A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, generates multiple protein-coding, subgenomic RNAs (sgRNAs) from a longer genomic RNA, all bearing identical termini with poorly understood roles in regulating viral gene expression. Insulin and interferon-gamma, two host-derived, stress-related agents, and virus spike protein, induce binding of glutamyl-prolyl-tRNA synthetase (EPRS1), within an unconventional, tetra-aminoacyl-tRNA synthetase complex, to the sgRNA 3'-end thereby enhancing sgRNA expression. We identify an EPRS1-binding sarbecoviral pan-end activating RNA (SPEAR) element in the 3'-end of viral RNAs driving agonist-induction. Translation of another co-terminal 3'-end feature, ORF10, is necessary for SPEAR-mediated induction, independent of Orf10 protein expression. The SPEAR element enhances viral programmed ribosomal frameshifting, thereby expanding its functionality. By co-opting noncanonical activities of a family of essential host proteins, the virus establishes a post-transcriptional regulon stimulating global viral RNA translation. A SPEAR-targeting strategy markedly reduces SARS-CoV-2 titer, suggesting a pan-sarbecoviral therapeutic modality.

An optimized circular polymerase extension reaction-based method for functional analysis of SARS-CoV-2.

BACKGROUND: Reverse genetics systems have been crucial for studying specific viral genes and their relevance in the virus lifecycle, and become important tools for the rational attenuation of viruses and thereby for vaccine design. Recent rapid progress has been made in the establishment of reverse genetics systems for functional analysis of SARS-CoV-2, a coronavirus that causes the ongoing COVID-19 pandemic that has resulted in detrimental public health and economic burden. Among the different reverse genetics approaches, circular polymerase extension reaction (CPER) has become one of the leading methodologies to generate recombinant SARS-CoV-2 infectious clones. Although CPER has greatly facilitated SARS-CoV-2 analysis, it still has certain intrinsic limitations that impede the efficiency and robustness of virus rescue. RESULTS: We developed an optimized CPER methodology which, through the use of a modified linker plasmid and by performing DNA nick ligation and direct transfection of permissive cells, overcomes certain intrinsic limitations of the 'traditional' CPER approaches for SARS-CoV-2, allowing for efficient virus rescue. CONCLUSIONS: The herein described optimized CPER system may facilitate research studies to assess the contribution of SARS-CoV-2 genes and individual motifs or residues to virus replication, pathogenesis and immune escape, and may also be adapted to other viruses.

Insights into pandemic respiratory viruses: manipulation of the antiviral interferon response by SARS-CoV-2 and influenza A virus.

The outbreak of the COVID-19 pandemic one year after the centennial of the 1918 influenza pandemic reaffirms the catastrophic impact respiratory viruses can have on global health and economy. A key feature of SARS-CoV-2 and influenza A viruses (IAV) is their remarkable ability to suppress or dysregulate human immune responses. Here, we summarize the growing knowledge about the interplay of SARS-CoV-2 and antiviral innate immunity, with an emphasis on the regulation of type-I or -III interferon responses that are critically implicated in COVID-19 pathogenesis. Furthermore, we draw parallels to IAV infection and discuss shared innate immune sensing mechanisms and the respective viral countermeasures.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

Actin cytoskeleton remodeling primes RIG-I-like receptor activation.

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.

ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity.

Activation of the RIG-I-like receptors, retinoic-acid inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15, the mechanistic roles of which in innate immunity still remain enigmatic. In the present study, we report that ISG15 conjugation is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISGylation of the caspase activation and recruitment domains of MDA5 promotes its oligomerization and thereby triggers activation of innate immunity against a range of viruses, including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease of SARS-CoV-2, a recently emerged coronavirus that has caused the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies a key immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19.

Viral Evasion of RIG-I-Like Receptor-Mediated Immunity through Dysregulation of Ubiquitination and ISGylation.

Viral dysregulation or suppression of innate immune responses is a key determinant of virus-induced pathogenesis. Important sensors for the detection of virus infection are the RIG-I-like receptors (RLRs), which, in turn, are antagonized by many RNA viruses and DNA viruses. Among the different escape strategies are viral mechanisms to dysregulate the post-translational modifications (PTMs) that play pivotal roles in RLR regulation. In this review, we present the current knowledge of immune evasion by viral pathogens that manipulate ubiquitin- or ISG15-dependent mechanisms of RLR activation. Key viral strategies to evade RLR signaling include direct targeting of ubiquitin E3 ligases, active deubiquitination using viral deubiquitinating enzymes (DUBs), and the upregulation of cellular DUBs that regulate RLR signaling. Additionally, we summarize emerging new evidence that shows that enzymes of certain coronaviruses such as SARS-CoV-2, the causative agent of the current COVID-19 pandemic, actively deISGylate key molecules in the RLR pathway to escape type I interferon (IFN)-mediated antiviral responses. Finally, we discuss the possibility of targeting virally-encoded proteins that manipulate ubiquitin- or ISG15-mediated innate immune responses for the development of new antivirals and vaccines.