The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

Miorin, Lisa; Mire, Chad E.; Hume, Adam J.; Huang, Jessie; Crossland, Nicholas A.; White, Kris M.; Laporte, Manon; Kehrer, Thomas; Haridas, Viraga; Moreno, Elena; Nambu, Aya; Jangra, Sonia; Cupic, Anastasija; Déjosez, Marion; Abo, Kristine A; Tseng, Anna; Werder, Rhiannon B.; Rathnasinghe, Raveen; Mutetwa, Tinaye; Ramos, Irene; Aja, Julio Sainz de; Rivas, C. Garcia de Alba; Schotsaert, Michael; Corley, Ronald B.; Falvo, James V.; Fernández-Sesma, Ana; Kim, Carla F.; Rossignol, J. E.; Wilson, Andrew; Zwaka, Thomas P.; Kotton, Darrell N.; M&uumlhlberger, Elke; Garc&iacutea-Sastre, Adolfo; Goldfeld, Anne E.

doi:10.1101/2022.02.08.479634

Cited by 15 publications

(22 citation statements)

References 65 publications

(88 reference statements)

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“…Vero E6 (ATCC, CRL-1586) and TMPRSS2-Vero E6 (BPS Bioscience Cat# 78081) were maintained in Dulbecco’s modified Eagle’s medium (Corning) supplemented with 10% fetal bovine serum (Peak Serum), 1% non-essential amino acids (Gibco), 1% HEPES (Gibco) and 1% penicillin/streptomycin (Corning) at 37 °C and 5% CO 2 . HEK293T (ATCC, CRL-3216), A549-ACE2 (previously described in (42, 43) , and BHK-21 (ATCC, CCL-10) were maintained in Dulbecco’s modified Eagle’s medium (Corning) supplemented with 10% fetal bovine serum (Peak Serum) and penicillin/streptomycin (Corning) at 37 °C and 5% CO 2 . All cell lines used in this study were regularly screened for Mycoplasma contamination, using the Universal Mycoplasma Detection Kit (ATCC, 30-1012K).…”

Section: Methodsmentioning

confidence: 99%

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

Kehrer

Cupic

et al. 2022

Preprint

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We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro, and in the Syrian golden hamster model in vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants.

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Section: Methodsmentioning

confidence: 99%

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

Kehrer

Cupic

et al. 2022

Preprint

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“…Cell culture Vero E6 (ATCC, CRL-1586) and Vero TMPRSS2 cells (BPS Bioscience Cat# 78081) were maintained in Dulbecco's modified Eagle's medium (Corning) supplemented with 10% fetal bovine serum (Peak Serum), non-essential amino acids (Gibco), HEPES (Gibco) and penicillin/streptomycin (Corning) at 37 °C and 5% CO2. A549-ACE2 (80) were maintained in Dulbecco's modified Eagle's medium (Corning) supplemented with 10% fetal bovine serum (Peak Serum) and penicillin/streptomycin (Corning) at 37 °C and 5% CO2. All cell lines used in this study were regularly screened for Mycoplasma contamination, using the Universal Mycoplasma Detection Kit (ATCC, 30-1012K).…”

Section: Infection Of A549-ace2 With Mutant Rsars-cov-2 N D3l and -3 ...mentioning

confidence: 99%

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

Bouhaddou

Reuschl

Polacco

et al. 2022

Preprint

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A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic—Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

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“…sgRNA sequences are shown in Table S1. Generation of CRISPR IFNAR1 KO A549 cells has been previously described 39 . For the generation of IFNLR1 KO A549 cells, CRISPR-Cas9 ribonucleoprotein (RNP) complex (IDT) were transfected using the Nucleofector system (Lonza Bioscience).…”

Section: Crispr/cas9 Komentioning

confidence: 99%

CNBP restricts SARS-CoV2 by regulating IFN and disrupting RNA-protein condensates

Fitzgerald

Chen

Lei

et al. 2022

Preprint

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades antiviral immunity through the expression of viral proteins that block detection, signaling, interferon (IFN) induction, and IFN-stimulated gene (ISG) expression. Weak induction of type I IFNs is associated with a hyperinflammatory response in patients that develop severe COVID-19. Here we uncover a role for cellular nucleic acid-binding protein (CNBP) in restricting SARS-CoV-2. Typically, CNBP resides in the cytosol and, in response to RNA sensing pathways, undergoes phosphorylation, nuclear translocation, and IFNβ enhancer DNA binding to turn on IFNβ gene transcription. In SARS-CoV-2-infected cells CNBP coordinates IFNβ gene transcription. In addition, CNBP binds SARS-CoV-2 viral RNA directly. CNBP competes with the nucleocapsid (N) protein and prevents viral RNA and nucleocapsid protein from undergoing liquid-liquid phase separation (LLPS) forming condensates critical for viral replication. Consequently, cells and animals lacking CNBP have higher viral loads and CNBP-deficient mice succumb rapidly to infection. Altogether, these findings identify CNBP as a key antiviral factor for SARS-CoV-2, functioning both as a regulator of antiviral IFN gene expression and a cell intrinsic restriction factor that disrupts LLPS to limit viral replication and spread.

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The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters

Cited by 15 publications

References 65 publications

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

CNBP restricts SARS-CoV2 by regulating IFN and disrupting RNA-protein condensates

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