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2.
Am J Transplant ; 2022 Apr 05.
Article in English | MEDLINE | ID: covidwho-1807004

ABSTRACT

Seroconversion after COVID-19 vaccination is impaired in kidney transplant recipients. Emerging variants of concern such as the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants pose an increasing threat to these patients. In this observational cohort study, we measured anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies three weeks after a third mRNA vaccine dose in 49 kidney transplant recipients and compared results to 25 age-matched healthy controls. In addition, vaccine-induced neutralization of SARS-CoV-2 wild-type, the B.1.617.2 (delta), and the B.1.1.529 (omicron) variants was assessed using a live-virus assay. After a third vaccine dose, anti-S1 IgG, surrogate neutralizing, and anti-receptor-binding domain antibodies were significantly lower in kidney transplant recipients compared to healthy controls. Only 29/49 (59%) sera of kidney transplant recipients contained neutralizing antibodies against the SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant and neutralization titers were significantly reduced compared to healthy controls (p < 0.001). Vaccine-induced cross-neutralization of the B.1.1.529 (omicron) variants was detectable in 15/35 (43%) kidney transplant recipients with seropositivity for anti-S1 IgG, surrogate neutralizing, and/or anti-RBD antibodies. Neutralization of the B.1.1.529 (omicron) variants was significantly reduced compared to neutralization of SARS-CoV-2 wild-type or the B.1.617.2 (delta) variant for both, kidney transplant recipients and healthy controls (p < .001 for all).

3.
J Clin Med ; 11(6)2022 Mar 21.
Article in English | MEDLINE | ID: covidwho-1753642

ABSTRACT

BACKGROUND: To characterize humoral response after standard anti-SARS-CoV-2 vaccination in Rituximab-treated patients and to determine the optimal time point after last Rituximab treatment for appropriate immunization. METHODS: Sixty-four patients who received Rituximab within the last seven years prior to the first anti-SARS-CoV-2 vaccination were recruited in a prospective observational study. Anti-S1 IgG, SARS-CoV-2 specific neutralization, and various SARS-CoV-2 target antibodies were determined. A live virus assay was used to assess neutralizing antibody activity against B.1.617.2 (delta). In Rituximab-treated patients, CD19+ peripheral B-cells were quantified using flow cytometry. RESULTS: After second vaccination, all antibodies were significantly reduced compared to healthy controls. Neutralizing antibody activity against B.1.617.2 (delta) was detectable with a median (IQR) ID50 of 0 (0-1:20) compared to 1:320 (1:160-1:320) in healthy controls (for all p < 0.001). Longer time period since last Rituximab administration correlated with higher anti-SARS-CoV-2 antibody levels and a stronger neutralization of B.1.617.2 (delta). With one exception, only patients with a CD19+ cell proportion ≥ 1% had detectable neutralizing antibodies. CONCLUSION: Our data indicate that a reconstitution of the B-cell population to >1% seems crucial in developing neutralizing antibodies against SARS-CoV-2. We suggest that anti-SARS-CoV-2 vaccination should be administered at least 8-12 months after the last Rituximab treatment for sufficient humoral responses.

4.
Front Immunol ; 13: 840136, 2022.
Article in English | MEDLINE | ID: covidwho-1753372

ABSTRACT

Hemodialysis patients are at high risk for severe COVID-19, and impaired seroconversion rates have been demonstrated after COVID-19 vaccination. Humoral immunity wanes over time and variants of concern with immune escape are posing an increasing threat. Little is known about protection against the B.1.617.2 (delta) variant of concern in hemodialysis patients before and after third vaccination. We determined anti-S1 IgG, surrogate neutralizing, and IgG antibodies against different SARS-CoV-2 epitopes in 84 hemodialysis patients directly before and three weeks after a third vaccine dose with BNT162b2. Third vaccination was performed after a median (IQR) of 119 (109-165) days after second vaccination. In addition, neutralizing activity against the B.1.617.2 (delta) variant was assessed in 31 seroconverted hemodialysis patients before and after third vaccination. Triple seropositivity for anti-S1 IgG, surrogate neutralizing, and anti-RBD antibodies increased from 31/84 (37%) dialysis patients after second to 80/84 (95%) after third vaccination. Neutralizing activity against the B.1.617.2 (delta) variant was significantly higher after third vaccination with a median (IQR) ID50 of 1:320 (1:160-1:1280) compared with 1:20 (0-1:40) before a third vaccine dose (P<0.001). The anti-S1 IgG index showed the strongest correlation with the ID50 against the B.1.617.2 (delta) variant determined by live virus neutralization (r=0.91). We demonstrate low neutralizing activity against the B.1.617.2 (delta) variant in dialysis patients four months after standard two-dose vaccination but a substantial increase after a third vaccine dose. Booster vaccination(s) should be considered earlier than 6 months after the second vaccine dose in immunocompromised individuals.


Subject(s)
COVID-19 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Immunoglobulin G , Renal Dialysis , SARS-CoV-2
5.
mBio ; 13(2): e0370521, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1714363

ABSTRACT

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.


Subject(s)
COVID-19 , Hepatitis C, Chronic , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cobicistat , Cricetinae , Disease Progression , Humans , Mesocricetus , Pandemics , SARS-CoV-2 , Viral Load
6.
Biospektrum (Heidelb) ; 28(1): 47-49, 2022.
Article in German | MEDLINE | ID: covidwho-1694279

ABSTRACT

The severe acute respiratory syndrome Coronavirus type 2 (SARS-CoV-2) has caused a pandemic with major impact on human society, the economy, and our daily life. SARS-CoV-2 is a plus-strand RNA virus causing death of infected cells and an inflammation-dominated immune response. Replication of the virus occurs in the cytoplasm in distinct membranous compartments designated replication organelles, providing a shielded environment for synthesis of viral RNAs. Here, I will briefly summarize key aspects of the SARS-CoV-2 replication cycle.

7.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-321964

ABSTRACT

Background: Since SARS-CoV-2 is a highly contagious virus without an available disease-specific medication, the hope is focused on a sustained immunity after SARS-CoV-2 infection and a near-term successful vaccination therapy. A sufficient anti-SARS-CoV-2 antibody production with neutralizing antibodies is crucial to prevent further viral spreading and for protection against prospective reinfection. Kidney transplant recipients may have a potentially aggravated risk for COVID-19 complications as well as a reduced vaccine response due to the allograft protecting immunosuppressive therapy. However, little is known about the strength and duration of their immunological response upon SARS-CoV-2 infection. Case presentation Here we report on 4 kidney transplant recipients proven to have SARS-CoV-2 infection by positive PCR testing, focusing on their immunological response with the production of disease-specific neutralizing antibodies. All kidney transplant recipients developed a sufficient antibody response including specific neutralizing antibodies against SARS-CoV-2 within 2 to 3 weeks after the first onset of symptoms that sustained during the follow-up of 15 weeks. After 6 weeks, the virus was eliminated in all patients. Most important, the serological response and viral shedding were achieved and sustained in the presence of immunosuppression. Acute kidney graft deterioration was common but reconstituted in all transplant recipients during follow-up. Conclusions: Immunocompromised kidney transplant recipients showed a functional serological response with disease-specific neutralizing antibodies upon SARS-CoV-2 infection, a basic prerequisite for a prospective successful vaccination response.

8.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-321044

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS-CoV-2 hijacks host cellular machineries on a systems-wide scale so that potential host-directed therapy can be developed. In situ proteome-wide abundance and thermal stability measurements using thermal proteome profiling (TPP), can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS-CoV-2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and regulation of RNA splicing. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS-CoV-2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS-CoV-2 infection.

9.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-307867

ABSTRACT

Coronavirus 2 (SARS-CoV-2) infection and the resulting COVID-19 illness vary from asymptomatic disease, mild upper respiratory tract infection, pneumonia 1 , to a life-threatening multi-organ failure with case fatality rates ranging from 0.27–13.4% 2,3 . Despite increasing knowledge of the clinical and immunological features underlying COVID-19 1,4−6 , biological variables explaining the course of infection and its severity remain elusive. At the entry site of SARS-CoV2, the oropharyngeal microbiome represents a hub integrating viral and immune signals at the start of the infection 7–10 . To evaluate the role of the oropharyngeal microbiome in COVID-19, we performed a multi-center, cross-sectional clinical study analyzing the oropharyngeal microbial metagenomes in healthy adults, patients with non-SARS-CoV-2 infections, or with mild, moderate and severe COVID-19 encompassing a total of 345 participants. Significantly reduced microbiome diversity and high dysbiosis were observed in hospitalized patients with severe COVID-19, which was further associated with a loss of microbial genes and metabolic pathways. In this cohort, diversity measures were also associated with need for intensive care treatments as major clinical parameters in COVID-19. We further applied random forest machine learning to unravel microbial features for segregating clinical outcomes in hospitalized cases, and observed oropharyngeal microbiome abundances of Haemophilus or Streptococcu s species as most important features. These findings provide insights into the role of the oropharyngeal microbiome in SARS-CoV-2 infection, and may suggest new biomarkers for COVID-19 severity.

11.
Clin Microbiol Infect ; 28(7): 1024.e7-1024.e12, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1664807

ABSTRACT

OBJECTIVES: Humoral immunity wanes over time after two-dose BNT162b2 vaccination. Emerging variants of concern, such as the B.1.617.2 (delta) variant, are increasingly responsible for breakthrough infections owing to their higher transmissibility and partial immune escape. Longitudinal data on neutralization against the B.1.617.2 (delta) variant are urgently needed to guide vaccination strategies. METHODS: In this prospective longitudinal observational study, anti-S1 IgG and surrogate neutralizing antibodies were measured in 234 collected samples from 60 health care workers after two-dose vaccination with BNT162b2 at five different time points over an 8-month period. In addition, antibodies against various severe acute respiratory syndrome coronavirus 2 epitopes, neutralization against wild-type, and cross-neutralization against the B.1.617.2 (delta) variant using a live virus assay were measured 6 weeks (second time point) and 8 months (last time point) after first vaccine dose. RESULTS: Median (interquartile range) anti-S1 IgG, surrogate neutralizing, and receptor-binding domain antibodies decreased significantly from a maximum level of 147 (102-298), 97 (96-98), and 20 159 (19 023-21 628) to 8 (4-13), 92 (80-96), and 15 324 (13 055-17 288) at the 8-month follow-up, respectively (p < 0.001 for all). Neutralization against the B.1.617.2 (delta) variant was detectable in all 36 (100%) participants at 6 weeks and in 50 of 53 (94%) participants 8 months after first vaccine dose. Median (interquartile) ID50 as determined by a live virus assay decreased from 160 (80-320) to 40 (20-40) (p < 0.001). DISCUSSION: Although humoral immunity wanes over time after two-dose BNT162b2 vaccination in healthy individuals, most individuals still had detectable neutralizing activity against the B.1.617.2 (delta) variant after 8 months.


Subject(s)
Antibodies, Neutralizing , COVID-19 , Antibodies, Viral , COVID-19/prevention & control , Health Personnel , Humans , Immunoglobulin G , Neutralization Tests , Prospective Studies , SARS-CoV-2 , Vaccination
12.
Cell Rep ; 38(7): 110387, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1654154

ABSTRACT

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation. All four SARS-CoV-2 variants induce pDC depletion in the lungs, paralleled by reduced interferon responses. Remarkably, VOCs also use the murine ACE2 receptor for infection to replicate in the lungs of wild-type animals, which induce cellular and innate immune responses that apparently curtail the spread of overt disease. VOCs thus display distinct intrinsic pathogenic properties with broadened tissue and host range. The enhanced pathogenicity of VOCs and their potential for reverse zoonotic transmission pose challenges to clinical and pandemic management.


Subject(s)
COVID-19/virology , Disease Models, Animal , SARS-CoV-2/physiology , SARS-CoV-2/pathogenicity , Animals , COVID-19/immunology , Cytokines/metabolism , Host Specificity , Immunity, Cellular , Immunity, Innate , Lung/immunology , Lung/virology , Mice , Species Specificity , Viral Load , Viral Tropism , Virulence , Virus Replication
13.
Commun Biol ; 5(1): 45, 2022 01 12.
Article in English | MEDLINE | ID: covidwho-1625575

ABSTRACT

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to long-lasting lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses, which can cause systemic complications. Here, we have evaluated transcriptional and cytokine secretion profiles and detected a distinct upregulation of inflammatory cytokines in infected cell cultures and samples taken from infected patients. Building on these observations, we found a specific activation of NF-κB and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-κB response was mediated by cGAS-STING activation and could be attenuated through several STING-targeting drugs. Our results show that SARS-CoV-2 directs a cGAS-STING mediated, NF-κB-driven inflammatory immune response in human epithelial cells that likely contributes to inflammatory responses seen in patients and could be therapeutically targeted to suppress severe disease symptoms.


Subject(s)
COVID-19/metabolism , Cytokine Release Syndrome , Inflammation Mediators/metabolism , Membrane Proteins/metabolism , NF-kappa B/metabolism , Nucleotidyltransferases/metabolism , COVID-19/virology , Humans , SARS-CoV-2/isolation & purification , Signal Transduction
14.
Clin J Am Soc Nephrol ; 17(1): 98-106, 2022 01.
Article in English | MEDLINE | ID: covidwho-1581489

ABSTRACT

BACKGROUND AND OBJECTIVES: Antibody response after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is impaired in kidney transplant recipients. Emerging variants, such as B.1.617.2 (δ), are of particular concern because of their higher transmissibility and partial immune escape. Little is known about protection against these variants in immunocompromised patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this prospective two-center study, antispike 1 IgG and surrogate neutralizing antibodies were measured in 173 kidney transplant recipients and 166 healthy controls with different vaccination schedules. In addition, different SARS-CoV-2 epitope antibodies from 135 vaccinated kidney transplant recipients were compared with antibodies in 25 matched healthy controls after second vaccination. In 36 kidney transplant recipients with seroconversion, neutralization against B.1.1.7 (α), B.1.351 (ß), and B.1.617.2 (δ) was determined on VeroE6 cells and compared with neutralization in 25 healthy controls. RESULTS: Kidney transplant recipients had significantly lower seroconversion rates compared with healthy controls. After the second vaccination, antispike 1, antireceptor-binding domain, and surrogate neutralizing antibodies were detectable in 30%, 27%, and 24% of kidney transplant recipients, respectively. This compares with 100%, 96%, and 100% in healthy controls, respectively (P<0.001). Neutralization against B.1.1.7 was detectable in all kidney transplant recipients with seroconversion, with a median serum dilution that reduces infection of cells by 50% of 80 (interquartile range, 80-320). In contrast, only 23 of 36 (64%) and 24 of 36 (67%) kidney transplant recipients showed neutralization against B.1.351 and B.1.617.2, respectively, with median serum dilutions that reduce infection of cells by 50% of 20 (interquartile range, 0-40) and 20 (interquartile range, 0-40), respectively. Neutralization against different variants was significantly higher in healthy controls (P<0.001), with all patients showing neutralization against all tested variants. CONCLUSIONS: Seroconverted kidney transplant recipients show impaired neutralization against emerging variants of concern after standard two-dose vaccination. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Observational study to assess the SARS-CoV-2 specific immune response in kidney transplant recipients (COVID-19 related immune response), DRKS00024668.


Subject(s)
COVID-19 Vaccines , COVID-19/prevention & control , Kidney Transplantation , SARS-CoV-2 , Adult , Female , Humans , Male , Middle Aged , Prospective Studies
16.
Nat Commun ; 12(1): 7276, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1575708

ABSTRACT

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.


Subject(s)
Hepacivirus/genetics , Phosphatidic Acids/metabolism , SARS-CoV-2/genetics , Virus Replication/physiology , 1-Acylglycerol-3-Phosphate O-Acyltransferase , Acyltransferases , Autophagosomes/metabolism , Autophagy , COVID-19/virology , Cell Line , Cell Survival , Dengue Virus , HEK293 Cells , Humans , Membrane Proteins , Spike Glycoprotein, Coronavirus , Viral Nonstructural Proteins , Viral Proteins , Zika Virus
17.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-295255

ABSTRACT

Genetic variation in RNA viruses is generated by point mutation and recombination as well as reassortment in the case of viruses with segmented genomes. While point mutation concerns only few sites per genome copy, recombination and reassortment can affect large genome regions, possibly facilitating the sudden emergence of novel traits. The contribution of recombination and reassortment to genomic plasticity and their rates remain poorly understood and might be underappreciated because of the lack of a comprehensive description of the virosphere. Here we employed a computational approach that directly queries primary sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy. By screening more than 213,000 data sets from the Sequence Read Archive repository and using two metrics that quantitatively assess assembly quality we discovered 25 novel nidoviruses from a wide range of vertebrate hosts. These include eight fish coronaviruses with bipartite genomes, a giant 36.1 kilobase coronavirus genome with a duplicated Spike glycoprotein (S) gene, and 16 additional so far undescribed vertebrate nidoviruses. Some of these novel virus genomes encode protein domains that have not been described for nidoviruses. We provide evidence for a possible inter-family homologous recombination event involving S between ancestral bipartite coronaviruses and unsegmented tobaniviruses and report a case example of an individual fish simultaneously infected with members from both virus families. Our results shed light on the evolution and genomic plasticity of coronaviruses and identify recombinants with a possibly improved ability to cross species barriers, which might elevate their pandemic potential.

18.
Cell Rep ; 37(8): 110049, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1509642

ABSTRACT

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.


Subject(s)
Autophagy/physiology , Hepacivirus/physiology , SARS-CoV-2/physiology , Viral Replication Compartments/metabolism , Autophagosomes/metabolism , Carrier Proteins/metabolism , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class III Phosphatidylinositol 3-Kinases/metabolism , Humans , Phosphatidylinositol Phosphates/metabolism , RNA, Viral/biosynthesis , Viral Nonstructural Proteins/metabolism , Virus Replication
19.
ACS Infect Dis ; 7(6): 1457-1468, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1493012

ABSTRACT

Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Humans , Peptide Hydrolases , Protease Inhibitors
20.
Cell Rep Methods ; 1(7): 100117, 2021 Nov 22.
Article in English | MEDLINE | ID: covidwho-1487678

ABSTRACT

High-resolution and rapid imaging of host cell ultrastructure can generate insights toward viral disease mechanism, for example for a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Here, we employ full-rotation soft X-ray tomography (SXT) to examine organelle remodeling induced by SARS-CoV-2 at the whole-cell level with high spatial resolution and throughput. Most of the current SXT systems suffer from a restricted field of view due to use of flat sample supports and artifacts due to missing data. In this approach using cylindrical sample holders, a full-rotation tomogram of human lung epithelial cells is performed in less than 10 min. We demonstrate the potential of SXT imaging by visualizing aggregates of SARS-CoV-2 virions and virus-induced intracellular alterations. This rapid whole-cell imaging approach allows us to visualize the spatiotemporal changes of cellular organelles upon viral infection in a quantitative manner.

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