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Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome., (© 2024. The Author(s).)

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'- O -methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'- O -MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo , using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro . Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'- O -methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo , our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'- O -methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants., Competing Interests: Declaration of interests Eli Lilly and Company provided resources for this study. AbCellera Biologics Inc. received funding from the U.S. Department of Defense, Defense Advanced Research Projects Agency (DARPA) Pandemic Prevention Platform, agreement D18AC00002. This research was funded in part by the U.S. Government (the views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the U.S. Government). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357 (https://www.aps.anl.gov/Science/Publications/Acknowledgment-Statement-for-Publications). Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly and Company, which operates the facility (http://lrlcat.lilly.com/). This work was supported by the Intramural Program at the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Vaccine Research Center (to B.S.G. and J.R.M.). Operations support of the Galveston National Laboratory was supported by NIAID/NIH grant UC7AI094660. D.F., P.V., A.P., J.H., J.M.S., R.W.S., J.C., I. H., J.J.F., S.H., H.C.P., B.R., B.A.H., R.W.S., J.C., J.M.S., R.E.H., N.K., and B.E.J. are employees and/or stockholders of Eli Lilly and Company. K.W., S.Ž., M.W., E.L., L.K., Y.H., K.J., R.G., M.A.S., D.W.C., D.P., P.X., V.d.P., R.v.d.L., M.R., L.D., C.P., I.L., L.A., P.S., T.L.F., C.L.H., E.F., and B.C.B. are employees and stockholders of AbCellera Biologics Inc. AbCellera Biologics Inc. and the National Institutes of Health have filed patent applications related to the work described herein (US patent application 17/192243 and international patent application PCT/US21/20843, both titled “Anti-Coronavirus Antibodies and Methods of Use”)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2-infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: XX, P-YS, and VDM have filed a patent on the reverse genetic system and reporter SARS-CoV-2. Other authors declare no competing interests.

Coronaviruses have large positive-strand RNA genomes that are 5' capped and 3' polyadenylated. The 5'-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15-16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3'-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5' leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells., (© 2016 Elsevier Inc. All rights reserved.)

nsp1 protein of severe acute respiratory syndrome coronavirus (SARS-CoV), a group 2b CoV, suppresses host gene expression by promoting host mRNA degradation and translation inhibition. The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1, 133, and HKU9-1, belonging to groups 2b, 2c, and 2d, respectively. The host mRNA degradation and translational suppression activities of nsp1 of SARS-CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9-1. Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon (IFN-I) and IFN-stimulated genes in cells infected with an IFN-inducing SARS-CoV mutant, while 133 and HKU9-1 nsp1 proteins had relatively moderate IFN-inhibitory activities. The results of our studies suggested a conserved function among nsp1 proteins of SARS-CoV and group 2 bat CoVs.

The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.

Hantaviruses are causative agents of some severe human illnesses, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The viruses are maintained by rodent hosts, and humans acquire infection by inhaling virus-contaminated excreta from infected animals. To examine the epidemiology of hantavirus infections in Japan and Far East Russia, we conducted epidemiological surveys in these regions. In Japan, anti-hantavirus antibodies were found in four rodent species, Clethrionomys rufocanus, Rattus norvegicus, R. rattus, and Apodemus speciosus. Although no new HFRS cases have been officially reported over the past 20 years in Japan, one member of the Japan Ground Self-Defense Force did test positive for hantavirus antibody. Repeated surveys in Far East Russia have revealed that two distinct hantavirus types cause severe HFRS in this region. Hantavirus sequences identified from A. peninsulae, fetal HFRS cases in Vladivostok, and Amur virus are highly similar to each other (> 92% identity), but they are less similar (approximately 84% identity) to the prototypical Hantaan virus, which is carried by A. agrarius. Phylogenetic analysis also indicates that Amur and A. peninsulae-associated viruses are distinct from Hantaan virus, suggesting that A. peninsulae is the reservoir animal for Amur virus, which causes severe HFRS. From HFRS patients in the Khabarovsk region, we identified viruses with nucleotide sequences that are more similar to Far East virus (> 96%identity) than to the Hantaan (88-89% identity) or Amur (81-83% identity) viruses. Phylogenetic analysis also indicates that the viruses from Khabarovsk HFRS patients are closely related to the Far East virus, and distinct from Amur virus.

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) causes a recently emerged human disease associated with pneumonia. The 5' end two-thirds of the single-stranded positive-sense viral genomic RNA, gene 1, encodes 16 mature proteins. Expression of nsp1, the most N-terminal gene 1 protein, prevented Sendai virus-induced endogenous IFN-beta mRNA accumulation without inhibiting dimerization of IFN regulatory factor 3, a protein that is essential for activation of the IFN-beta promoter. Furthermore, nsp1 expression promoted degradation of expressed RNA transcripts and host endogenous mRNAs, leading to a strong host protein synthesis inhibition. SCoV replication also promoted degradation of expressed RNA transcripts and host mRNAs, suggesting that nsp1 exerted its mRNA destabilization function in infected cells. In contrast to nsp1-induced mRNA destablization, no degradation of the 28S and 18S rRNAs occurred in either nsp1-expressing cells or SCoV-infected cells. These data suggested that, in infected cells, nsp1 promotes host mRNA degradation and thereby suppresses host gene expression, including proteins involved in host innate immune functions. SCoV nsp1-mediated promotion of host mRNA degradation may play an important role in SCoV pathogenesis.

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissue of a striped-field mouse (Apodemus agrarius) captured in Songnae-ri, Gyeonggi Province, Republic of Korea. Found primarily in mountainous areas, the Korean field mouse (A. peninsulae) is the second-most dominant field rodent species found throughout Korea. A new hantavirus, designated Soochong (SOO), was isolated in Vero E6 cells from four A. peninsulae captured in August 1997 at Mt. Gyebang in Hongcheon-gun, Mt. Gachil, Inje-gun, Gangwon Province, and in September 1998 at Mt. Deogyu, Muju-gun, Jeollabuk Province. The entire S, M, and L genomic segments of SOO virus, amplified by RT-PCR from lung tissues of seropositive A. peninsulae and from virus-infected Vero E6 cells, diverged from HTN virus (strain 76-118) by 15.6%, 22.8%, and 21.7% at the nucleotide level and 3.5%, 9.5%, and 4.6% at the amino acid level, respectively. Phylogenetic analyses of the nucleotide and deduced amino acid sequences, using the maximum parsimony and neighbor-joining methods, indicated that SOO virus was distinct from A. agrarius-borne HTN virus. SOO virus shared a common ancestry with Amur virus from Far East Russia, as well as with H5 and B78 hantaviruses, previously isolated from HFRS patients in China. Cross-focus-reduction neutralizating antibody tests showed that SOO virus, which is the first hantavirus isolated in cell culture from A. peninsulae, could be classified as a new hantavirus serotype., (Copyright 2005 Wiley-Liss, Inc.)

The genetic and antigenic characteristics of the Amur (AMR) and Far East (FE) virus lineages, which are both within the genus Hantavirus, were studied. Representative viruses, H5 and B78 for AMR and Bao 14 for FE, were used. The entire small (S) and medium (M) segments, except for the 3'- and 5'-ends, were sequenced. The deduced amino acid sequences of AMR had 96.7 and 92.0-92.2% identities with the Hantaan (HTN) virus in the S and M segments, respectively. The amino acid sequences of FE had 99.1 and 97.9% identities in the S and M segments, respectively. The three viral strains and HTN virus had similar binding patterns to a panel of monoclonal antibodies (MAbs), except that one MAb did not bind AMR. However, sera from Apodemus peninsulae, naturally infected with AMR virus, neutralized homologous viruses at 1:160 to 1:320 dilutions and HTN at 1:20 to 1:40 dilutions. The anti-AMR serum neutralized homologous viruses at a 1:80 dilution and HTN at a 1:40 dilution. The anti-HTN serum did not neutralize AMR (<1:40 dilution), although it had a high neutralizing titer (1:320) against the homologous virus. Therefore, we suggest that AMR virus may constitute a distinct serotype within the genus Hantavirus.

The virulence of hantaviruses that are antigenically related but have different genetic characteristics from the prototype of hantavirus, Hantaan (HTN) virus, was examined in newborn mice. The H5 and B78 strains of the Amur (AMR) genotype, the Bao14 strain of the Far East (FE) genotype, and the 76-118 strain of HTN virus were inoculated subcutaneously (1focus-forming unit; FFU) into newborn mice. All of the AMR and FE genotype viruses inoculated mice were died by 16 days post-infection (dpi) and 21 dpi, respectively, while 50% of the HTN virus inoculated mice survived until 30 dpi. The AMR and FE genotype viruses inoculated mice had high viral titers in the lung (1.3x10(6) to 1.3x10(8) FFU/gram [g] tissue) , brain (2.1x10(7) to 1.2x10(9) FFU/g tissue), and kidney(2.5x10(5) to 1.6x10(7) FFU/g tissue), and showed a detectable level of antibodies (titers 1:16-1:32) at 14 dpi. In contrast, the HTN virus infected mice had viruses only in the lungs at low titers (1.1-5.3x10(5) FFU/g tissue). Observations of body-weight changes revealed that the AMR and FE genotype viruses inoculated mice had lower growth rates than the HTN virus inoculated mice. These data suggest that the AMR and FE genotype viruses are more virulent than the HTN virus in newborn mice.

Epizootiological surveys on hantavirus infections in rodents were carried out in various areas of Japan, including the four major islands of Hokkaido, Honshu, Shikoku, and Kyushu from 2000 to 2003. A total of 1,221 rodents and insectivores were captured. Seropositive animals were found in Apodemus (A.) speciosus (5/482, 1.0%), Rattus (R.) norvegicus (4/364, 1.1%), R. rattus (3/45, 6.7%), and Clethrionomys (C.) rufocanus (7/197, 3.6%). The partial S segment was amplified from one seropositive R. rattus captured at Hakodate. The nucleotide sequence showed 96% identity with the Seoul virus (SEOV) prototype strain SR-11. In addition, we conducted an epidemiological survey on human hantavirus infection in a high-risk population, the personnel of the Japan Ground Self-defense Force on Hokkaido. One out of 207 human blood samples was positive for anti-hantavirus antibody by IFA, ELISA, and WB analysis. The result of the serotype specific ELISA indicates that this individual acquired SEOV infection. This study indicates that A. speciosus, R. norvegicus, R. rattus, and C. rufocanus carry hantaviruses as the reservoir animals in Japan. Infected R. rattus and R. norvegicus in port areas could be the sources of human SEOV infection and a threat to travelers and individuals working in seaports.

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.

In an epizootiologic survey of 122 rodents captured in Vladivostok, Russia, antibodies positive for hantavirus were found in Apodemus peninsulae (4/70), A. agrarius (1/39), and Clethrionomys rufocanus (1/8). The hantavirus sequences identified in two seropositive A. peninsulae and two patients with hemorrhagic fever with renal syndrome (HFRS) from the Primorye region of Far East Russia were designated as Solovey and Primorye, respectively. The nucleotide sequences of the Solovey, Primorye, and Amur (obtained through GenBank) sequences were closely related (>92% identity). Solovey and Primorye sequences shared 84% nucleotide identity with the prototype Hantaan 76-118. Phylogenetic analysis also indicated a close relationship between Solovey, Primorye, Amur, and other viruses identified in Russia, China, and Korea. Our findings suggest that the Korean field mouse (A. peninsulae) is the reservoir for a hantavirus that causes HFRS over a vast area of east Asia, including Far East Russia.

Hantaviral antibodies were detected in the sera from Apodemus (A.) agrarius and A. peninsulae captured in Ningxia province, China by several different serological diagnostic methods. A total of 409 sera from rodent and insectivore species were collected in 1999 and examined by indirect immunofluorescent antibody assay (IFA). Among them, 19 of 191 (9.9%) sera of A. agrarius and 1 of 13 (7.7%) sera of A. peninsulae were positive for hantaviral antibodies. The other species (Rattus norvegicus, Mus musculus, Cricetulus triton, and Sorex cylindricauda) were negative. The reaction pattern of positive serum was characterized as scattered and granular virus antigens in the cytoplasm of hantavirus infected Vero E6 cells. Some of the A. agrarius sera positive for hantavirus were further examined by Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and the focus reduction neutralization test (FRNT). By WB, positive sera showed the same specific reaction pattern of baculovirus-expressed recombinant hantaviral nucleocapsid protein, as shown in hantavirus-immune serum. By ELISA, IFA-positive sera showed significantly higher optical densities (around 1.0) than the negative A. agrarius sera. Hantaan type hantavirus was neutralized with the positive sera. These results suggest that A. agrarius have hantavirus infection and may play a role as a reservoir animal for hantavirus in Ningxia Hui Autonomous Province, China.