Editor's evaluation: SARS-CoV-2 host-shutoff impacts innate NK cell functions, but antibody-dependent NK activity is strongly activated through non-spike antibodies
Year: 2021
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design. Editor's evaluation By using a systematic proteomics approach, the authors demonstrated that SARS-CoV2 remodels the plasma membrane of infected human epithelial cells. Although the study indicates the manipulation of different immune response pathways, it seems that in the focus of viral immunoevasion are natural killer (NK) cells, which play a crucial role in controlling early viral infection. However, antibody-dependent NK cell activation was observed later in the disease process. These findings could have implications for the understanding of SARS-CoV-2 control by the immune system and vaccine development. https://doi.org/10.7554/eLife.74489.sa0 Decision letter eLife's review process Introduction COVID-19 studies have focused on interferon responses during the early innate phase of infection, and neutralising antibodies and virus-specific T-cells during the adaptive phase. In contrast, and despite their importance in antiviral protection (Pierce et al., 2020), considerably less is known about the role of Natural Killer (NK) cells in infection. NK cells bridge the innate and adaptive responses, and individuals with NK cell deficiencies suffer severe viral infections (Orange, 2013). NK cells respond rapidly to viruses, directly killing infected cells by releasing cytotoxic granules. They promote inflammation through the release of TNFα and IFNγ, and influence the induction of both B- and T-cell responses. Once virus-specific antibodies are induced, antibody-dependent NK activation (ADNKA) is generated through antibody linking cell-surface viral antigens with NK cell Fc receptors and subsequent antibody-dependent cellular cytotoxicity (ADCC). The majority of studies on NK cells have focussed on their adaptive roles in established COVID-19 infection, with highly activated NK cells seen in both the periphery and the lung (Maucourant et al., 2020). The genetic loss of NKG2C (a hallmark of adaptive NK cells, capable of enhanced ADCC) is also a risk factor for severe disease (Vietzen et al., 2021b), implying a protective role in COVID-19 infection. In support of this, in animal models, Fc-mediated functions correlate with protection in vaccination (Gorman et al., 2021), and monoclonal antibody-mediated control of SARS-CoV-2 infection is enhanced by Fc-dependent mechanisms (Chan et al., 2020; Schäfer et al., 2021; Winkler et al., 2020; Suryadevara et al., 2021; Winkler et al., 2021; Yamin et al., 2021), which enable control of disease even in the absence of neutralising activity (Beaudoin-Bussières et al., 2021). Furthermore, although neutralising monoclonal antibodies are effective when administered prophylactically, Fc-activity is required for efficacy when administered to animals therapeutically (Yamin et al., 2021). These animal studies demonstrate potentially important roles for adaptive NK cell responses in COVID-19. Despite this, our understanding of ADCC following infection and vaccination in humans remains limited. Studies to date have assumed spike protein is the dominant ADCC target, and have tested plate-bound protein or transfected cells (Barrett et al., 2021; Herman et al., 2021; Zohar et al., 2020; Tauzin et al., 2021; Anand et al., 2021; Lee et al., 2020; Dufloo et al., 2020; Tortorici et al., 2020; McCallum et al., 2021; Cathcart et al., 2021). Using these systems, they have shown that ADNKA-inducing antibodies are generated following SARS-CoV-2 infection (Herman et al., 2021; Anand et al., 2021; Lee et al., 2020; Dufloo et al., 2020), are induced by vaccination in humans (Barrett et al., 2021; Tauzin et al., 2021), and can be mediated by a subset of neutralising monoclonal antibodies targeting the spike protein (Tortorici et al., 2020; McCallum et al., 2021; Cathcart et al., 2021). However, these responses have not been tested against SARS-CoV-2 infected cells; the presence, abundance, accessibility, and conformation of viral glycoproteins can be very different during productive infection. Furthermore, although viral entry glycoproteins such as spike can be found on the infected cell surface, and can mediate ADCC, other viral proteins can be the dominant mediators of ADCC during infection; indeed, we have shown that in other viruses, non-structural proteins are often the dominant targets (Vlahava et al., 2021). In addition, there is little information on the NK cell response to SARS-CoV-2 infected cells during the innate phase of infection, despite this process being important to virus pathogenesis; successful viruses must counteract robust NK activation to establish infection, and their ability to do this is critical to their ability to cause disease (Patel et al., 2018; Berry et al., 2020). Whether SARS-CoV-2’s ability to infect humans is dependent on viral evasion of innate cellular immunity, is unknown. NK cell activation is complex and depends on the balance of ligands for a wide range of inhibitory or activating receptors (Kumar, 2018). These ligands can be induced on the surface of target cells in response to stress, infection, or transformation. For example, the stress ligands MICA, MICB, and ULBP1-6, all bind to the ubiquitously expressed activating receptor NKG2D. To limit NK cell activation, many viruses manipulate these NK activating ligands, reducing NK-cell-mediated control (Patel et al., 2018). Whether similar manipulations underlie the ability of SARS-CoV-2 to cause disease in humans remains unclear. To address these critical gaps in our understanding of the SARS-CoV-2-specific NK cell response, we used quantitative proteomics to determine how SARS-CoV-2 infection affected cell surface protein expression (Rihn et al., 2021). We found that SARS-CoV-2-infected cells downregulate multiple activating NK ligands, resulting in a reduced NK cell response to infected cells. This suggests a novel mechanism by which SARS-CoV-2 counteracts innate immunity early in infection, to establish disease. The development of humoral immunity following natural infection resulted in robust ADNKA against infected cells, and high levels of proinflammatory cytokines; these responses persisted for at least 6 months in most patients. Proteomics identified four cell-surface viral proteins capable of driving this response, leading to the surprising discovery that ADNKA is not primarily driven by spike antibody, but was dominated by antibody to other viral proteins such as nucleocapsid, membrane and ORF3a; this defines a novel role for antibodies targeting the immunodominant nucleocapsid protein. In agreement with these findings, and in contrast to plate-bound protein or transfected cells, spike was a poor activator of ADNKA following vaccination when tested using infected cells. This implies that the breadth and magnitude of ADNKA responses is limited in current vaccination strategies. The inclusion of antigens such as nucleocapsid would recruit additional effector mechanisms, and may help maintain vaccine efficacy following mutation of spike in naturally circulating virus variants. Results SARS-CoV-2 remodels the plasma membrane proteome Immune cells recognise and interact with infected cells through cell surface ligands. We therefore wanted to gain a comprehensive and unbiased overview of how SARS-CoV-2 infection changes the plasma membrane protein landscape. We performed plasma membrane enrichment through selective aminooxy-biotinylation (Plasma Membrane Profiling; PMP), a methodology that we previously developed (Weekes et al., 2010; Weekes et al., 2014) and have validated extensively against human immunodeficiency virus (HIV) (Matheson et al., 2015), human cytomegalovirus (HCMV) (Vlahava et al., 2021; Weekes et al., 2014; Fielding et al., 2017; Hsu et al., 2015; Weekes et al., 2013), and herpes simplexvirus (HSV) (Soh et al., 2020) infection. High infection rates are critical to minimise confounding effects from bystander cells. We therefore applied PMP to SARS-CoV-2 infected A549-ACE2-TMPRSS2 (AAT) cells, which are highly permissive to SARS-CoV-2 (Rihn et al., 2021) and infection at a high multiplicity of infection (MOI) results in over 90% infection by 24 hr (Figure 1A). We utilised TMT-based quantification to compare plasma membrane (PM) protein abundance in uninfected cells, and cells infected for 12, 24, and 48 hr (Figure 1B). In total, 4953 proteins were quantitated, including 2227 proteins with gene ontology annotation associated with the cell surface and plasma membrane (Figure 1—figure supplement 1A). Importantly however, the plasma membrane annotated proteins made up nearly 80% of the total protein abundance, indicating a high degree of enrichment for plasma membrane proteins, comparable with our previous PMP datasets (Vlahava et al., 2021; Weekes et al., 2010; Weekes et al., 2014; Matheson et al., 2015; Fielding et al., 2017; Hsu et al., 2015; Weekes et al., 2013; Soh et al., 2020). The detection of low abundance proteins that are not plasma-membrane annotated reflects a combination of incorrect annotations, and contaminating intracellular proteins that were incompletely removed following PMP. Figure 1 with 3 supplements see all Download asset Open asset SARS-CoV-2 remodels the plasma membrane proteome. (A) AAT cells were infected with SARS-CoV-2 in biological triplicate (MOI = 5). Twenty-four hr later, they were detached with trypsin, fixed and permeabilised, stained for Spike protein, and analysed by flow cytometry. (B) Schematic of plasma membrane profiling and analysis pathway. (C). Examples of temporal profiles of viral and cellular genes, fold change is compared to 0 hr timepoint. Data points show mean ± SD. (D). Left – heat map of the 914 significantly changing proteins clustered by k-mean, colour indicates log2 fold change compared to 0 hr, right, Z-score normalised temporal profiles of proteins within each cluster. Five SARS-CoV-2 viral proteins were detected (see later), including spike, ORF3A, and membrane, which have previously been annotated as having a cell surface localisation (Figure 1C). Consistent with prior reports, the viral receptor ACE2 was strongly downregulated after infection (Patra et al., 2020), while TMPRSS2 expression was unaffected (Figure 1C). Overall, of the 2227 PM proteins quantified with more than one peptide, 914 showed changes which were both significant and with a greater than 1.5-fold alteration over the course of the experiment. Clustering of proteins by their temporal pattern of change has previously shown utility in defining classes of regulated proteins and predicting underlying mechanisms (Matheson et al., 2015; Greenwood et al., 2016). Here, we used K-means clustering to define five groups of significantly altered proteins by their temporal profile. To assess the innate immune response triggered by SARS-CoV-2 infection, the clusters were interrogated for the presence of a predefined list of interferon alpha-inducible genes. Thirteen of the 23 detected PM genes associated with the interferon response fell into cluster 5 (Figure 1—figure supplement 1B, Supplementary file 2), which describes proteins with an increase in expression at 48 hr, examples shown in Figure 1—figure supplement 1C. As the SARS-CoV-2 viral replication cycle is completed in less than 24 hr (Lei et al., 2020), SARS-CoV-2 is able to evade innate immune recognition and/or suppress IFN signalling until long after virus egress has been achieved. To identify potential innate and adaptive immune evasion strategies employed by SARS-CoV-2, we focused on cluster 1, which describes proteins downregulated from early in the time course. This cluster was subject to gene ontology and pathway analysis to define proteins related by functional and structural similarities (Figure 2). Several classifications enriched in this cluster are particularly relevant, including the downregulation of several cytokine receptors, including IFNAR1. Much of the SARS-CoV-2 genome is dedicated to antagonising a type I interferon response within an infected cell (Lei et al., 2020) and the plasma membrane downregulation of IFNAR1 presents a previously undescribed strategy to prevent signalling from exogenous IFN. Figure 2 with 1 supplement see all Download asset Open asset SARS-CoV-2 manipulates multiple classes of plasma membrane proteins. Enriched gene ontology and pathway annotations of proteins downregulated in temporal cluster 1 were condensed to groups of related terms, bar chart shows the enrichment score of these groups. Examples of annotation terms falling within each group are shown underlined with Bejamani-Hochberg adjusted p-values in parentheses, alongside four examples of proteins for each annotation. Also of note is the downregulation of heparan sulfate proteoglycans, the syndecan (SDC), glypican (GPC), and neurophillin (NRP) proteins, which together encompass the families of heparan sulfate modified proteins expressed on epithelial cells (Sarrazin et al., 2011). NRP1 is a cell entry factor for SARS-CoV-2 (Cantuti-Castelvetri et al., 2020; Daly et al., 2020), and SARS-CoV-2 spike is also reported to bind heparan sulphated proteins (Clausen et al., 2020). The decrease in cell surface expression of heparan sulfated proteins may therefore enable the egress of viral particles and prevent super-infection, and is reminiscent of the capacity of other viruses to downregulate both primary viral receptors and co-receptors (Landi et al., 2011). SARS-CoV-2 inhibits the synthesis of multiple NK cell ligands and reduces NK activation Our experience with PMP has emphasised the modulation of NK ligands as an important immune evasion mechanism (Fielding et al., 2017). As these ligands are poorly annotated in public databases, we investigated the PMP dataset for previously described ligands (Kumar, 2018), of which 18 were detected (Figure 2—figure supplement 1). MHC-I proteins HLA-B to HLA-E were upregulated in cluster 5, likely reflecting a late IFN-mediated upregulation, while HLA-A was unchanged. By contrast, the activating NK-cell ligands MICA, ULBP2, B7-H6 (NCR3LG1), and Nectin1 (which is inhibitory in mice, but recently reported as activating in humans Holmes, 2019) were significantly downregulated in cluster 1. This was confirmed by flow cytometry in multiple cell types (Figure 3A, Figure 3—figure supplement 1A). These responses were dependent on virus replication, because no alterations in ligand abundance were observed when virus was heat-inactivated (Figure 3—figure supplement 2A). Figure 3 with 2 supplements see all Download asset Open asset SARS-CoV-2 infection inhibits the expression of multiple NK ligands. (A) AAT cells were either mock infected, or infected with SARS-CoV-2 for 24 hr (MOI = 5), detached using TrypLE, then stained for the indicated NK cell receptors before being analysed by flow cytometry (bottom). Plots for the same proteins from PMP are included for reference (top) (B) AAT cells were infected with RAd-MICA, RAd-ULBP2, or RAd-B7-H6, then after 48 hr either mock infected, or infected with SARS-CoV-2 (SCV2) for a further 24 hr (MOI = 5). Lysates were either kept undigested, or digested with EndoH (E), or PNGaseF (P), then western blotted for the indicated proteins. Mature cell surface glycoproteins are resistant to EndoH, while immature (ER resident) forms are digested and therefore run with a smaller mass following EndoH digestion (C) AAT cells were infected with RAd-MICA or RAd-B7-H6, then after 24 hr either mock infected, or infected with SARS-CoV-2 (SCV2), and treated with MG132 (M) or Leupeptin (L).Twenty-four hour later lysates were made, and analysed by western blot for the indicated proteins. (Note: The additional band seen when staining for actin in the presence of SARS-CoV-2 may be non-specific). Many of the best characterised mechanisms by which viruses inhibit the function of NK ligands involve inhibition of cell-surface transport and/or proteasomal or lysosomal degradation of the mature protein (Patel et al., 2018; Berry et al., 2020). To determine whether SARS-CoV-2 targeted NK ligands in the same way we measured their abundance, and their sensitivity to digestion with EndoH, which cleaves N-linked glycans from proteins that have not yet transited (or have been retained in) the endoplasmic reticulum (ER). Digestion with PNGaseF (which cleaves N-linked glycans irrespective of subcellular trafficking) served as a control to show the effect of complete deglycosylation. We were unable to detect endogenous NK ligands by western blot (data not shown), and therefore overexpressed MICA, ULBP2, and B7-H6 using replication deficient adenovirus vectors (RAd). Following infection with SARS-CoV-2, levels of all three NK ligands were substantially reduced, however there was no alteration in the pattern of EndoH sensitivity (Figure 3B). Thus, SARS-CoV-2 infection does not result in intracellular retention of NK ligands, but instead reduces the overall abundance of protein. To determine whether this occurred by inhibition of synthesis or degradation of mature protein, we treated cells with proteasomal (MG132) or lysosomal (Leupeptin) inhibitors after infection (Figure 3C). When tested with overexpressed MICA or B7-H6 (ULBP2 was not tested), MG132 did not result in recovery of NK ligand levels. Leupeptin resulted in a small recovery of B7-H6; however, this was seen in both Mock and infected cells, implying that it reflects constitutive turnover of the ligand rather than virus-specific targeting, and recovery in infected cells remained reduced compared to untreated mock infected cells. Overall, these results are consistent with the virus targeting NK ligands at the level of synthesis, rather than post-translationally. We therefore investigated whether NK ligands were particularly sensitive to inhibition of synthesis, by treating cells expressing MICA, B7-H6, or a control protein (GFP) with the translation inhibitor cycloheximide (ULBP2 was not tested). This resulted in rapid loss of MICA, ULBP2, and B7-H6, but not GFP (Figure 4A). Finally, to determine which viral proteins were responsible for these effects, we transfected cells with a library of previously validated plasmids expressing all 29 SARS-CoV-2 ORFs individually (Gordon et al., 2020), and assessed them for levels of NK ligands on the cell surface. Only two SARS-CoV-2 ORFs resulted in a reduction of staining; Nsp1 and (Figure For MICA and ULBP2, resulted in the with Nsp1 being For Nectin1 and B7-H6, effects were but both Nsp1 and resulted in similar Figure with 1 supplement see all Download asset Open asset SARS-CoV-2 infection the expression of multiple NK ligands, NK activation. (A) AAT cells were infected with RAd-MICA, RAd-B7-H6, or then after 24 hr either mock infected, infected with SARS-CoV-2 (SCV2), or treated with Twenty-four hr later, cells were and analysed by western blot for the indicated proteins (Note: The additional band seen when staining for actin in the presence of SARS-CoV-2 may be non-specific). (B) cells were transfected with expression plasmids for Nsp1 or Nsp14, or GFP as a Twenty-four hr later, cells were detached with and stained for the indicated proteins. AAT cells were either mock infected, or infected with SARS-CoV-2 for 24 (MOI = 5), detached with and with interferon for 5 hr in the presence of and antibody, before staining for and were then permeabilised, and stained for TNFα and were on NK cells, and the of cells for and were run in with data shown from four using different Nsp1 and are in the ability of SARS-CoV-2 to and inhibit protein translation and and these have been in their ability to counteract the interferon response within the infected cell et al., 2021; Hsu et al., 2021). with our this data suggests that NK ligands are highly to inhibition of synthesis, and that SARS-CoV-2 manipulation of protein could the NK response in to the interferon To determine whether this was the we SARS-CoV-2 infected cells with and measured of NK cells (Figure Figure 3—figure supplement of (Figure and TNFα (Figure Figure 3—figure supplement Consistent with data using other viruses et al., was on a of NK cells than and all three of NK activation were significantly reduced in SARS-CoV-2 infected mock infected cells, that the virus has to inhibit NK cell responses. results were seen at both 24 hr and 48 hr (data not NK-cell evasion is overcome by antibody-dependent activation In to their role in innate immunity through with activating and inhibitory NK receptors early in infection, NK cells play additional roles following the development of adaptive immunity in which with the Fc of antibodies to targets on the surface of infected cells. To this of NK were with infected cells in the presence of from individuals that were or for NK cell more in response to antibody These NK cells can be by the presence of NKG2C and on the cell surface and 2020). However, NKG2C is only found in a subset of are and a of these individuals are to a in the As a NKG2C be used as a in all We have previously shown that is to identify NK cells in all and activation of this with the ability of antibodies to mediate ADCC (Fielding et al., 2021; et al., 2018). We therefore focussed on this of activation were observed in the of smaller magnitude activation was for by against cells (Figure as as of (Figure and TNFα (Figure were all significantly in the presence of a range of different NK (Figure ADNKA was dependent on virus replication, heat virus did not to NK (Figure supplement 1A). NK cells were activated whether NK cells or were used (Figure supplement these responses are to of NK cells, rather than other cells to antibodies and releasing NK immune evasion can therefore be overcome through antibody dependent mechanisms, that in to neutralising SARS-CoV-2 antibodies may virus through Figure 5 with 1 supplement see all Download asset Open asset SARS-CoV-2 inhibition of NK activation can be overcome AAT cells were either mock infected, or infected with SARS-CoV-2 for 24 hr (MOI = 5), detached using TrypLE, then with in the presence of antibody, and from were or for 5 hr, cells were stained for and and stained for and then analysed by flow cytometry. were performed using and the of cells for TNFα and were were performed in triplicate from multiple were also compared were performed using a of from then the the from or severe were In to for ADNKA were tested for their ability to SARS-CoV-2 infection of cells, and the then compared to the for analysis is The magnitude of the neutralising antibody response to SARS-CoV-2 with of individuals with more severe disease levels of neutralising antibody et al., 2020; et al., 2021; et al., 2020). Although responses levels of circulating antibody can decrease over time following of such that in infected they after a months et al., 2020; et al., 2021; et al., 2020). To determine whether the same was of ADCC responses, we assessed ADNKA in a of with known disease for were (Figure ADNKA responses were of greater magnitude in individuals with more severe with responses during the with strong responses were over while weaker responses by was a the ability of antibodies to SARS-CoV-2, and to ADNKA in response to SARS-CoV-2 (Figure with a of Thus, although the antibodies these may be induced in a similar the antibodies the two responses may not be SARS-CoV-2 ORFs mediate ADCC has been a focus on spike as an activator of ADCC (Barrett et al., 2021; Herman et al., 2021; Zohar et al., 2020; Tauzin et al., 2021; Anand et al., 2021; Lee et al., 2020; Dufloo et al., 2020; Tortorici et al., 2020; McCallum et al., 2021; Cathcart et al., 2021). is a potential target during infection despite the that particles rather than from the cell membrane, both our PMP and flow analysis detected levels of spike protein on the infected cell surface (Figure Figure supplement However, our studies with other viruses have demonstrated that although viral proteins in and entry are found on the cell surface, and can be by antibody, they are not the mediators of ADCC activity (Vlahava et al., 2021). PMP also detected membrane nucleocapsid ORF3a, and on the cell surface. is expressed as a which is into multiple non-structural proteins of of the did not Furthermore, the abundance of viral proteins in was only at very low levels of all viral proteins, significantly than other SARS-CoV-2 it is that are we performed analysis of all viral proteins for the presence of potential or
Source: eLife
Author: Stipan Jonjic
Institution: University of Rijeka
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4285323372