Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
1.
Curr Opin Virol ; 54: 101229, 2022 Jun.
Article in English | MEDLINE | ID: covidwho-1881820
2.
Viruses ; 14(2)2022 02 10.
Article in English | MEDLINE | ID: covidwho-1715773

ABSTRACT

Understanding the magnitude of responses to vaccination during the ongoing SARS-CoV-2 pandemic is essential for ultimate mitigation of the disease. Here, we describe a cohort of 102 subjects (70 COVID-19-naïve, 32 COVID-19-experienced) who received two doses of one of the mRNA vaccines (BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna)). We document that a single exposure to antigen via infection or vaccination induces a variable antibody response which is affected by age, gender, race, and co-morbidities. In response to a second antigen dose, both COVID-19-naïve and experienced subjects exhibited elevated levels of anti-spike and SARS-CoV-2 neutralizing activity; however, COVID-19-experienced individuals achieved higher antibody levels and neutralization activity as a group. The COVID-19-experienced subjects exhibited no significant increase in antibody or neutralization titer in response to the second vaccine dose (i.e., third antigen exposure). Finally, we found that COVID-19-naïve individuals who received the Moderna vaccine exhibited a more robust boost response to the second vaccine dose (p = 0.004) as compared to the response to Pfizer-BioNTech. Ongoing studies with this cohort will continue to contribute to our understanding of the range and durability of responses to SARS-CoV-2 mRNA vaccines.


Subject(s)
/immunology , Antibodies, Viral/blood , COVID-19/prevention & control , Immunogenicity, Vaccine , SARS-CoV-2/immunology , Vaccination/statistics & numerical data , /administration & dosage , Adult , Antibodies, Viral/immunology , Antibody Formation , COVID-19/immunology , Cohort Studies , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged
3.
Viruses ; 13(9)2021 09 01.
Article in English | MEDLINE | ID: covidwho-1390786

ABSTRACT

The pandemic of COVID-19 caused by SARS-CoV-2 infection continues to spread around the world. Vaccines that elicit protective immunity have reduced infection and mortality, however new viral variants are arising that may evade vaccine-induced immunity or cause disease in individuals who are unable to develop robust vaccine-induced responses. Investigating the role of viral variants in causing severe disease, evading vaccine-elicited immunity, and infecting vulnerable individuals is important for developing strategies to control the pandemic. Here, we report fourteen breakthrough infections of SARS-CoV-2 in vaccinated individuals with symptoms ranging from asymptomatic/mild (6/14) to severe disease (8/14). High viral loads with a median Ct value of 19.6 were detected in the nasopharyngeal specimens from subjects regardless of disease severity. Sequence analysis revealed four distinct virus lineages, including alpha and gamma variants of concern. Immunosuppressed individuals were more likely to be hospitalized after infection (p = 0.047), however no specific variant was associated with severe disease. Our results highlight the high viral load that can occur in asymptomatic breakthrough infections and the vulnerability of immunosuppressed individuals to post-vaccination infections by diverse variants of SARS-CoV-2.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , Immunocompromised Host , SARS-CoV-2 , Aged , COVID-19/diagnosis , COVID-19/immunology , Female , Genome, Viral , Genomics/methods , Humans , Male , Middle Aged , Mutation , SARS-CoV-2/immunology , Severity of Illness Index , Vaccines/immunology , Viral Load
4.
Science ; 373(6557): 931-936, 2021 08 20.
Article in English | MEDLINE | ID: covidwho-1319369

ABSTRACT

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus OC43, Human/drug effects , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Thiazoles/pharmacology , A549 Cells , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Benzamides , COVID-19/virology , Catalytic Domain , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Coronavirus OC43, Human/physiology , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , HEK293 Cells , Humans , Inhibitory Concentration 50 , Mice , Mice, Transgenic , Microbial Sensitivity Tests , Piperidines , Pyridines , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Thiazoles/chemistry , Thiazoles/metabolism , Thiazoles/therapeutic use , Viral Load/drug effects , Virus Replication/drug effects
5.
J Virol ; 95(19): e0086221, 2021 09 09.
Article in English | MEDLINE | ID: covidwho-1309804

ABSTRACT

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.


Subject(s)
COVID-19/metabolism , Host Microbial Interactions/physiology , Janus Kinases/metabolism , SARS-CoV-2/metabolism , Cell Line , Gene Expression Regulation , Humans , Immune Evasion , Immunity, Innate , Interferon Type I/metabolism , Janus Kinase 1/metabolism , Myocytes, Cardiac , Receptor, Interferon alpha-beta/metabolism , STAT1 Transcription Factor/metabolism , Signal Transduction , TYK2 Kinase/metabolism , Virus Replication
6.
Virology ; 556: 73-78, 2021 04.
Article in English | MEDLINE | ID: covidwho-1049897

ABSTRACT

The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors.


Subject(s)
Biosensing Techniques/methods , Coronavirus 3C Proteases/metabolism , Immune Sera/immunology , Luciferases/metabolism , SARS-CoV-2/metabolism , Animals , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/immunology , Cross Reactions , Luciferases/genetics , Protease Inhibitors/pharmacology , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS Virus/immunology , SARS Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
7.
Virology ; 556: 9-22, 2021 04.
Article in English | MEDLINE | ID: covidwho-985483

ABSTRACT

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.


Subject(s)
Coronavirus Infections/virology , Endoplasmic Reticulum/metabolism , Murine hepatitis virus/physiology , Viral Replication Compartments/metabolism , Animals , Cell Line , Electron Microscope Tomography , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Lysosomes/metabolism , Lysosomes/ultrastructure , Lysosomes/virology , Mice , Viral Proteins/metabolism , Viral Replication Compartments/ultrastructure , Virion/metabolism , Virus Assembly , Virus Replication
8.
Virology ; 553: 35-45, 2021 01 15.
Article in English | MEDLINE | ID: covidwho-922156

ABSTRACT

We report the generation of a full-length infectious cDNA clone for porcine deltacoronavirus strain USA/IL/2014/026. Similar to the parental strain, the infectious clone virus (icPDCoV) replicated efficiently in cell culture and caused mild clinical symptoms in piglets. To investigate putative viral interferon (IFN) antagonists, we generated two mutant viruses: a nonstructural protein 15 mutant virus that encodes a catalytically-inactive endoribonuclease (icEnUmut), and an accessory gene NS6-deletion virus in which the NS6 gene was replaced with the mNeonGreen sequence (icDelNS6/nG). By infecting PK1 cells with these recombinant PDCoVs, we found that icDelNS6/nG elicited similar levels of type I IFN responses as icPDCoV, however icEnUmut stimulated robust type I IFN responses, demonstrating that the deltacoronavirus endoribonuclease, but not NS6, functions as an IFN antagonist in PK1 cells. Collectively, the construction of a full-length infectious clone and the identification of an IFN-antagonistic endoribonuclease will aid in the development of live-attenuated deltacoronavirus vaccines.


Subject(s)
DNA, Complementary/isolation & purification , Deltacoronavirus/genetics , Swine/virology , Animals , Clone Cells , Coronavirus Infections/pathology , Deltacoronavirus/pathogenicity , Deltacoronavirus/physiology , Endoribonucleases/physiology , Interferons/antagonists & inhibitors , Virus Replication
9.
J Virol ; 94(17)2020 08 17.
Article in English | MEDLINE | ID: covidwho-601769

ABSTRACT

Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.


Subject(s)
Coronavirus Infections/immunology , Coronavirus/immunology , Interferons/immunology , Porcine epidemic diarrhea virus/immunology , Vaccines, Attenuated/immunology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Betacoronavirus/immunology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/prevention & control , Diarrhea/pathology , Diarrhea/virology , Disease Models, Animal , Endoribonucleases/antagonists & inhibitors , Feces/virology , Ileum/pathology , Immunity, Innate , Jejunum/pathology , Pandemics , Pneumonia, Viral/immunology , Porcine epidemic diarrhea virus/genetics , RNA, Viral , RNA-Dependent RNA Polymerase , SARS-CoV-2 , Swine , Swine Diseases/virology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology
10.
Proc Natl Acad Sci U S A ; 117(14): 8094-8103, 2020 04 07.
Article in English | MEDLINE | ID: covidwho-11430

ABSTRACT

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.


Subject(s)
Coronavirus Infections/immunology , Coronavirus Infections/virology , Coronavirus/metabolism , Endoribonucleases/metabolism , Poly U/metabolism , Viral Nonstructural Proteins/metabolism , Animals , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Coronavirus/enzymology , Coronavirus/immunology , Endoribonucleases/genetics , Host Microbial Interactions/physiology , Humans , Interferons/pharmacology , Poly U/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , Uridine/chemistry , Vero Cells , Viral Nonstructural Proteins/genetics , Virus Replication/physiology
11.
J Virol ; 94(11)2020 05 18.
Article in English | MEDLINE | ID: covidwho-10361

ABSTRACT

Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (P < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis.IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.


Subject(s)
Coronavirus Infections/virology , Murine hepatitis virus/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Host-Pathogen Interactions , Interferon Type I/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/virology , Mice , Models, Molecular , Murine hepatitis virus/pathogenicity , Mutagenesis , Protein Conformation , Structure-Activity Relationship , Ubiquitination , Viral Proteins/chemistry , Virulence , Virus Replication
12.
J Virol ; 94(11)2020 05 18.
Article in English | MEDLINE | ID: covidwho-9937

ABSTRACT

Coronaviruses (CoVs) encode multiple interferon (IFN) antagonists that modulate the host response to virus replication. Here, we evaluated the host transcriptional response to infection with murine coronaviruses encoding independent mutations in one of two different viral antagonists, the deubiquitinase (DUB) within nonstructural protein 3 or the endoribonuclease (EndoU) within nonstructural protein 15. We used transcriptomics approaches to compare the scope and kinetics of the host response to the wild-type (WT), DUBmut, and EndoUmut viruses in infected macrophages. We found that the EndoUmut virus activates a focused response that predominantly involves type I interferons and interferon-related genes, whereas the WT and DUBmut viruses more broadly stimulate upregulation of over 2,800 genes, including networks associated with activating the unfolded protein response (UPR) and the proinflammatory response associated with viral pathogenesis. This study highlights the role of viral interferon antagonists in shaping the kinetics and magnitude of the host response during virus infection and demonstrates that inactivating a dominant viral antagonist, the coronavirus endoribonuclease, dramatically alters the host response in macrophages.IMPORTANCE Macrophages are an important cell type during coronavirus infections because they "notice" the infection and respond by inducing type I interferons, which limits virus replication. In turn, coronaviruses encode proteins that mitigate the cell's ability to signal an interferon response. Here, we evaluated the host macrophage response to two independent mutant coronaviruses, one with reduced deubiquitinating activity (DUBmut) and the other containing an inactivated endoribonuclease (EndoUmut). We observed a rapid, robust, and focused response to the EndoUmut virus, which was characterized by enhanced expression of interferon and interferon-related genes. In contrast, wild-type virus and the DUBmut virus elicited a more limited interferon response and ultimately activated over 2,800 genes, including players in the unfolded protein response and proinflammatory pathways associated with progression of significant disease. This study reveals that EndoU activity substantially contributes to the ability of coronaviruses to evade the host innate response and to replicate in macrophages.


Subject(s)
Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus/physiology , Endoribonucleases/metabolism , Interferons/metabolism , Macrophages/metabolism , Macrophages/virology , Viral Nonstructural Proteins/metabolism , Virus Replication , Animals , Computational Biology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Cytokines/metabolism , Gene Expression Profiling , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Inflammation Mediators/metabolism , Macrophages/immunology , Mice , Models, Biological , Mutation , RNA, Viral , Unfolded Protein Response
SELECTION OF CITATIONS
SEARCH DETAIL