable beyond 12 months. Children thus distinctly generate robust, cross-reactive and sustained immune responses after SARS-CoV-2 infection with focussed specificity against spike protein. These observations demonstrate novel features of SARS-CoV-2-specific immune responses in children and may provide insight into their relative clinical protection. Furthermore, this information will help to guide the introduction of vaccination regimens in the paediatric population.
ABSTRACT
Remdesivir (RDV) is used widely for COVID-19 patients despite varying results in recent clinical trials. Here, we show how serially passaging SARS-CoV-2 in vitro in the presence of RDV selected for drug-resistant viral populations. We determined that the E802D mutation in the RNA-dependent RNA polymerase was sufficient to confer decreased RDV sensitivity without affecting viral fitness. Analysis of more than 200,000 sequences of globally circulating SARS-CoV-2 variants show no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we also observed changes in the Spike (i.e., H69 E484, N501, H655) corresponding to mutations identified in emerging SARS-CoV-2 variants indicating that they can arise in vitro in the absence of immune selection. This study illustrates SARS-CoV-2 genome plasticity and offers new perspectives on surveillance of viral variants. One Sentence SummarySARS-CoV-2 drug resistance & genome plasticity
ABSTRACT
The clinical outcome of COVID-19 has an extreme age, genetic and comorbidity bias that is thought to be driven by an impaired immune response to SARS-CoV-2, the causative agent of the disease. The unprecedented impact of COVID-19 on global health has resulted in multiple studies generating extensive gene expression datasets in a relatively short period of time. In order to better understand the immune dysregulation induced by SARS-CoV-2, we carried out a meta-analysis of these transcriptomics data available in the published literature. Datasets included both those available from SARS-CoV-2 infected cell lines in vitro and those from patient samples. We focused our analysis on the identification of viral perturbed host functions as captured by co-expressed gene module analysis. Transcriptomics data from lung biopsies and nasopharyngeal samples, as opposed to those available from other clinical samples and infected cell lines, provided key signatures on the role of the hosts immune response on COVID-19 pathogenesis. For example, severity of infection and patients age are linked to the absence of stimulation of the RIG-I-like receptor signaling pathway, a known critical immediate line of defense against RNA viral infections that triggers type-I interferon responses. In addition, co-expression analysis of age-stratified transcriptional data provided evidence that signatures of key immune response pathways are perturbed in older COVID-19 patients. In particular, dysregulation of antigen-presenting components, down-regulation of cell cycle mechanisms and signatures of hyper-enriched monocytes were strongly correlated with the age of older individuals infected with SARS-CoV-2. Collectively, our meta-analysis highlights the ability of transcriptomics and gene-module analysis of aggregated datasets to aid our improved understanding of the host-specific disease mechanisms underpinning COVID-19.
ABSTRACT
SARS-CoV-2, the causative agent of COVID-19, emerged in Wuhan, China in December 2019 and spread rapidly throughout the world. Understanding the introductions of this new coronavirus in different settings may assist control efforts and the establishment of frameworks to support rapid response in future infectious disease outbreaks. We investigated the first four weeks of emergence of the SARS-CoV-2 virus in Scotland after the first case reported on the 1st March 2020. We obtained full genome sequences from 452 individuals with a laboratory-confirmed diagnosis of COVID-19, representing 20% of all cases until 1st April 2020 (n=2310). This permitted a genomic epidemiology approach to study the introductions and spread of the SARS-2 virus in Scotland. From combined phylogenetic and epidemiological analysis, we estimated at least 113 introductions of SARS-CoV-2 into Scotland during this period. Clusters containing multiple sequences suggestive of onward transmission occurred in 48/86 (56%). 42/86 (51%) clusters had no known international travel history indicating undetected introductions. The majority of viral sequences were most closely related to those circulating in other European countries, including Italy, Austria and Spain. Travel-associated introductions of SARS-CoV-2 into Scotland predated travel restrictions in the UK and other European countries. The first local transmission occurred three days after the first case. A shift from travel-associated to sustained community transmission was apparent after only 11 days. Undetected introductions occurred prior to the first known case of COVID-19. Earlier travel restrictions and quarantine measures might have resulted in fewer introductions into Scotland, thereby reducing the number of cases and the subsequent burden on health services. The high number of introductions and transmission rates were likely to have impacted on national contact tracing efforts. Our results also demonstrate that local real-time genomic epidemiology can be used to monitor transmission clusters and facilitate control efforts to restrict the spread of COVID-19. FundingMRC (MC UU 1201412), UKRI/Wellcome (COG-UK), Wellcome Trust Collaborator Award (206298/Z/17/Z - ARTIC Network; TCW Wellcome Trust Award 204802/Z/16/Z Research in contextO_ST_ABSEvidence before this studyC_ST_ABSCoronavirus disease-2019 (COVID-19) was first diagnosed in Scotland on the 1st of March 2020 following the emergence of the causative severe acute respiratory system coronavirus 2 (SARS-CoV-2) virus in China in December 2019. During the first month of the outbreak in Scotland, 2310 positive cases of COVID-19 were detected, associated with 1832 hospital admissions, 207 intensive care admissions and 126 deaths. The number of introductions into Scotland and the source of those introductions was not known prior to this study. Added value of this studyUsing a combined phylogenetic and epidemiological approach following real-time next generation sequencing of 452 SARS-CoV-2 samples, it was estimated that the virus was introduced to Scotland on at least 113 occasions, mostly from other European countries, including Italy, Austria and Spain. Localised outbreaks occurred in the community across multiple Scottish health boards, within healthcare facilities and an international conference and community transmission was established rapidly, before local and international lockdown measures were introduced.
