Spike N354 glycosylation augments SARS-CoV-2 fitness for human adaptation through multiple mechanisms (preprint)

Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity could be recovered in the presence of heparin sulfate, which targets the N354 pocket to ease restrictions of conformational transition resulting in a RBD-up state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms.

Enhanced transmissibility of XBB.1.5 is contributed by both strong ACE2 binding and antibody evasion (preprint)

SARS-CoV-2 recombinant subvariant XBB.1.5 is growing rapidly in the United States, carrying an additional Ser486Pro substitution compared to XBB.1 and outcompeting BQ.1.1 and other XBB sublineages. The underlying mechanism for such high transmissibility remains unclear. Here we show that XBB.1.5 exhibits a substantially higher hACE2-binding affinity compared to BQ.1.1 and XBB/XBB.1. Convalescent plasma samples from BA.1, BA.5, and BF.7 breakthrough infection are significantly evaded by both XBB.1 and XBB.1.5, with XBB.1.5 displaying slightly weaker immune evasion capability than XBB.1. Evusheld and Bebtelovimab could not neutralize XBB.1/XBB.1.5, while Sotrovimab remains its weak reactivity and notably, SA55 is still highly effective. The fact that XBB.1 and XBB.1.5 showed comparable antibody evasion but distinct transmissibility suggests enhanced receptor-binding affinity would indeed lead to higher growth advantages. The strong hACE2 binding of XBB.1.5 could also enable its tolerance of further immune escape mutations, which should be closely monitored.

Neutralizing antibody evasion and receptor binding features of SARS-CoV-2 Omicron BA.2.75 (preprint)

The Omicron subvariants BA.2.75 is rapidly raising in India. BA.2.75 also shows a local growth advantage compared to BA.2.38 and BA.4/BA.5. Its immune evasion capability and receptor binding affinity is unclear and requires investigation. Here, we show that BA.2.75 is more neutralization evasive than BA.2.12.1 against the plasma from post-vaccination BA.2 infection, but less compared to BA.4/BA.5. However, as shown in a small sample of plasma from post-vaccination Delta infection, BA.2.75 seems to be more immune evasive than BA.4/BA.5 in Delta-stimulated immune background, which may explain BA. 2.75's growth advantage over BA.4/BA.5 in India. The additional N460K, G446S, D339H and R493Q mutations carried by BA.2.75 allows it to escape BA.2-effective neutralizing antibodies of different RBD epitopes, and BA.2.75 has a distinct antibody escaping profile from BA.4/BA.5. Compared to BA.2, REGN10933 and COV2-2196 partially recovered neutralization against BA.2.75 due to R493Q reversion. However, the efficacy of their corresponding cocktail was not significantly changed, since REGN10987 and COV2-2130 showed reduced neutralizing activity due to G446S. BA.2.75 exhibits higher ACE2-binding affinity than BA.4/BA.5, which should be contributed by R493Q and N460K, according to deep mutational scanning (DMS) results. This affinity-strengthening feature is being further examined and verified, which will be updated soon.

BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection (preprint)

Recent emergence of SARS-CoV-2 Omicron sublineages BA.2.12.1, BA.2.13, BA.4 and BA.5 all contain L452 mutations and show potential higher transmissibility over BA.2. The new variants' receptor binding and immune evasion capability require immediate investigation, especially on the role of L452 substitutions. Herein, coupled with structural comparisons, we showed that BA.2 sublineages, including BA.2.12.1 and BA.2.13, exhibit increased ACE2-binding affinities compared to BA.1; while BA.4/BA.5 shows the weakest receptor-binding activity due to F486V and R493Q reversion. Importantly, compared to BA.2, BA.2.12.1 and BA.4/BA.5 exhibit stronger neutralization escape from the plasma of 3-dose vaccinees and, most strikingly, from vaccinated BA.1 convalescents. To delineate the underlying evasion mechanism, we determined the escaping mutation profiles, epitope distribution and Omicron sublineage neutralization efficacy of 1640 RBD-directed neutralizing antibodies (NAbs), including 614 isolated from BA.1 convalescents. Interestingly, post-vaccination BA.1 infection mainly recalls wildtype (WT) induced humoral memory and elicits antibodies that neutralize both WT and BA.1. These cross-reactive NAbs are significantly enriched on non-ACE2-competing epitopes; and surprisingly, the majority are undermined by R346 and L452 substitutions, namely R346K (BA.1.1), L452M (BA.2.13), L452Q (BA.2.12.1) and L452R (BA.4/BA.5), suggesting that R346K and L452 mutations appeared under the immune pressure of Omicron convalescents. Nevertheless, BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1 but do not respond to WT SARS-CoV-2, due to the high susceptibility to N501, N440, K417 and E484. However, these NAbs are largely escaped by BA.2 sublineages and BA.4/BA.5 due to D405N and F486V, exhibiting poor neutralization breadths. As for therapeutic NAbs, LY-CoV1404 (Bebtelovimab) and COV2-2130 (Cilgavimab) can still effectively neutralize BA.2.12.1 and BA.4/BA.5, while the S371F, D405N and R408S mutations carried by BA.2/BA.4/BA.5 sublineages would undermine most broad sarbecovirus NAbs. Together, our results indicate that Omicron can evolve mutations to specifically evade humoral immunity elicited by BA.1 infection. The continuous evolution of Omicron poses great challenges to SARS-CoV-2 herd immunity and suggests that BA.1-derived vaccine boosters may not be ideal for achieving broad-spectrum protection.

BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron BA.1 infection (preprint)

Recent emergence of SARS-CoV-2 Omicron sublineages BA.2.12.1, BA.2.13, BA.4 and BA.5 all contain L452 mutations and show potential higher transmissibility over BA.2. The new variants’ receptor binding and immune evasion capability require immediate investigation, especially on the role of L452 substitutions. Herein, coupled with structural comparisons, we showed that BA.2 sublineages, including BA.2.12.1 and BA.2.13, exhibit increased ACE2-binding affinities compared to BA.1; while BA.4/BA.5 shows the weakest receptor-binding activity due to F486V and R493Q reversion. Importantly, compared to BA.2, BA.2.12.1 and BA.4/BA.5 exhibit stronger neutralization escape from the plasma of 3-dose vaccinees and, most strikingly, from vaccinated BA.1 convalescents. To delineate the underlying evasion mechanism, we determined the escaping mutation profiles, epitope distribution and Omicron sub-lineage neutralization efficacy of 1640 RBD-directed neutralizing antibodies (NAbs), including 614 isolated from BA.1 convalescents. Interestingly, post-vaccination BA.1 infection mainly recalls wildtype-induced humoral memory and elicits antibodies that neutralize both wild-type and BA.1. These cross-reactive NAbs are significantly enriched on non-ACE2-competing epitopes; and surprisingly, the majority are undermined by R346 and L452 substitutions, namely R346K (BA.1.1), L452M (BA.2.13), L452Q (BA.2.12.1) and L452R (BA.4/BA.5), suggesting that R346K and L452 mutations appeared under the immune pressure of Omicron convalescents. Nevertheless, BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1 but do not respond to wild-type SARS-CoV-2, due to the high susceptibility to N501, N440, K417 and E484. However, these NAbs are largely escaped by BA.2 sublineages and BA.4/BA.5 due to D405N and F486V, exhibiting poor neutralization breadths. As for therapeutic NAbs, LY-CoV1404 (Bamlanivimab) and COV2-2130 (Cilgavimab) can still effectively neutralize BA.2.12.1 and BA.4/BA.5, while the S371F, D405N and R408S mutations carried by BA.2/BA.4/BA.5 sublineages would undermine most broad sarbecovirus NAbs. Together, our results indicate that Omicron can evolve mutations to specifically evade humoral immunity elicited by BA.1 infection. The continuous evolution of Omicron poses great challenges to SARS-CoV-2 herd immunity and suggests that BA.1-derived vaccine boosters may not be ideal for achieving broad-spectrum protection.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors (preprint)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) and several bat coronaviruses employ Dipeptidyl peptidase-4 (DPP4) as their functional receptors. However, the receptor for NeoCoV, the closest MERS-CoV relative yet discovered in bats, remains enigmatic. In this study, we unexpectedly found that NeoCoV and its close relative, PDF-2180-CoV, can efficiently use some types of bat Angiotensin-converting enzyme 2 (ACE2) and, less favorably, human ACE2 for entry. The two viruses use their spikes' S1 subunit carboxyl-terminal domains (S1-CTD) for high-affinity and species-specific ACE2 binding. Cryo-electron microscopy analysis revealed a novel coronavirus-ACE2 binding interface and a protein-glycan interaction, distinct from other known ACE2-using viruses. We identified a molecular determinant close to the viral binding interface that restricts human ACE2 from supporting NeoCoV infection, especially around residue Asp338. Conversely, NeoCoV efficiently infects human ACE2 expressing cells after a T510F mutation on the receptor-binding motif (RBM). Notably, the infection could not be cross-neutralized by antibodies targeting SARS-CoV-2 or MERS-CoV. Our study demonstrates the first case of ACE2 usage in MERS-related viruses, shedding light on a potential bio-safety threat of the human emergence of an ACE2 using 'MERS-CoV-2' with both high fatality and transmission rate.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors (preprint)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) and several bat coronaviruses employ Dipeptidyl peptidase-4 (DPP4) as their functional receptors. However, the receptor for NeoCoV, the closest MERS-CoV relative yet discovered in bats, remains enigmatic. In this study, we unexpectedly found that NeoCoV and its close relative, PDF-2180-CoV, can efficiently use some types of bat Angiotensin-converting enzyme 2 (ACE2) and, less favorably, human ACE2 for entry. The two viruses use their spikes' S1 subunit carboxyl-terminal domains (S1-CTD) for high-affinity and species-specific ACE2 binding. Cryo-electron microscopy analysis revealed a novel coronavirus-ACE2 binding interface and a protein-glycan interaction, distinct from other known ACE2-using viruses. We identified a molecular determinant close to the viral binding interface that restricts human ACE2 from supporting NeoCoV infection, especially around residue Asp338. Conversely, NeoCoV efficiently infects human ACE2 expressing cells after a T510F mutation on the receptor-binding motif (RBM). Notably, the infection could not be cross-neutralized by antibodies targeting SARS-CoV-2 or MERS-CoV. Our study demonstrates the first case of ACE2 usage in MERS-related viruses, shedding light on a potential bio-safety threat of the human emergence of an ACE2 using "MERS-CoV-2" with both high fatality and transmission rate.

Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant (preprint)

The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging new interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Apart from already existing mutations, we have identified three new immune escape sites: 1) Q493R, 2) G446S and 3) S371L/S373P/S375F that confers greater resistance to five of the six classes of RBD-antibodies. Structure of the Omicron S bound with human ACE2, together with analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron (preprint)

Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine. One sentence summary A sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.

B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes (preprint)

The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) and humoral immunity requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs identified from SARS-CoV/SARS-CoV-2 convalescents and vaccinees. Based on the results, NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlaps with ACE2-binding motif, are largely escaped by K417N, N440K, G446S, E484A, Q493K, and G496S. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, S371L, and S375F. Furthermore, B.1.1.529 pseudovirus neutralization and RBD binding assay showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, LY-CoV016/LY-CoV555 cocktail, REGN-CoV2 cocktail, AZD1061/AZD8895 cocktail, and BRII-196 were escaped by Omicron, while VIR7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron could cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes (preprint)

The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) and humoral immunity requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs identified from SARS-CoV/SARS-CoV-2 convalescents and vaccinees. Based on the results, NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlaps with ACE2-binding motif, are largely escaped by K417N, N440K, G446S, E484A, Q493K, and G496S. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, S371L, and S375F. Furthermore, B.1.1.529 pseudovirus neutralization and RBD binding assay showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, LY-CoV016/LY-CoV555 cocktail, REGN-CoV2 cocktail, AZD1061/AZD8895 cocktail, and BRII-196 were escaped by Omicron, while VIR7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron could cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

A third dose of inactivated vaccine augments the potency, breadth, and duration of anamnestic responses against SARS-CoV-2 (preprint)

Emergence of variants of concern (VOC) with altered antigenic structures and waning humoral immunity to SARS-CoV-2 are harbingers of a long pandemic. Administration of a third dose of an inactivated virus vaccine can boost the immune response. Here, we have dissected the immunogenic profiles of antibodies from 3-dose vaccinees, 2-dose vaccinees and convalescents. Better neutralization breadth to VOCs, expeditious recall and long-lasting humoral response bolster 3-dose vaccinees in warding off COVID-19. Analysis of 171 complex structures of SARS-CoV-2 neutralizing antibodies identified structure-activity correlates, revealing ultrapotent, VOCs-resistant and broad-spectrum antigenic patches. Construction of immunogenic and mutational heat maps revealed a direct relationship between "hot" immunogenic sites and areas with high mutation frequencies. Ongoing antibody somatic mutation, memory B cell clonal turnover and antibody composition changes in B cell repertoire driven by prolonged and repeated antigen stimulation confer development of monoclonal antibodies with enhanced neutralizing potency and breadth. Our findings rationalize the use of 3-dose immunization regimens for inactivated vaccines. One sentence summaryA third booster dose of inactivated vaccine produces a highly sifted humoral immune response via a sustained evolution of antibodies capable of effectively neutralizing SARS-CoV-2 variants of concern.

Nanometer resolution in situ structure of SARS-CoV-2 post-fusion spike (preprint)

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of viral genome into host cell. To understand its detailed entry mechanism and develop specific entry inhibitor, the in situ structural information of SARS-CoV-2 spikes in different states are urgently important. Here, by using the cryo-electron microscopic tomograms, we observed spikes of inactivated SARS-CoV-2 virions in both pre-fusion and post-fusion states and solved the nanometer resolution structure of in situ post-fusion spike. With a more complete model compared to previous reports, the relative spatial position between fusion peptide and transmembrane domain was discovered. Novel oligomerizations of spikes on viral membrane were observed, likely suggesting a new mechanism of fusion pore formation.

The architecture of the SARS-CoV-2 RNA genome inside virion (preprint)

SARS-CoV-2 carries the largest single-stranded RNA genome and is the causal pathogen of the ongoing COVID-19 pandemic. How the SARS-CoV-2 RNA genome is folded in the virion remains unknown. To fill the knowledge gap and facilitate structure-based drug development, we developed a virion RNA in situ conformation sequencing technology, named vRIC-seq, for probing viral RNA genome structure unbiasedly. Using vRIC-seq data, we reconstructed the tertiary structure of the SARS-CoV-2 genome and revealed a surprisingly "unentangled globule" conformation. We uncovered many long-range duplexes and higher-order junctions, both of which were under purifying selections and contributed to the sequential package of the SARS-CoV-2 genome. Unexpectedly, the D614G and the other two accompanying mutations might remodel duplexes into more stable forms. Lastly, the structure-guided design of potent small interfering RNAs could obliterate the SARS-CoV-2 in Vero cells. Overall, our work provides a framework for studying the genome structure, function, and dynamics of emerging deadly RNA viruses.

SARS-CoV-2 spike protein interacts with and activates TLR4 (preprint)

The onset of sepsis is an important feature of COVID19 and a main cause of death. It is unknown how SARS-CoV-2 infection results in viral sepsis in human. We recently found that SARS-CoV-2 provoked an anti-bacterial like response and activation of TLR4 pathway at the very early stage of infection in animal models. This abnormal immune response led to emergency granulopoiesis and sepsis. However, the original trigger of TLR4 signaling by SARS-CoV-2 is unknown. We here identified that the trimeric spike protein of SARS-CoV-2 could bind to TLR4 directly and robustly activate downstream signaling in monocytes and neutrophils. Moreover, specific TLR4 or NFKB inhibitor, or knockout of MyD88 could significantly block IL-1B induction by spike protein. We thus reveal that spike protein of SARS-CoV-2 functions as a potent stimulus causing TLR4 activation and sepsis related abnormal responses.

Double Lock of a Potent Human Monoclonal Antibody against SARS-CoV-2 (preprint)

Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10-fold of effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the RBD, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19.

Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2 (preprint)

The ongoing SARS-CoV-2 pandemic has brought an urgent need for animal models to study the pathogenicity of the virus. Herein, we generated and characterized a novel mouse-adapted SARS-CoV-2 strain named MASCp36 that causes acute respiratory symptoms and mortality in standard laboratory mice. Particularly, this model exhibits age and gender related skewed distribution of mortality akin to severe COVID-19, and the 50% lethal dose (LD50) of MASCp36 was ~100 PFU in aged, male BALB/c mice. Deep sequencing identified three amino acid mutations, N501Y, Q493H, and K417N, subsequently emerged at the receptor binding domain (RBD) of MASCp36, which significantly enhanced the binding affinity to its endogenous receptor, mouse ACE2 (mACE2). Cryo-electron microscopy (cryo-EM) analysis of mACE2 in complex with the RBD of MASCp36 at 3.7-angstrom resolution elucidates molecular basis for the receptor-binding switch driven by amino acid substitutions. Our study not only provides a robust platform for studying the pathogenesis of severe COVID-19 and rapid evaluation of coutermeasures against SARS-CoV-2, but also unveils the molecular mechanism for the rapid adaption and evolution of SARS-CoV-2 in mice.

Ozone exposure upregulates the expression of host susceptibility protein TMPRSS2 to SARS-CoV-2 (preprint)

Background: SARS-CoV-2, a novel coronavirus, and the etiologic agent for the current global health emergency, causes acute infection of the respiratory tract leading to severe disease and significant mortality. Ever since the start of SARS-CoV-2, also known as COVID-19 pandemic, countless uncertainties have been revolving around the pathogenesis and epidemiology of the SARS-CoV-2 infection. While air pollution has been shown to be strongly correlated to increased SARS-CoV-2 morbidity and mortality, whether environmental pollutants such as ground level ozone affects the susceptibility of individuals to SARS-CoV-2 is not yet established. Objective: To investigate the impact of ozone inhalation on the expression levels of signatures associated with host susceptibility to SARS-CoV-2. Methods: We analyzed lung tissues collected from mice that were sub-chronically exposed to air or 0.8ppm ozone for three weeks (4h/night, 5 nights/week), and analyzed the expression of signatures associated with host susceptibility to SARS-CoV-2. Results: SARS-CoV-2 entry into the host cells requires proteolytic priming by the host-derived protease, transmembrane protease serine 2 (TMPRSS2). The TMPRSS2 protein and Tmprss2 transcripts were significantly elevated in the extrapulmonary airways, parenchyma, and alveolar macrophages from ozone-exposed mice. A significant proportion of additional known SARS-CoV-2 host susceptibility genes were upregulated in alveolar macrophages and parenchyma from ozone-exposed mice. Conclusions: Our data indicate that the unhealthy levels of ozone in the environment may predispose individuals to severe SARS-CoV-2 infection. Given the severity of this pandemic, and the challenges associated with direct testing of host-environment interactions in clinical settings, we believe that this mice-ozone-exposure based study informs the scientific community of the potentially detrimental effects of the ambient ozone levels determining the host susceptibility to SARS-CoV-2.

Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination (preprint)

The Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by binding to the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. We show the direct influence of Nsp1 on translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we reveal that Nsp1 stimulates translation termination in the stop codon recognition stage. We identify that activity of Nsp1 in translation termination is localized in its N-terminal domain. The data obtained will enable an investigation of new classes of potential therapeutic agents from coronavirus infection competing with Nsp1 for binding with the termination complex.

Network-based Virus-Host Interaction Prediction with Application to SARS-CoV-2 (preprint)

COVID-19, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has quickly become a global health crisis since the first report of infection in December of 2019. However, the infection spectrum of SARS-CoV-2 and its comprehensive protein-level interactions with hosts remain unclear. There is a massive amount of under-utilized data and knowledge about RNA viruses highly relevant to SARS-CoV-2 and their hosts' proteins. More in-depth and more comprehensive analyses of that knowledge and data can shed new insight into the molecular mechanisms underlying the COVID-19 pandemic and reveal potential risks. In this work, we constructed a multi-layer virus-host interaction network to incorporate these data and knowledge. A machine learning-based method, termed Infection Mechanism and Spectrum Prediction (IMSP), was developed to predict virus-host interactions at both protein and organism levels. Our approach revealed five potential infection targets of SARS-CoV-2, which deserved public health attention, and eight highly possible interactions between SARS-CoV-2 proteins and human proteins. Given a new virus, IMSP can utilize existing knowledge and data about other highly relevant viruses to predict multi-scale interactions between the new virus and potential hosts.