Determinants of species-specific utilization of ACE2 by human and animal coronaviruses.

Utilization of human ACE2 allowed several bat coronaviruses (CoVs), including the causative agent of COVID-19, to infect humans directly or via intermediate hosts. However, the determinants of species-specific differences in ACE2 usage and the frequency of the ability of animal CoVs to use human ACE2 are poorly understood. Here we applied VSV pseudoviruses to analyze the ability of Spike proteins from 26 human or animal CoVs to use ACE2 receptors across nine reservoir, potential intermediate and human hosts. We show that SARS-CoV-2 Omicron variants evolved towards more efficient ACE2 usage but mutation of R493Q in BA.4/5 and XBB Spike proteins disrupts utilization of ACE2 from Greater horseshoe bats. Variations in ACE2 residues 31, 41 and 354 govern species-specific differences in usage by coronaviral Spike proteins. Mutation of T403R allows the RaTG13 bat CoV Spike to efficiently use all ACE2 orthologs for viral entry. Sera from COVID-19 vaccinated individuals neutralize the Spike proteins of various bat Sarbecoviruses. Our results define determinants of ACE2 receptor usage of diverse CoVs and suggest that COVID-19 vaccination may protect against future zoonoses of bat coronaviruses.

Endogenous IFITMs boost SARS-coronavirus 1 and 2 replication whereas overexpression inhibits infection by relocalizing ACE2.

Opposing effects of interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) on SARS-CoV-2 infection have been reported. The reasons for this are unclear and the role of IFITMs in infection of other human coronaviruses (hCoVs) remains poorly understood. Here, we demonstrate that endogenous expression of IFITM2 and/or IFITM3 is critical for efficient replication of SARS-CoV-1, SARS-CoV-2 and hCoV-OC43 but has little effect on MERS-, NL63-and 229E-hCoVs. In contrast, overexpression of IFITMs inhibits all these hCoVs, and the corresponding spike-containing pseudo-particles, except OC43, which is enhanced by IFITM3. We further demonstrate that overexpression of IFITMs impairs cell surface expression of ACE2 representing the entry receptor of SARS-CoVs and hCoV-NL63 but not hCoV-OC43. Our results explain the inhibitory effects of artificial IFITM overexpression on ACE2-tropic SARS-CoVs and show that three hCoVs, including major causative agents of severe respiratory disease, hijack IFITMs for efficient infection of human cells.

The Role of Long Noncoding RNA BST2-2 in the Innate Immune Response to Viral Infection.

Long noncoding RNAs (lncRNAs) widely exist in the cells and play important roles in various biological processes. The role of lncRNAs in immunity remains largely unknown. lncRNA BST2-2 (lncBST2-2) was upregulated upon viral infection and dependent on the interferon (IFN)/JAK/STAT signaling pathway. There was no coding potential found in the lncBST2-2 transcript. Overexpression of lncBST2-2 inhibited the replication of hepatitis C virus (HCV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), and herpes simplex virus (HSV), while knockdown of lncBST2-2 facilitated viral replication. Further studies showed that lncBST2-2 promoted the phosphorylation, dimerization, and nuclear transport of IRF3, promoting the production of IFNs. Importantly, lncBST2-2 interacted with the DNA-binding domain of IRF3, which augmented TBK1 and IRF3 interaction, thereby inducing robust production of IFNs. Moreover, lncBST2-2 impaired the interaction between IRF3 and PP2A-RACK1 complex, an essential step for the dephosphorylation of IRF3. These data shown that lncBST2-2 promotes the innate immune response to viral infection through targeting IRF3. Our study reveals the lncRNA involved in the activation of IRF3 and provides a new insight into the role of lncRNA in antiviral innate immunity. IMPORTANCE Innate immunity is an important part of the human immune system to resist the invasion of foreign pathogens. IRF3 plays a critical role in the innate immune response to viral infection. In this study, we demonstrated that lncBST2-2 plays an important role in innate immunity. Virus-induced lncBST2-2 positively regulates innate immunity by interacting with IRF3 and blocking the dephosphorylation effect of RACK1-PP2A complex on IRF3, thus inhibiting viral infection. Our study provides a new insight into the role of lncBST2-2 in the regulation of IRF3 signaling activation.

Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination Boosts Neutralizing Activity Against Seasonal Human Coronaviruses.

BACKGROUND: Most of the millions of people that are vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), have previously been infected by related circulating human coronaviruses (hCoVs) causing common colds and will experience further encounters with these viruses in the future. Whether COVID-19 vaccinations impact neutralization of seasonal coronaviruses is largely unknown. METHODS: We analyzed the capacity of sera derived from 24 individuals before and after heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination to neutralize genuine OC43, NL63, and 229E hCoVs, as well as viral pseudoparticles carrying the SARS-CoV-1, SARS-CoV-2, Middle East Respiratory Syndrome (MERS)-CoV, and hCoV-OC43, hCoV-NL63, and hCoV-229E spike proteins. Genuine hCoVs or spike containing pseudovirions were incubated with different concentrations of sera and neutralization efficiencies were determined by measuring viral RNA yields, intracellular viral nucleocapsid expression, or reporter gene expression in Huh-7 cells. RESULTS: All individuals showed strong preexisting immunity against hCoV-OC43. Neutralization of hCoV-NL63 was more variable and all sera showed only modest inhibitory activity against genuine hCoV-229E. SARS-CoV-2 vaccination resulted in efficient cross-neutralization of SARS-CoV-1 but not of MERS-CoV. On average, vaccination significantly increased the neutralizing activity against genuine hCoV-OC43, hCoV-NL63, and hCoV-229E. CONCLUSIONS: Heterologous COVID-19 vaccination may confer some cross-protection against endemic seasonal coronaviruses.

Spike residue 403 affects binding of coronavirus spikes to human ACE2.

The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses.

Long Noncoding RNA ITPRIP-1 Positively Regulates the Innate Immune Response through Promotion of Oligomerization and Activation of MDA5.

Emerging evidence indicates that long noncoding RNAs (lncRNAs) regulate various biological processes, especially innate and adaptive immunity. However, the relationship between lncRNAs and the interferon (IFN) pathway remains largely unknown. Here, we report that lncRNA ITPRIP-1 (lncITPRIP-1) is involved in viral infection and plays a crucial role in the virus-triggered IFN signaling pathway through the targeting of melanoma differentiation-associated gene 5 (MDA5). LncITPRIP-1 can be induced by viral infection, which is not entirely dependent on the IFN signal. Besides, there is no coding potential found in the lncITPRIP-1 transcript. LncITPRIP-1 binds to the C terminus of MDA5, and it possesses the ability to boost the oligomerization of both the full length and the 2 caspase activation and recruitment domains of MDA5 in a K63-linked polyubiquitination-independent manner. Amazingly, we also found that MDA5 can suppress hepatitis C virus (HCV) replication independently of IFN signaling through its C-terminal-deficient domain bound to viral RNA, in which lncITPRIP-1 plays a role as an assistant. In addition, the expression of lncITPRIP-1 is highly consistent with MDA5 expression, indicating that lncITPRIP-1 may function as a cofactor of MDA5. All the data suggest that lncITPRIP-1 enhances the innate immune response to viral infection through the promotion of oligomerization and activation of MDA5. Our study discovers the first lncRNA ITPRIP-1 involved in MDA5 activation.IMPORTANCE Hepatitis C virus infection is a global health issue, and there is still no available vaccine, which makes it urgent to reveal the underlying mechanisms of HCV and host factors. Although RIG-I has been recognized as the leading cytoplasmic sensor against HCV for a long time, recent findings that MDA5 regulates the IFN response to HCV have emerged. Our work validates the significant role of MDA5 in IFN signaling and HCV infection and proposes the first lncRNA inhibiting HCV replication by promoting the activation of MDA5 and mediating the association between MDA5 and HCV RNA, the study of which may shed light on the MDA5 function and treatment for hepatitis C patients. Our suggested model of how lncITPRIP-1 orchestrates signal transduction for IFN production illustrates the essential role of lncRNAs in virus elimination.

NLRX1 Mediates MAVS Degradation To Attenuate the Hepatitis C Virus-Induced Innate Immune Response through PCBP2.

Activation of innate immunity is essential for host cells to restrict the spread of invading viruses and other pathogens. However, attenuation or termination of signaling is also necessary for preventing immune-mediated tissue damage and spontaneous autoimmunity. Here, we identify nucleotide binding oligomerization domain (NOD)-like receptor X1 (NLRX1) as a negative regulator of the mitochondrial antiviral signaling protein (MAVS)-mediated signaling pathway during hepatitis C virus (HCV) infection. The depletion of NLRX1 enhances the HCV-triggered activation of interferon (IFN) signaling and causes the suppression of HCV propagation in hepatocytes. NLRX1, a HCV-inducible protein, interacts with MAVS and mediates the K48-linked polyubiquitination and subsequent degradation of MAVS via the proteasomal pathway. Moreover, poly(rC) binding protein 2 (PCBP2) interacts with NLRX1 to participate in the NLRX1-induced degradation of MAVS and the inhibition of antiviral responses during HCV infection. Mutagenic analyses further revealed that the NOD of NLRX1 is essential for NLRX1 to interact with PCBP2 and subsequently induce MAVS degradation. Our study unlocks a key mechanism of the fine-tuning of innate immunity by which NLRX1 restrains the retinoic acid-inducible gene I-like receptor (RLR)-MAVS signaling cascade by recruiting PCBP2 to MAVS for inducing MAVS degradation through the proteasomal pathway. NLRX1, a negative regulator of innate immunity, is a pivotal host factor for HCV to establish persistent infection.IMPORTANCE Innate immunity needs to be tightly regulated to maximize the antiviral response and minimize immune-mediated pathology, but the underlying mechanisms are poorly understood. In this study, we report that NLRX1 is a proviral host factor for HCV infection and functions as a negative regulator of the HCV-triggered innate immune response. NLRX1 recruits PCBP2 to MAVS and induces the K48-linked polyubiquitination and degradation of MAVS, leading to the negative regulation of the IFN signaling pathway and promoting HCV infection. Overall, this study provides intriguing insights into how innate immunity is regulated during viral infection.

ISG12a Restricts Hepatitis C Virus Infection through the Ubiquitination-Dependent Degradation Pathway.

UNLABELLED: Interferons (IFNs) restrict various kinds of viral infection via induction of hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high-IFN-inducible gene, ISG12a (also known as IFI27), exhibits a nonapoptotic antiviral effect on hepatitis C virus (HCV) infection. Viral NS5A protein is targeted specifically by ISG12a, which mediates NS5A degradation via a ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. S-phase kinase-associated protein 2 (SKP2) is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity. IMPORTANCE: Upon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadly unclear. The present study shows a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase, SKP2, for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity.