The SARS-CoV-2 variant, Omicron, shows rapid replication in human primary nasal epithelial cultures and efficiently uses the endosomal route of entry.

At the end of 2021 a new SARS-CoV-2 variant, Omicron, emerged and quickly spread across the world. It has been demonstrated that Omicrons high number of Spike mutations lead to partial immune evasion from even polyclonal antibody responses, allowing frequent re-infection and vaccine breakthroughs. However, it seems unlikely these antigenic differences alone explain its rapid growth; here we show Omicron replicates rapidly in human primary airway cultures, more so even than the previously dominant variant of concern, Delta. Omicron Spike continues to use human ACE2 as its primary receptor, to which it binds more strongly than other variants. Omicron Spike mediates enhanced entry into cells expressing several different animal ACE2s, including various domestic avian species, horseshoe bats and mice suggesting it has an increased propensity for reverse zoonosis and is more likely than previous variants to establish an animal reservoir of SARS-CoV-2. Unlike other SARS-CoV-2 variants, however, Omicron Spike has a diminished ability to induce syncytia formation. Furthermore, Omicron is capable of efficiently entering cells in a TMPRSS2-independent manner, via the endosomal route. We posit this enables Omicron to infect a greater number of cells in the respiratory epithelium, allowing it to be more infectious at lower exposure doses, and resulting in enhanced intrinsic transmissibility.

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.

Receptor binding and escape from Beta antibody responses drive Omicron-B.1.1.529 evolution

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced. Omicron contains a total of 30 substitutions plus deletions and an insertion in Spike, far more than any previously reported variant. The mutations include those previously identified by In-vitro evolution to contribute to high-affinity binding to ACE2, including mutations Q498R and N501Y critical in forming additional interactions in the interface. Together with increased charge complementarity between the RBD and ACE2, these substantially increase affinity and potentially virus transmissibility through increased syncytia formation. Further mutations promote immune evasion. We have studied the binding of a large panel of potent monoclonal antibodies generated from early pandemic or Beta infected cases. Mutations in Omicron will likely compromise the binding of many of these and additionally, the binding of antibodies under commercial development, however residual binding should provide protection from severe disease.

Inhibiting ACSL1-Related Ferroptosis Restrains Murine Coronavirus Infection

Murine hepatitis virus strain A59 (MHV-A59) was shown to induce pyroptosis, apoptosis, and necroptosis of infected cells, especially in the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59 is unknown. We utilized murine macrophages and a C57BL/6 mice intranasal infection model to address this. In primary macrophages, the ferroptosis inhibitor inhibited viral propagation, inflammatory cytokines released, and cell syncytia formed after MHV-A59 infection. In the mouse model, we found that in vivo administration of liproxstatin-1 ameliorated lung inflammation and tissue injuries caused by MHV-A59 infection. To find how MHV-A59 infection influenced the expression of ferroptosis-related genes, we performed RNA-seq in primary macrophages and found that MHV-A59 infection upregulates the expression of the acyl-CoA synthetase long-chain family member 1 (ACSL1), a novel ferroptosis inducer. Using ferroptosis inhibitors and a TLR4 inhibitor, we showed that MHV-A59 resulted in the NF-kB-dependent, TLR4-independent ACSL1 upregulation. Accordingly, ACSL1 inhibitor Triacsin C suppressed MHV-A59-infection-induced syncytia formation and viral propagation in primary macrophages. Collectively, our study indicates that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serve as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.

Human Cytomegalovirus Replication and Infection-Induced Syncytia Formation in Labial, Foreskin, and Fetal Lung Fibroblasts

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell–cell fusion, and ensuing viral spread and pathogenesis in different organs.

SARS-CoV-2 Delta Spike Protein Enhances the Viral Fusogenicity and Inflammatory Cytokine Production

The Delta variant is now the most dominant and virulent SARS-CoV-2 variant of concern (VOC). In this study, we investigated several virological features of Delta spike protein (SPDelta), including protein maturation and its impact on viral entry of cell-free pseudotyped virus, cell-cell fusion ability and its induction of inflammatory cytokine production in human macrophages and dendritic cells. The results showed that SPΔCDelta exhibited enhanced S1/S2 cleavage in cells and pseudotyped virus-like particles (PVLPs). We further showed that SPΔCDelta elevated pseudovirus infection in human lung cell lines and mediated significantly enhanced syncytia formation. Furthermore, we revealed that SPΔCDelta-PVLPs had stronger effects on stimulating NF-κB and AP-1 signaling in human monocytic THP1 cells and induced significantly higher levels of pro-inflammatory cytokine, such as TNF-α, IL-1β and IL-6, released from human macrophages and dendritic cells. Overall, these studies provide evidence to support the important role of SPΔCDelta during virus infection, transmission and pathogenesis.

Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

The SARS-CoV-2 coronavirus responsible for the global pandemic contains a novel furin cleavage site in the spike protein (S) that increases viral infectivity and syncytia formation in cells. Here, we show that O-glycosylation near the furin cleavage site is mediated by members of the GALNT enzyme family, resulting in decreased furin cleavage and decreased syncytia formation. Moreover, we show that O-glycosylation is dependent on the novel proline at position 681 (P681). Mutations of P681 seen in the highly transmissible alpha and delta variants abrogate O-glycosylation, increase furin cleavage, and increase syncytia formation. Finally, we show that GALNT family members capable of glycosylating S are expressed in human respiratory cells that are targets for SARS-CoV-2 infection. Our results suggest that host O-glycosylation may influence viral infectivity/tropism by modulating furin cleavage of S and provide mechanistic insight into the role of the P681 mutations found in the highly transmissible alpha and delta variants.

Trypsin Enhances SARS-CoV-2 Infection by Facilitating Viral Entry

Coronavirus infects the cell by cytoplasmic or endosomal membrane fusion driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and S2′ site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we showed that trypsin mediated the enhancement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 entered the cells via either a non-endosomal or an endosomal fusion pathway depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection efficiency than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger high replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytia formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin for the cell-adapted SARS-CoV-2 strains. During 50 serial passages without trypsin addition, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but with several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that S1/S2 FCS is essential for S protein proteolytic priming and fusion activity for SARS-CoV-2 entry but not for SARS-CoV-2 replication. Our data can contribute to the improvement of SARS-CoV-2 production to develop vaccines or antivirals and motivate further investigations into the explicit functions of cell adaptation-related genetic drift in SARS-CoV-2 pathogenesis.

Inhibiting ACSL1 related ferroptosis restrains MHV-A59 infection

Murine hepatitis virus strain A59 (MHV-A59) belongs to the β-coronavirus and is considered as a representative model for studying coronavirus infection. MHV-A59 was shown to induce pyroptosis, apoptosis and necroptosis of infected cells, especially the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59, is unknown. Here, we demonstrate inhibiting ferroptosis suppresses MHV-A59 infection. MHV-A59 infection upregulates the expression of Acsl1, a novel ferroptosis inducer. MHV-A59 upregulates Acsl1 expression depending on the NF-kB activation, which is TLR4-independent. Ferroptosis inhibitor inhibits viral propagation, inflammatory cytokines release and MHV-A59 infection induced cell syncytia formation. ACSL1 inhibitor Triacsin C suppresses MHV-A59 infection induced syncytia formation and viral propagation. In vivo administration of liproxstatin-1 ameliorates lung inflammation and tissue injuries caused by MHV-A59 infection. Collectively, these results indicate that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serves as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.