SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2–6). The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7–10).Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11–15). However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17). S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20–22).Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24). Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33–37) and may have contributed to the COVID-19 pandemic and disease progression (38–43).In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.     

Targeting stem-loop 1 of the SARS-CoV-2 5′ UTR to suppress viral translation and Nsp1 evasion

SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5′ untranslated region (5′ UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5′ UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2–induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus’ own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the infectious disease COVID-19, is a highly contagious and deadly virus with fast person-to-person transmission and potent pathogenicity (1, 2). It is an enveloped, single-stranded betacoronavirus that contains a positive-sense RNA genome of about 29.9 kb (3–5). The SARS-CoV-2 genome codes for two large overlapping open reading frames (ORF1a and ORF1b) and a variety of structural and nonstructural accessory proteins (6). Upon infection, the polyproteins ORF1a and ORF1b are synthesized by host machinery and proteolytically cleaved into 16 mature nonstructural proteins, namely Nsp1 to Nsp16 (1, 6, 7).Nsp1 is a critical virulence factor of coronaviruses and plays key roles in suppressing host gene expression, which facilitates viral replication and immune evasion, presumably by repurposing the host translational machinery for viral production and preventing the induction of type I interferons (IFNs) (8–11). It has been shown that SARS-CoV Nsp1 effectively suppresses the translation of host messenger RNAs (mRNAs) by directly binding to the 40S small ribosomal subunit (12, 13). Recent cryoelectron microscopy (cryo-EM) structures of SARS-CoV-2 Nsp1 indeed reveal the binding of its C-terminal domain (CT) to the mRNA entry channel of the 40S subunit (14–17), which contributes to blocking translation. These structural data are further supported by experiments demonstrating that Nsp1 binding to the 40S ribosome requires an open-head conformation induced by core elongation initiation factors and that Nsp1 cannot bind to a 40S with an mRNA already occupying the entry channel (18).Besides directly inhibiting mRNA translation, Nsp1 has also been shown to reduce the available pool of host cytosolic mRNAs by both promoting their degradation and inhibiting their nuclear export (19–21). Mutants of Nsp1 that disrupt ribosome binding also abolish mRNA degradation, suggesting that the degradation is likely downstream of the Nsp1 translational block, and these two processes likely synergize to shut off host protein expression (12, 22).Previous studies on SARS-CoV and SARS-CoV-2 have implicated stem-loop 1 (SL1) in the leader region of the 5′ untranslated region (5′ UTR) in protecting the virus against Nsp1-mediated mRNA translation inhibition (9, 17, 22, 23). However, how SARS-CoV-2 overcomes Nsp1-mediated translation suppression for its replication and whether this mechanism can be targeted for therapeutic intervention remain open questions. Here, we show that SARS-CoV-2 depends on SL1 to escape Nsp1 suppression to effectively switch the translational machinery from synthesizing host proteins to making viral proteins, and that both the CT and the N-terminal domain (NT) are required for the transition from host to viral translation. The latter is supported by complementary experiments in a study released while this paper was in preparation (24). We further show that SL1 can be targeted by locked nucleic acid (LNA) antisense oligonucleotides to prevent the SARS-CoV-2 5′ UTR from evading its own translational suppression to potently inhibit viral replication.     

QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site—the FCS, loop length, and glycosylation—are required for efficient SARS-CoV-2 replication and pathogenesis.

SARS-CoV-2 emerged in late 2019 and has caused the largest pandemic since the 1918 influenza outbreak (1). An unusual feature of SARS-CoV-2 is the presence of a furin cleavage site (FCS) in its spike protein (2). The CoV spike is a trimer of spike proteins composed of the S1 and S2 subunits, responsible for receptor binding and membrane fusion, respectively (1). After receptor binding, the spike protein is proteolytically cleaved at the S1/S2 and S2′ sites to activate the fusion machinery. For SARS-CoV-2, the spike protein contains a novel cleavage motif recognized by the host cell furin protease (PRRAR) directly upstream of the S1/S2 cleavage site that facilitates cleavage prior to virion release from the producer cell. This FCS, not found in other group 2B CoVs, plays a key role in spike processing, infectivity, and pathogenesis as shown by our group and others (3, 4).Importantly, another novel amino acid motif, QTQTN, is found directly upstream of the FCS. This QTQTN motif, also absent in other group 2B CoVs, is often deleted and has been pervasive in cultured virus stocks of the alpha, beta, and delta variants (5–8). In addition, the QTQTN deletion has been observed in a small subset of patient samples as well (9–11). Because this deletion has been frequently identified, we set out to characterize it and determine whether it has consequences for viral replication and virulence. Using our infectious clone (12, 13), we demonstrated that the loss of this motif attenuates SARS-CoV-2 replication in respiratory cells in vitro and pathogenesis in hamsters. The QTQTN deletion results in reduced spike cleavage and diminished capacity to use serine proteases on the cell surface for entry. Importantly, mutations of glycosylation-enabling residues in the QTQTN motif results in similar replication attenuation despite intact spike processing. Together, our results highlight elements in the SARS-CoV-2 spike in addition to the FCS that contribute to increased replication and pathogenesis.     

A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2′-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer–spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2020–2021 has launched a global quest to find new molecular tools for the detection of the virus and treatment of the potential deadly disease it causes, COVID-19. Despite the exceptional efforts worldwide for containment and unprecedented technological progress in vaccine development, the challenge to find an effective cure remains, due to the limited access to SARS-CoV-2 vaccines, particularly in developing countries, and the emergence of new viral strains that can evade immune responses and potentially compromise the efficacy of current vaccines. Therefore, it is of utmost importance to focus efforts on developing affordable and easy-to-produce antiviral molecules against SARS-CoV-2 infection.Like other coronaviruses, SARS-CoV-2 expresses a surface spike (S) glycoprotein which is composed of two domains (S1 and S2) (1) and forms a trimeric structure capable of interacting with human cells (2, 3). In particular, the receptor binding domain (RBD) located on the S1 subunit of the spike protein binds with high affinity to human angiotensin-converting enzyme 2 (ACE2) (4, 5), which, in conjunction with the associated transmembrane protease, serine 2 (TMPRSS2), facilitates viral uptake. Efforts to neutralize viral infection have therefore mainly focused on inhibiting the spike–ACE2 interaction. Antibodies (Abs) have been developed and are currently used for SARS-CoV-2 detection, and some, primarily those targeting RBD, show therapeutic potential due to their potent neutralizing effect (6–13). However, the high costs of Ab production, the use of animals to generate them, and their poor stability at ambient temperatures remain a disadvantage. Moreover, Ab immunogenicity and the risk of Ab-dependent enhancement of infection associated with Fc-containing Abs put their therapeutic potential at risk (14).V_HH Abs or nanobodies raised to the spike protein may overcome some of these drawbacks (15–20) but are more prone to immunological response (21, 22). Interesting alternatives such as de novo proteins based on the host ACE2 receptor (23) and other synthetic molecules (24) have been investigated and may, if potential immunogenicity and stability problems are solved, help develop efficient detection methods and drugs.Nucleic acid-based aptamers have gained increased attention as alternatives to Abs due to their ease of production, low immunogenicity, high thermal and chemical stability, and smaller size, while they still retain comparable target binding and specificity. Aptamers are short single-stranded oligonucleotides, developed through an in vitro selection process termed SELEX (systematic evolution of ligands by exponential enrichment), that bind with high affinity and selectivity to cognate targets (25–27). During the last few decades, a wide variety of aptamers binding to diverse biologically relevant targets (28), including viruses (29, 30), have been identified. However, selection of aptamers targeting spike protein has proven difficult. An explanation for this may be that highly glycosylated proteins such as SARS-CoV-2 spike are challenging to target with nucleic acid-based binders. Indeed, to date, there are only a few reports on DNA aptamers targeting SARS-CoV-2 spike where the authors report leading aptamers with affinities in the nanomolar range (31–35).Here we report the selection and characterization of a serum-stable RNA aptamer, RBD-PB6, that binds with nanomolar affinity to the RBD of SARS-CoV-2 spike protein and neutralizes viral infectivity. The aptamer contains 2′-fluoro pyrimidine modifications to increase its chemical stability and resistance to nucleases (36, 37), and it shows high selectivity to SARS-CoV-2 and related strains, including alpha and beta. Aptamer multimerization strongly enhances its affinity to the picomolar range as well as its SARS-CoV-2 neutralizing potency. These unique features open avenues for developing inexpensive, fast, and reliable detection platforms for SARS-CoV-2 and therapeutic application for COVID-19.     

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV−like Sarbecovirus vaccine development.

The COVID-19 pandemic, precipitated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to threaten global public health and economies. Threats of future outbreaks also loom, as evidenced by three emergent SARS-like diseases caused by zoonotic Betacoronaviruses in the last two decades. While several emergency use authorized (EUA) vaccines currently in use are expected to curb both disease and transmission of SARS-CoV-2 (1–6), the emergence of circulating variants of concern (VOCs) less sensitive to vaccine-elicited immunity has raised concerns for sustained vaccine efficacy (7). Logistical challenges of vaccine production, distribution, storage, and access for these vaccines must be resolved to achieve resolution to the pandemic (8, 9). The rapid and unparalleled spread of SARS-CoV-2 has driven an urgent need to deploy scalable vaccine platforms to combat the ongoing pandemic and mitigate future outbreaks.Current vaccines primarily focus the immune response on the spike glycoprotein (S) as it mediates host cell viral fusion and entry. The receptor-binding domain (RBD) of S engages the primary host cell receptor, angiotensin-converting enzyme 2 (ACE2), for both SARS-CoV-2 and SARS-CoV-1, making RBD a promising domain for vaccine-elicited immune focus (10–12). Moreover, many of the potently neutralizing monoclonal antibodies isolated against SARS-CoV-2 target the RBD (13, 14). Vaccination of nonhuman primates (NHPs) with RBD-encoding RNA or DNA protects against respiratory tract challenge, indicating that immune responses to the RBD can prevent viral replication (15, 16). RBD vaccination also elicits cross-reactive responses to circulating SARS-CoV-2 VOCs in both animals and humans (17, 18), with decrements against the B.1.351 variant similar to that seen with S immunogens (19). The breadth of RBD immunogenicity is further supported by the ability of RBD-specific monoclonal antibodies isolated from SARS-CoV-1 convalescent individuals to cross-neutralize SARS-CoV-2 (20, 21). These findings suggest potential for RBD-based vaccines being efficacious against SARS-CoV-2 variants and other related coronavirus species.Approaches to improve immunogenicity of S or RBD protein vaccines include optimizing antigen presentation and coformulating with adjuvants to enhance the protective immunity. One common approach to enhance the induction of adaptive immune responses is the multimeric presentation of antigen, for example, on the surface of nanoparticles or virus-like particles (22). Presenting RBD in ordered, multivalent arrays on the surface of self-assembling protein nanoparticles is immunogenic and efficacious in animals (23–28), with improved immunogenicity relative to monomeric soluble RBD and cross-reactive responses to variants (17, 24, 26). However, it is unknown whether RBD nanoparticle vaccines protect against infection in primates, which have become a standard model for benchmarking performance of vaccine candidates by virological and immunologic endpoints. Liposomal adjuvants incorporating QS-21, such as that used in the efficacious varicella zoster vaccine, SHINGRIX, may augment protective immunity to SARS-CoV-2 vaccines. Such adjuvants have superior humoral and cellular immunogenicity relative to conventional adjuvants (29, 30).Here, we evaluate the use of a ferritin nanoparticle vaccine presenting the SARS-CoV-2 RBD (RFN) adjuvanted with the Army Liposomal Formulation QS-21 (ALFQ) (31). Both ferritin nanoparticles and ALFQ have been evaluated for vaccination against multiple pathogens in humans in phase 1 clinical trials (32–34). We demonstrate, in an NHP model, that immunization with RFN induces robust and broad antibody and T cell responses, as well as protection against viral replication and lung pathology following high-dose respiratory tract challenge with SARS-CoV-2.     

SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell–cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2′ fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2′ cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2′ cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has exceeded 240 million cases across the globe, but the molecular mechanisms of viral infection and host pathogenesis remain elusive. The SARS-CoV-2 spike (S) glycoprotein is a class I fusion protein decorated on the viral lipid envelope and is a key determinant of viral entry (1). The SARS-CoV-2 spike monomer contains two fragments: The amino terminus S1 subunit contains a receptor binding domain (RBD) (2–5), which recognizes the host receptor angiotensin-converting enzyme 2 (ACE2) for initial docking, while the carboxyl terminus S2 subunit catalyzes the fusion of viral and cell membranes (6, 7), enabling the subsequent release of viral RNA genome and downstream replication within the infected cells (8). Although many studies have captured the stationary phases of spike binding to human ACE2 (9–11), key molecular and cellular processes downstream of receptor recognition have not been explored.Spike can be proteolytically processed (12). SARS-CoV-2 spike encodes a polybasic cleavage site at its S1/S2 junction, and is posttranslationally processed by the endopeptidase furin (13, 14); cleaved S1 and S2 subunits remain noncovalently attached and fusion competent (15). Furin-cleaved S1 also exposes a C-terminal motif recognized by the host receptor neuropilin-1 (NRP1) (16, 17), which can facilitate SARS-CoV-2 entry. Although spike protein is autoprocessed, additional proteolytic cleavage event within the S2 subunit is proposed to be responsible for the subsequent membrane fusion (18, 19). This cleavage can be mediated at the plasma membrane by the type II transmembrane serine proteases (TMPRSS2) (20–23), or processed by the lysosomal cathepsins during the endocytosis of viral particles (24). Secreted tissue proteases, such as elastase and trypsin, can also facilitate this cleavage event and promote infection (25). As a result, this proteolytic event within the S2 subunit induces the release of a highly conserved hydrophobic region, known as the fusion peptide (18), which subsequently anchors the target host cell membrane (6, 26). A conformational reconfiguration within the S2 subunit then pulls the viral and host membranes into close proximity, allowing lipid membranes to fuse (7, 27–29). The unilateral change of the S2 subunit is of the utmost importance during viral entry, but molecular events regulating the spike processing and activation have not been demonstrated.Cells infected with SARS-CoV-2 drive the fusion with adjacent ACE2-expressing cells, producing morphologically distinct multinuclear giant cells, also known as syncytia (2, 30, 31). Spike-mediated syncytia have been reported in the postmortem lung samples of severe COVID-19 patients (32, 33). Apart from virus to cell transmission, spike-driven syncytia formation may provide an additional route for cell–cell transmission of SARS-CoV-2. Here, by using a cell–cell fusion system, in complement with a pseudoviral particle infection model, we study the functional and molecular requirements of spike activation. Through analyzing the prefusion and postfusion spike protein products, we show that proteolytic cleavage at the S2′ site is triggered by human cell receptor recognition in a range of immortalized cell lines and humanized primary cells. Generation of the S2′ fragment specifically requires spike recognition of functional host ACE2 and is conserved in the several variants of concern. We highlight that arginine 815, but not arginine residues at the S1/S2 cleavage site, is indispensable for the S2′ cleavage and syncytia formation in wild type (WT), as well as the more infectious Alpha, Beta, and Delta spike variants. Hence, these data highlight that both receptor recognition and proteolytic event at the S2′ site are functionally important for spike-mediated membrane fusion and SARS-CoV-2 infection.     

Correlated substitutions reveal SARS-like coronaviruses recombine frequently with a diverse set of structured gene pools

Quantifying SARS-like coronavirus (SL-CoV) evolution is critical to understanding the origins of SARS-CoV-2 and the molecular processes that could underlie future epidemic viruses. While genomic analyses suggest recombination was a factor in the emergence of SARS-CoV-2, few studies have quantified recombination rates among SL-CoVs. Here, we infer recombination rates of SL-CoVs from correlated substitutions in sequencing data using a coalescent model with recombination. Our computationally-efficient, non-phylogenetic method infers recombination parameters of both sampled sequences and the unsampled gene pools with which they recombine. We apply this approach to infer recombination parameters for a range of positive-sense RNA viruses. We then analyze a set of 191 SL-CoV sequences (including SARS-CoV-2) and find that ORF1ab and S genes frequently undergo recombination. We identify which SL-CoV sequence clusters have recombined with shared gene pools, and show that these pools have distinct structures and high recombination rates, with multiple recombination events occurring per synonymous substitution. We find that individual genes have recombined with different viral reservoirs. By decoupling contributions from mutation and recombination, we recover the phylogeny of non-recombined portions for many of these SL-CoVs, including the position of SARS-CoV-2 in this clonal phylogeny. Lastly, by analyzing >400,000 SARS-CoV-2 whole genome sequences, we show current diversity levels are insufficient to infer the within-population recombination rate of the virus since the pandemic began. Our work offers new methods for inferring recombination rates in RNA viruses with implications for understanding recombination in SARS-CoV-2 evolution and the structure of clonal relationships and gene pools shaping its origins.

Recombination can enable viruses to rapidly adapt to selective pressures (1–4) and to avoid accumulation of deleterious mutations that can lead to viral decline and extinction (5–7). Positive-sense single-stranded RNA ((+)ssRNA) viruses display highly variable levels of recombination (8, 9), with some species such as West Nile and Yellow fever viruses showing scant evidence of recombination (10) and others such as those of the Coronaviridae family showing evidence of frequent recombination (11). During the ongoing COVID-19 pandemic, population genomics has played an invaluable role in tracking the spread of SARS-CoV-2 and its variants (12–14), as well as understanding correlations between genomic substitutions and transmission patterns (15–19). However, a quantitative, population genomics-based understanding of the relative contributions of recombination and mutation to the evolution of SARS-CoV-2 and other SARS-like coronaviruses (SL-CoVs) is still being developed (20–27). Such knowledge will be important to understand the emergence of past and future viruses at the source of major epidemics.The majority of tools for studying recombination in RNA viruses are phylogeny-based, where recombination breakpoints are assessed by examining phylogenetic incongruence and Bayesian and Markov chain Monte Carlo techniques are used to infer recombination parameters (20–25, 28). These approaches have been successful at identifying instances of recombination, yet their application to large-scale population genomics data remains challenging due to the computational demands of these methods. Importantly, the inferred recombination parameters rely only on the observed (i.e., sampled) sequences, while recombination within the much larger, unobserved gene pools with which these branches interact is not captured by these models. Here, to infer the recombination parameters of (+)ssRNA viruses, we adapt our non-phylogenetic, computationally-efficient mcorr method, which we originally developed to measure homologous recombination rates in bacteria (29–31). In contrast to previous approaches which focus on recombination within sampled sequences, we infer recombination parameters for both sampled sequences and the larger gene pools they recombine with, revealing that SL-CoVs recombine with a diverse set of gene pools which have high levels of recombination.     

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic coronavirus disease (COVID-19) (1–3), resulting in more than 209 million infections worldwide since December 2019. SARS-CoV-2 is the seventh coronavirus (CoV) infecting humans beyond endemic human CoVs (HCoV-HKU1, -NL63, -OC43, and -229E), SARS-CoV, and Middle East respiratory syndrome CoV (MERS-CoV). SARS-CoV-2 preferentially infects the airway epithelial cells (1, 4), although viral infection has been detected in a variety of human organs, including the lungs, pharynx, heart, liver, brain, kidneys, and digestive system organs (5–7), causing upper respiratory diseases, fever, and severe pneumonia in humans.CoVs were not considered to be highly pathogenic to humans until the outbreak of SARS-CoV started in 2002 in Guangdong province, China (8–10). The primary determinant of CoV tropism is their spike (S) glycoproteins, which mediate the viral infection by binding to membrane receptors on the host cells and could be cleaved to the N-terminal S1 and C-terminal S2 subunit by the host proteases such as transmembrane protease serine 2 (TMPRSS2) and Furin (11). Studies have shown that ACE2, a cellular receptor for SARS-CoV, also binds SARS-CoV-2 S and serves as the entry point for SARS-CoV-2 (3, 12). However, ACE2 cannot fully explain the tissue tropism of SARS-CoV-2 because of the virus detection in tissues with little ACE2 expression, such as the liver, brain, and blood, and even in the lung, only a small subset of cells expresses ACE2 (SI Appendix, Fig. 1B) (13–16). Recently, neuropilin-1 (NRP1) (17, 18) and heparan sulfate (19, 20) have been identified as cofactors implicated in enhancing ACE2-dependent SARS-CoV-2 infection, while tyrosine-protein kinase receptor (AXL) (21) and CD147 (22) were identified as receptors involved in SARS-CoV-2 cell entry independently of ACE2. These highlight the multiple routes of SARS-CoV-2 infection, likely contributing to high infectivity and spread of COVID-19. Hence, SARS-CoV-2 is assumed to be dependent on other potential receptors or coreceptors to facilitate its infection in humans.Here, we applied engineered ascorbate peroxidase APEX2-based subcellular proteomics (23–25) to capture spatiotemporal membrane protein complexes interacting with SARS-CoV-2 S in living cells. We found that the nonmuscle myosin heavy chain IIA (MYH9) specifically interacts with SARS-CoV-2 S protein. The C-terminal domain (designated as PRA) of MYH9 interacts with the S2 subunit and the N-terminal domain (NTD) of the S1 subunit, and PRA overexpression facilitates a pan-CoV entry into host cells. In addition, depletion of MYH9 significantly reduced authentic SARS-CoV-2 infection in human lung cell lines. Mechanistically, a myosin inhibitor but not TMPRSS2 and CatB/L inhibitors blocked SARS-CoV-2 infection in PRA-A549 cells, and the presence of ACE2 is required for MYH9 mediated SARS-CoV-2 entry. Collectively, our results suggest that MYH9 is a coreceptor of ACE2 for SARS-CoV-2 infection in human pulmonary cells.     

The neutralizing breadth of antibodies targeting diverse conserved epitopes between SARS-CoV and SARS-CoV-2

Antibody therapeutics for the treatment of COVID-19 have been highly successful. However, the recent emergence of the Omicron variant has posed a challenge, as it evades detection by most existing SARS-CoV-2 neutralizing antibodies (nAbs). Here, we successfully generated a panel of SARS-CoV-2/SARS-CoV cross-neutralizing antibodies by sequential immunization of the two pseudoviruses. Of the potential candidates, we found that nAbs X01, X10, and X17 offer broad neutralizing potential against most variants of concern, with X17 further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-electron microscopy structures of the three antibodies together in complex with each of the spike proteins of the prototypical SARS-CoV, SARS-CoV-2, and Delta and Omicron variants of SARS-CoV-2 defined three nonoverlapping conserved epitopes on the receptor-binding domain. The triple-antibody mixture exhibited enhanced resistance to viral evasion and effective protection against infection of the Beta variant in hamsters. Our findings will aid the development of antibody therapeutics and broad vaccines against SARS-CoV-2 and its emerging variants.

By June 2022, the COVID-19 pandemic, caused by SARS-CoV-2, had resulted in more than 6 million deaths worldwide (1–3). Monoclonal antibodies (mAbs) isolated from SARS-CoV-2–infected individuals were effective as both therapeutic and prophylactic agents against SARS-CoV-2 (4–6), with several neutralizing antibodies (nAbs), including sotrovimab (7) and bamlanivimab (8), and nAb mixtures, including casirivimab–imdevimab (9) and bamlanivimab–etesevimab (10), approved under Emergency Use Authorization (EUA) for the treatment of patients with COVID-19. However, the constant evolution and genetic drift of SARS-CoV-2 has resulted in the emergence of many variants of concern (VOCs) depending on the main protein of the SARS-CoV-2 prototype strain, including the Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2), and Omicron (B.1.1.529) variants, the latter of which has become the major concern. Indeed, the Omicron variant harbors numerous residue substitutions in the spike (S) protein, with at least 15 mutations highly intertwined with common neutralizing epitopes in the receptor-binding domain (RBD) (11, 12). Various studies have reported that critical mutations within these VOCs prohibit the potent mAb neutralization that works against the ancestral isolate, leading to a much-diminished protective efficacy of antibody therapeutics against SARS-CoV-2 (13–20). Therefore, there is still a pressing need for nAbs with broader neutralizing breadth against current VOCs and future emerging variants.The trimeric S protein mediates SARS-CoV-2 entry into host cells via the RBD, which binds to the angiotensin-converting enzyme 2 (ACE2) receptor (1, 21, 22). Given its role, the RBD is regarded as a critical target for the development of therapeutics and vaccines against COVID-19. Indeed, numerous potently neutralizing mAbs are shown to target the receptor-binding motif (RBM) on the RBD, thereby efficiently inhibiting the S protein from binding to ACE2 to minimize or prohibit infection (4, 23, 24). However, VOCs frequently possess mutations within the RBM, which significantly reduces the neutralization breadth of mAbs that recognize this site (13–18, 25). Nevertheless, of the five classes of RBD-targeting nAbs (4, 26), three classes—represented by S309 (27), S2X259 (28), and S2H97 (26)—offer cross-neutralization against SARS-CoV-2 and SARS-CoV and thus can also inhibit infection from most VOCs. Consequently, it is assumed that epitopes within these sites are highly conserved among Sarbecoviruses and that antibody mixtures comprising representative nAbs that bind to these conserved epitopes may be able to prevent SARS-CoV-2 variants and other zoonotic “spillover” SARS-like viruses. In addition, under the selective pressure of antibody therapeutics, such as screening, the emergence of avoidance mutations becomes an important issue that should be considered. Such antibody avoidance studies in vitro have strongly supported the rationale of antibody mixtures consisting of noncompeting antibodies to avoid the development of resistance (13, 15, 29).nAbs reported to date have been primarily obtained from the human humoral immune response induced by vaccination or natural infection of SARS-CoV or SARS-CoV-2. The singular exposure of Sarbecoviruses at a time has hindered the generation of cross-neutralizing mAbs (26–28). Based on influenza virus research (30–32), the development of cross-neutralizing antibodies may benefit from the combined immunization of SARS-CoV and SARS-CoV-2 in sequence, offering insight into immune-focusing on conserved epitopes between the two virus strains.In this study, we focus on the conserved epitopes between SARS-CoV-2 and SARS-CoV. To this end, we generated a panel of broad-neutralizing antibodies (bnAbs) against SARS-CoV, SARS-CoV-2, and VOCs from sequentially immunized mice. Three representative bnAbs, X01, X10, and X17, were further identified to offer potent cross-neutralizing activity against most VOCs but with a decreased neutralization breadth against Omicron. High-resolution cryo-electron microscopy (cryo-EM) structures revealed three nonoverlapping conserved epitopes and defined the structural basis for the neutralization breadth of the three bnAbs. Using these three bnAbs in a mixture efficiently resisted viral escape and protected Syrian hamsters against challenge with the SARS-CoV-2 Beta variant. Thus, by taking advantage of conserved epitopes, our results expand upon the current therapeutic strategy and offer a way to cope with circulating and future emerging SARS-CoV-2 VOCs as well as any potential spillover zoonotic SARS-like viruses. This study thus highlights the potential utility of diverse, conserved epitopes for effective vaccine design.