Identification of a 1.4-kb-Long Sequence Located in the nsp12 and nsp13 Coding Regions of SARS-CoV-2 Genomic RNA That Mediates Efficient Viral RNA Packaging.

The specific packaging of the viral RNA genome into virus particles is an essential step in the replication cycle of coronaviruses (CoVs). Using a single-cycle, replicable severe acute respiratory syndrome CoV-2 (SARS-CoV-2) mutant, we demonstrated the preferential packaging of the SARS-CoV-2 genomic RNA into purified virus particles. Furthermore, based on the sequence of an efficiently packaged defective interfering RNA of SARS-CoV, a closely related CoV, that was generated after serial passages of SARS-CoV in cell culture, we designed a series of replication-competent SARS-CoV-2 minigenome RNAs to identify the specific viral RNA region that is important for SARS-CoV-2 RNA packaging into virus particles. We showed that a 1.4-kb-long sequence, derived from the nsp12 and nsp13 coding regions of the SARS-CoV-2 genomic RNA, is required for the efficient packaging of SARS-CoV-2 minigenome RNA into SARS-CoV-2 particles. In addition, we also showed that the presence of possibly the entire 1.4-kb-long sequence is important for the efficient packaging of SARS-CoV-2 RNA. Our findings highlight the differences between the RNA packaging sequence identified in SARS-CoV-2, a Sarbecovirus, and the packaging signal of mouse hepatitis virus (MHV), an Embecovirus, which is a 95-nt-long sequence located at the nsp15 coding region of MHV genomic RNA. Collectively, our data imply that both the location and the sequence/structural features of the RNA element(s) that drives the selective and efficient packaging of viral genomic RNA are not conserved among the subgenera Embecovirus and Sarbecovirus within the Betacoronavirus genus. IMPORTANCE Elucidating the mechanism of SARS-CoV-2 RNA packaging into virus particles is important for the rational design of antiviral drugs that inhibit this vital step in the replication cycle of CoVs. However, our knowledge about the RNA packaging mechanism in SARS-CoV-2, including the identification of the viral RNA region important for SARS-CoV-2 RNA packaging, is limited, primarily due to the logistical challenges of handing SARS-CoV-2 in biosafety level 3 (BSL3) facilities. Our study, using a single-cycle, replicable SARS-CoV-2 mutant, which can be handled in a BSL2 lab, demonstrated the preferential packaging of full-length SARS-CoV-2 genomic RNA into virus particles and identified a specific 1.4-kb-long RNA region in SARS-CoV-2 genomic RNA that is required for the efficient packaging of SARS-CoV-2 RNA into virus particles. The information generated in our study could be valuable for clarifying the mechanisms of SARS-CoV-2 RNA packaging and for the development of targeted therapeutics against SARS-CoV-2 and other related CoVs.

Sequence basis for selectivity of ephrin-B2 ligand for Eph receptors and pathogenic henipavirus G glycoproteins.

IMPORTANCE: Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors. We then use this information to engineer EFNB2 as a soluble decoy receptor that specifically binds the attachment glycoproteins of the Nipah virus and other related henipaviruses to neutralize infection. These findings further mechanistic understanding of protein selectivity and may facilitate the development of diagnostics or therapeutics against henipavirus infection.

Characterization of the Molecular Interactions That Govern the Packaging of Viral RNA Segments into Rift Valley Fever Phlebovirus Particles.

Rift Valley fever phlebovirus (RVFV) has a single-stranded, negative-sense RNA genome, consisting of L, M, and S segments. The virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes (RNPs), composed of encapsidated genomes carrying N protein and the viral polymerase, L protein. A quantitative analysis of the profile of viral RNA segments packaged into RVFV particles showed that all three genomic RNA segments had similar packaging abilities, whereas among antigenomic RNA segments, the antigenomic S RNA, which serves as the template for the transcription of mRNA expressing the RVFV virulence factor, NSs, displayed a significantly higher packaging ability. To delineate the factor(s) governing the packaging of RVFV RNA segments, we characterized the interactions between Gn and viral RNPs in RVFV-infected cells. Coimmunoprecipitation analysis demonstrated the interaction of Gn with N protein, L protein, and viral RNAs in RVFV-infected cells. Furthermore, UV-cross-linking and immunoprecipitation analysis revealed, for the first time in bunyaviruses, the presence of a direct interaction between Gn and all the viral RNA segments in RVFV-infected cells. Notably, analysis of the ability of Gn to bind to RVFV RNA segments indicated a positive correlation with their respective packaging abilities and highlighted a binding preference of Gn for antigenomic S RNA, among the antigenomic RNA segments, suggesting the presence of a selection mechanism for antigenomic S RNA incorporation into infectious RVFV particles. Collectively, the results of our study illuminate the importance of a direct interaction between Gn and viral RNA segments in determining their efficiency of incorporation into RVFV particles. IMPORTANCE Rift Valley fever phlebovirus, a bunyavirus, is a mosquito-borne, segmented RNA virus that can cause severe disease in humans and ruminants. An essential step in RVFV life cycle is the packaging of viral RNA segments to produce infectious virus particles for dissemination to new hosts. However, there are key gaps in knowledge regarding the mechanisms that regulate viral RNA packaging efficiency in bunyaviruses. Our studies investigating the mechanism of RNA packaging in RVFV revealed the presence of a direct interaction between the viral envelope glycoprotein, Gn, and the viral RNA segments in infected cells, for the first time in bunyaviruses. Furthermore, our data strongly indicate a critical role for the direct interaction between Gn and viral RNAs in determining the efficiency of incorporation of viral RNA segments into RVFV particles. Clarifying the fundamental mechanisms of RNA packaging in RVFV would be valuable for the development of antivirals and live-attenuated vaccines.

An Enhanced Decoding Algorithm for Coded Compressed Sensing with Applications to Unsourced Random Access.

Unsourced random access (URA) has emerged as a pragmatic framework for next-generation distributed sensor networks. Within URA, concatenated coding structures are often employed to ensure that the central base station can accurately recover the set of sent codewords during a given transmission period. Many URA algorithms employ independent inner and outer decoders, which can help reduce computational complexity at the expense of a decay in performance. In this article, an enhanced decoding algorithm is presented for a concatenated coding structure consisting of a wide range of inner codes and an outer tree-based code. It is shown that this algorithmic enhancement has the potential to simultaneously improve error performance and decrease the computational complexity of the decoder. This enhanced decoding algorithm is applied to two existing URA algorithms, and the performance benefits of the algorithm are characterized. Findings are supported by numerical simulations.

Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Very little is known about the host pathways that regulate the stability of CoV mRNAs, which carry some unusual features. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation. Although CoV mRNAs are of cytoplasmic origin, the presence of several NMD-inducing features (including multiple ORFs with internal termination codons that create a long 3' untranslated region) in CoV mRNAs led us to explore the interplay between the NMD pathway and CoVs. Our study using murine hepatitis virus as a model CoV showed that CoV mRNAs are recognized by the NMD pathway as a substrate, resulting in their degradation. Furthermore, CoV replication induced the inhibition of the NMD pathway, and N protein (a viral structural protein) had an NMD inhibitory function that protected viral mRNAs from rapid decay. Our data further suggest that the NMD pathway interferes with optimal viral replication by degrading viral mRNAs early in infection, before sufficient accumulation of N protein. Our study presents clear evidence for the biological importance of the NMD pathway in controlling the stability of mRNAs and the efficiency of replication of a cytoplasmic RNA virus.

Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States.

The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into 2 virus repositories, making it broadly available to the public health and research communities. We hope that open access to this reagent will expedite development of medical countermeasures.

Inhibition of Stress Granule Formation by Middle East Respiratory Syndrome Coronavirus 4a Accessory Protein Facilitates Viral Translation, Leading to Efficient Virus Replication.

Stress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication. The MERS-CoV 4a accessory protein is known to inhibit SG formation in cells in which it was expressed by binding to double-stranded RNAs and inhibiting protein kinase R (PKR)-mediated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Replication of MERS-CoV lacking the genes for 4a and 4b (MERS-CoV-Δp4), but not MERS-CoV, induced SG accumulation in MERS-CoV-susceptible HeLa/CD26 cells, while replication of both viruses failed to induce SGs in Vero cells, demonstrating cell type-specific differences in MERS-CoV-Δp4-induced SG formation. MERS-CoV-Δp4 replicated less efficiently than MERS-CoV in HeLa/CD26 cells, and inhibition of SG formation by small interfering RNA-mediated depletion of the SG components promoted MERS-CoV-Δp4 replication, demonstrating that SG formation was detrimental for MERS-CoV replication. Inefficient MERS-CoV-Δp4 replication was not due to either the induction of type I and type III interferons or the accumulation of viral mRNAs in the SGs. Rather, it was due to the inefficient translation of viral proteins, which was caused by high levels of PKR-mediated eIF2α phosphorylation and likely by the confinement of various factors that are required for translation in the SGs. Finally, we established that deletion of the 4a gene alone was sufficient for inducing SGs in infected cells. Our study revealed that 4a-mediated inhibition of SG formation facilitates viral translation, leading to efficient MERS-CoV replication.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory failure with a high case fatality rate in patients, yet effective antivirals and vaccines are currently not available. Stress granule (SG) formation is one of the cellular stress responses to virus infection and is generally triggered as a result of stress-induced translation arrest. SGs can be beneficial or detrimental for virus replication, and the biological role of SGs in CoV infection is unclear. The present study showed that the MERS-CoV 4a accessory protein, which was reported to block SG formation in cells in which it was expressed, inhibited SG formation in infected cells. Our data suggest that 4a-mediated inhibition of SG formation facilitates the translation of viral mRNAs, resulting in efficient virus replication. To our knowledge, this report is the first to show the biological significance of SG in CoV replication and provides insight into the interplay between MERS-CoV and antiviral stress responses.

The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines.

Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN-ß and IFN-λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic Origin.

UNLABELLED: The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome CoV (SARS-CoV) represent highly pathogenic human CoVs that share a property to inhibit host gene expression at the posttranscriptional level. Similar to the nonstructural protein 1 (nsp1) of SARS-CoV that inhibits host gene expression at the translational level, we report that MERS-CoV nsp1 also exhibits a conserved function to negatively regulate host gene expression by inhibiting host mRNA translation and inducing the degradation of host mRNAs. Furthermore, like SARS-CoV nsp1, the mRNA degradation activity of MERS-CoV nsp1, most probably triggered by its ability to induce an endonucleolytic RNA cleavage, was separable from its translation inhibitory function. Despite these functional similarities, MERS-CoV nsp1 used a strikingly different strategy that selectively targeted translationally competent host mRNAs for inhibition. While SARS-CoV nsp1 is localized exclusively in the cytoplasm and binds to the 40S ribosomal subunit to gain access to translating mRNAs, MERS-CoV nsp1 was distributed in both the nucleus and the cytoplasm and did not bind stably to the 40S subunit, suggesting a distinctly different mode of targeting translating mRNAs. Interestingly, consistent with this notion, MERS-CoV nsp1 selectively targeted mRNAs, which are transcribed in the nucleus and transported to the cytoplasm, for translation inhibition and mRNA degradation but spared exogenous mRNAs introduced directly into the cytoplasm or virus-like mRNAs that originate in the cytoplasm. Collectively, these data point toward a novel viral strategy wherein the cytoplasmic origin of MERS-CoV mRNAs facilitates their escape from the inhibitory effects of MERS-CoV nsp1. IMPORTANCE: Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human CoV that emerged in Saudi Arabia in 2012. MERS-CoV has a zoonotic origin and poses a major threat to public health. However, little is known about the viral factors contributing to the high virulence of MERS-CoV. Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors. The nonstructural protein 1 (nsp1) of CoVs is one such protein that inhibits host gene expression and is a major virulence factor. This study presents evidence for a strategy used by MERS-CoV nsp1 to inhibit host gene expression that has not been described previously for any viral protein. The present study represents a meaningful step toward a better understanding of the factors and molecular mechanisms governing the virulence and pathogenesis of MERS-CoV.

Interplay between viruses and host mRNA degradation.

Messenger RNA degradation is a fundamental cellular process that plays a critical role in regulating gene expression by controlling both the quality and the abundance of mRNAs in cells. Naturally, viruses must successfully interface with the robust cellular RNA degradation machinery to achieve an optimal balance between viral and cellular gene expression and establish a productive infection in the host. In the past several years, studies have discovered many elegant strategies that viruses have evolved to circumvent the cellular RNA degradation machinery, ranging from disarming the RNA decay pathways and co-opting the factors governing cellular mRNA stability to promoting host mRNA degradation that facilitates selective viral gene expression and alters the dynamics of host-pathogen interaction. This review summarizes the current knowledge of the multifaceted interaction between viruses and cellular mRNA degradation machinery to provide an insight into the regulatory mechanisms that influence gene expression in viral infections. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation.

Severe acute respiratory syndrome (SARS) coronavirus nonstructural protein 1 (nsp1) binds to the 40S ribosomal subunit and inhibits translation, and it also induces a template-dependent endonucleolytic cleavage of host mRNAs. nsp1 inhibits the translation of cap-dependent and internal ribosome entry site (IRES)-driven mRNAs, including SARS coronavirus mRNAs, hepatitis C virus (HCV), and cricket paralysis virus (CrPV) IRES-driven mRNAs that are resistant to nsp1-induced RNA cleavage. We used an nsp1 mutant, nsp1-CD, lacking the RNA cleavage function, to delineate the mechanism of nsp1-mediated translation inhibition and identify the translation step(s) targeted by nsp1. nsp1 and nsp1-CD had identical inhibitory effects on mRNA templates that are resistant to nsp1-induced RNA cleavage, implying the validity of using nsp1-CD to dissect the translation inhibition function of nsp1. We provide evidence for a novel mode of action of nsp1. nsp1 inhibited the translation initiation step by targeting at least two separate stages: 48S initiation complex formation and the steps involved in the formation of the 80S initiation complex from the 48S complex. nsp1 had a differential, mRNA template-dependent, inhibitory effect on 48S and 80S initiation complex formation. nsp1 inhibited different steps of translation initiation on CrPV and HCV IRES, both of which initiate translation via an IRES-40S binary complex intermediate; nsp1 inhibited binary complex formation on CrPV IRES and 48S complex formation on HCV IRES. Collectively, the data revealed that nsp1 inhibited translation by exerting its effect on multiple stages of translation initiation, depending on the mechanism of initiation operating on the mRNA template.

Roles of the coding and noncoding regions of rift valley Fever virus RNA genome segments in viral RNA packaging.

We characterized the RNA elements involved in the packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5'-terminal 25 nucleotides of each RNA segment were equally competent for RNA packaging and carried an RNA packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA packaging.

SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

Mechanistic insight into the efficient packaging of antigenomic S RNA into Rift Valley fever virus particles.

Rift Valley fever virus (RVFV), a bunyavirus, has a single-stranded, negative-sense tri-segmented RNA genome, consisting of L, M and S RNAs. An infectious virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes composed of encapsidated viral RNA segments. The antigenomic S RNA, which serves as the template of the mRNA encoding a nonstructural protein, NSs, an interferon antagonist, is also efficiently packaged into RVFV particles. An interaction between Gn and viral ribonucleoprotein complexes, including the direct binding of Gn to viral RNAs, drives viral RNA packaging into RVFV particles. To understand the mechanism of efficient antigenomic S RNA packaging in RVFV, we identified the regions in viral RNAs that directly interact with Gn by performing UV-crosslinking and immunoprecipitation of RVFV-infected cell lysates with anti-Gn antibody followed by high-throughput sequencing analysis (CLIP-seq analysis). Our data suggested the presence of multiple Gn-binding sites in RVFV RNAs, including a prominent Gn-binding site within the 3' noncoding region of the antigenomic S RNA. We found that the efficient packaging of antigenomic S RNA was abrogated in a RVFV mutant lacking a part of this prominent Gn-binding site within the 3' noncoding region. Also, the mutant RVFV, but not the parental RVFV, triggered the early induction of interferon-ß mRNA expression after infection. These data suggest that the direct binding of Gn to the RNA element within the 3' noncoding region of the antigenomic S RNA promoted the efficient packaging of antigenomic S RNA into virions. Furthermore, the efficient packaging of antigenomic S RNA into RVFV particles, driven by the RNA element, facilitated the synthesis of viral mRNA encoding NSs immediately after infection, resulting in the suppression of interferon-ß mRNA expression.

The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins: Selective Ephrin-B2 Decoys for Nipah and Hendra Virus.

Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and functions as a cell entry receptor for several henipaviruses including Nipah virus (NiV), a pathogenic zoonotic virus with pandemic potential. To understand the sequence basis of promiscuity for EFNB2 binding to the attachment glycoprotein of NiV (NiV-G) and Eph receptors, we performed deep mutagenesis on EFNB2 to identify mutations that enhance binding to NiV-G over EphB2, one of the highest affinity Eph receptors. The mutations highlight how different EFNB2 conformations are selected by NiV-G versus EphB2. Specificity mutations are enriched at the base of the G-H binding loop of EFNB2, especially surrounding a phenylalanine hinge upon which the G-H loop pivots, and at a phenylalanine hook that rotates away from the EFNB2 core to engage Eph receptors. One EFNB2 mutant, D62Q, possesses pan-specificity to the attachment glycoproteins of closely related henipaviruses and has markedly diminished binding to the six Eph receptors. However, EFNB2-D62Q has high residual binding to EphB3 and EphB4. A second deep mutational scan of EFNB2 identified combinatorial mutations to further enhance specificity to NiV-G. A triple mutant of soluble EFNB2, D62Q-Q130L-V167L, has minimal binding to Eph receptors but maintains binding, albeit reduced, to NiV-G. Soluble EFNB2 decoy receptors carrying the specificity mutations were potent neutralizers of chimeric henipaviruses. These findings demonstrate how specific residue changes at the shared binding interface of a promiscuous ligand (EFNB2) can influence selectivity for multiple receptors, and may also offer insight towards the development of henipavirus therapeutics and diagnostics.

Alphacoronavirus transmissible gastroenteritis virus nsp1 protein suppresses protein translation in mammalian cells and in cell-free HeLa cell extracts but not in rabbit reticulocyte lysate.

The nsp1 protein of transmissible gastroenteritis virus (TGEV), an alphacoronavirus, efficiently suppressed protein synthesis in mammalian cells. Unlike the nsp1 protein of severe acute respiratory syndrome coronavirus, a betacoronavirus, the TGEV nsp1 protein was unable to bind 40S ribosomal subunits or promote host mRNA degradation. TGEV nsp1 also suppressed protein translation in cell-free HeLa cell extract; however, it did not affect translation in rabbit reticulocyte lysate (RRL). Our data suggested that HeLa cell extracts and cultured host cells, but not RRL, contain a host factor(s) that is essential for TGEV nsp1-induced translational suppression.

An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.2 sublineage of B.1.1.529/omicron escapes nearly all monoclonal antibodies currently authorized for therapeutic treatment of COVID-19. Decoy receptors, which are based on soluble forms of the host entry receptor ACE2, are an alternative strategy that broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE2 2 .v2.4-IgG1 was previously shown to be effective in vivo against SARS-CoV-2 variants when administered intravenously. Here, the inhalation of sACE2 2 .v2.4-IgG1 is found to increase survival and ameliorate lung injury in K18-hACE2 transgenic mice inoculated with a lethal dose of the virulent P.1/gamma virus. Loss of catalytic activity reduced the decoy’s therapeutic efficacy supporting dual mechanisms of action: direct blocking of viral S and turnover of ACE2 substrates associated with lung injury and inflammation. Binding of sACE2 2 .v2.4-IgG1 remained tight to S of BA.1 omicron, despite BA.1 omicron having extensive mutations, and binding exceeded that of four monoclonal antibodies approved for clinical use. BA.1 pseudovirus and authentic virus were neutralized at picomolar concentrations. Finally, tight binding was maintained against S from the BA.2 omicron sublineage, which differs from S of BA.1 by 26 mutations. Overall, the therapeutic potential of sACE2 2 .v2.4-IgG1 is further confirmed by inhalation route and broad neutralization potency persists against increasingly divergent SARS-CoV-2 variants.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo , in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.