Biology subjects

Debbink, K.

Publications and source records attributed to Debbink, K..

6 recordsLinked to original sources

bioRxiv2022

SARS-CoV-2 Uses Nonstructural Protein 16 to Evade Restriction by IFIT1 and IFIT3

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 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, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. ImportanceSimilar to other coronaviruses, disruption of 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.

microbiology↗

bioRxiv2021

QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing 1,2. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates 3. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif ({Delta}QTQTN). Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS, and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated4, and disruption its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site - the FCS, loop length, and glycosylation - are required for efficient SARS-CoV-2 replication and pathogenesis.

microbiology↗

bioRxiv2021

Mouse Adapted SARS-CoV-2 protects animals from lethal SARS-CoV challenge

The emergence of SARS-CoV-2 has resulted in a worldwide pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of COVID-19 disease have been hampered by the lack of robust mouse models. To overcome this barrier, we utilized a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARSCoV-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. Utilizing this model, we demonstrate that SARS-CoV-2 infected mice are protected from lethal challenge with the original SARS-CoV, suggesting immunity from heterologous CoV strains. Together, the results highlight the utility of this mouse model for further study of SARS-CoV-2 infection and disease.

microbiology↗

bioRxiv2020

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site ({Delta}PRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the {Delta}PRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the {Delta}PRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays. ImportanceAs COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis. Article SummaryA deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

microbiology↗

bioRxiv2017

MERS-CoV NSP16 necessary for IFN resistance and viral pathogenesis

Coronaviruses encode a mix of highly conserved and novel genes as well as genetic elements necessary for infection and pathogenesis, raising the possibility for common targets for attenuation and therapeutic design. In this study, we focus on the highly conserved nonstructural protein (NSP) 16, a viral 2O methyl-transferase (MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of MERS NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2O MTase activity had only marginal impact on propagation and replication in Vero cells, the MERS dNSP16 mutant demonstrated significant attenuation relative to control both in primary human airway cultures and in vivo. Further examination indicated the MERS dNSP16 mutant had a type I IFN based attenuation and was partially restored in the absence of IFIT molecules. Importantly, the robust attenuation permitted use of MERS dNSP16 as a live attenuated vaccine platform protecting from challenge with a mouse adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2O MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.\n\nSignificanceCoronavirus emergence in both human and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of SARS-CoV, MERS-CoV, PEDV and swine delta coronavirus in the 21st century. These studies describe an approach that effectively targets the highly conserved 2O methyl-transferase activity of coronaviruses for attenuation. With clear understanding of the IFN/IFIT based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-coronavirus functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2O MTase function across coronaviruses.

microbiology↗

bioRxiv2016

Vaccination has minimal impact on the intrahost diversity of H3N2 influenza viruses

While influenza virus diversity and antigenic drift have been well characterized on a global scale, the factors that influence the virus rapid evolution within and between human hosts are less clear. Given the modest effectiveness of seasonal vaccination, vaccine-induced antibody responses could serve as a potent selective pressure for novel influenza variants at the individual or community level. We used next generation sequencing of patient-derived viruses from a randomized, placebo-controlled trial of vaccine efficacy to characterize the diversity of influenza A virus and to define the impact of vaccine-induced immunity on within-host populations. Importantly, this study design allowed us to isolate the impact of vaccination while still studying natural infection. We used pre-season hemagglutination inhibition and neuraminidase inhibition titers to quantify vaccine-induced immunity directly and to assess its impact on intrahost populations. We identified 166 cases of H3N2 influenza over 3 seasons and 5119 person-years. We obtained whole genome sequence data for 119 samples and used a stringent and empirically validated analysis pipeline to identify intrahost single nucleotide variants at [≥]1% frequency. Phylogenetic analysis of consensus hemagglutinin and neuraminidase sequences showed no stratification by pre-season HAI and NAI titer, respectively. In our study population, we found that the vast majority of intrahost single nucleotide variants were rare and that very few were found in more than one individual. Most samples had fewer than 15 single nucleotide variants across the entire genome, and the level of diversity did not significantly vary with day of sampling, vaccination status, or pre-season antibody titer. Contrary to what has been suggested in experimental systems, our data indicate that seasonal influenza vaccination has little impact on intrahost diversity in natural infection and that vaccine-induced immunity may be only a minor contributor to antigenic drift at local scales.

microbiology↗