Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization. Respiratory syncytial virus (RSV) is the second most common infectious cause of infant death worldwide. Despite this great clinical impact, no effective antivirals or vaccines against RSV are available. Here we find that the RSV attachment (G) glycoprotein uses CX3CR1 as a receptor on primary human airway epithelial (HAE) cultures, an excellent model of RSV infection of the human lung. The G protein contains a CX3C motif and we find that this region is critical for its role in infection of HAE cultures, but not of immortalized cells. Furthermore, we find that antibodies against the G protein neutralize RSV infection of HAE cultures differently from immortalized cells. These insights suggest that HAE cultures should be used to quantify neutralizing antibodies, including during vaccine development, that the CX3CR1 interaction with the RSV G protein could be a target for antiviral drug development, and that the G protein should be considered for inclusion in vaccines.

Significance: It is currently unknown if SARS-CoV-2 can spread through cell–cell contacts, and if so, the underlying mechanisms and implications. In this work, we show, by using lentiviral pseudotyped virus, that the spike protein of SARS-CoV-2 mediates the viral cell-to-cell transmission, with an efficiency higher than that of SARS-CoV. We also find that cell–cell fusion contributes to cell-to-cell transmission, yet ACE2 is not absolutely required. While the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) differ in cell-free infectivity from wild type and from each other, these VOCs have similar cell-to-cell transmission capability and exhibit differential sensitivity to neutralization by vaccinee sera. Results from our study will contribute to a better understanding of SARS-CoV-2 spread and pathogenesis.

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

5-methylcytosine (m ⁵ C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m ⁵ C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m ⁵ C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m ⁵ C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m ⁵ C methylation of several abundant noncoding RNAs (ncRNAs). However, m ⁵ C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I–mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m ⁵ C methylation controls antiviral innate immunity through modulating the m ⁵ C methylome of ncRNAs and their expression.

Respiratory syncytial virus (RSV) is associated with major morbidity in infants, although most cases result in mild disease. The pathogenesis of the disease is incompletely understood, especially the determining factors of disease severity. A better characterization of these factors may help with development of RSV vaccines and antivirals. Hence, identification of a “safe and protective” immunoprofile induced by natural RSV infection could be used as a as a surrogate of ideal vaccine-elicited responses in future clinical trials. In this study, we integrated blood transcriptional and cell immune profiling, RSV loads, and clinical data to identify factors associated with a mild disease phenotype in a cohort of 190 children <2 years of age. Children with mild disease (outpatients) showed higher RSV loads, greater induction of interferon (IFN) and plasma cell genes, and decreased expression of inflammation and neutrophil genes versus children with severe disease (inpatients). Additionally, only infants with severe disease had increased numbers of HLA-DR^low monocytes, not present in outpatients. Multivariable analyses confirmed that IFN overexpression was associated with decreased odds of hospitalization, whereas increased numbers of HLA-DR^low monocytes were associated with increased risk of hospitalization. These findings suggest that robust innate immune responses are associated with mild RSV infection in infants.