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1.
Mol Cell ; 2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34687604

RESUMO

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

2.
Virologie (Montrouge) ; 25(4): 224-235, 2021 08 01.
Artigo em Francês | MEDLINE | ID: mdl-34468319

RESUMO

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.


Assuntos
Vírus de RNA de Cadeia Positiva , Recombinação Genética , Sequência de Bases , RNA Viral/genética , Recombinação Genética/genética , Replicação Viral/genética
3.
Virologie (Montrouge) ; 25(4): 62-73, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34468320

RESUMO

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and the frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.


Assuntos
Vírus de RNA de Cadeia Positiva , Recombinação Genética , Sequência de Bases , RNA Viral/genética , Recombinação Genética/genética , Replicação Viral/genética
4.
Nat Commun ; 12(1): 5553, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548480

RESUMO

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/metabolismo , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/tratamento farmacológico , Linhagem Celular , Dipeptídeos/farmacologia , Humanos , Mutação , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Proteólise , Proteômica , RNA Interferente Pequeno/farmacologia , SARS-CoV-2/genética , Proteases Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
5.
J Theor Biol ; 531: 110895, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34499915

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2), a causative agent of COVID-19 disease, poses a significant threat to public health. Since its outbreak in December 2019, Wuhan, China, extensive collection of diverse data from cell culture and animal infections as well as population level data from an ongoing pandemic, has been vital in assessing strategies to battle its spread. Mathematical modelling plays a key role in quantifying determinants that drive virus infection dynamics, especially those relevant for epidemiological investigations and predictions as well as for proposing efficient mitigation strategies. We utilized a simple mathematical model to describe and explain experimental results on viral replication cycle kinetics during SARS-CoV-2 infection of animal and human derived cell lines, green monkey kidney cells, Vero-E6, and human lung epithelium cells, A549-ACE2, respectively. We conducted cell infections using two distinct initial viral concentrations and quantified viral loads over time. We then fitted the model to our experimental data and quantified the viral parameters. We showed that such cellular tropism generates significant differences in the infection rates and incubation times of SARS-CoV-2, that is, the times to the first release of newly synthesised viral progeny by SARS-CoV-2-infected cells. Specifically, the rate at which A549-ACE2 cells were infected by SARS-CoV-2 was 15 times lower than that in the case of Vero-E6 cell infection and the duration of latent phase of A549-ACE2 cells was 1.6 times longer than that of Vero-E6 cells. On the other hand, we found no statistically significant differences in other viral parameters, such as viral production rate or infected cell death rate. Since in vitro infection assays represent the first stage in the development of antiviral treatments against SARS-CoV-2, discrepancies in the viral parameter values across different cell hosts have to be identified and quantified to better target vaccine and antiviral research.

6.
J Virol ; : JVI0097721, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34468175

RESUMO

Here we examine in-silico the infection dynamics and interactions of two ZIKV genomes: one is the full-length ZIKV genome (WT) and the other is one of the naturally occurring defective viral genomes (DVG), which can replicate in the presence of WT genome, appears under high MOI passaging conditions and carries a deletion encompassing part of the structural and NS1 protein-coding region. Ordinary differential equations (ODE) were used to simulate the infection of cells by virus particles and intra-cellular replication of the WT and DVG genomes that produces these particles. For each virus passage in Vero and C6/36 cell cultures, rates of the simulated processes were fitted to two types of observations: virus titer data and the assembled haplotypes of the replicate passage samples. We studied the consistency of the model with the experimental data across all passages of infection in each cell type separately, as well as sensitivity of model's parameters. We also determined which simulated processes of the virus evolution are most important for adaptation of the WT and DVG interplay in these two disparate cell culture environments. Our results demonstrate that in majority of passages, the rates of DVG-production are higher in the C6/36 cells compared to Vero cells, which might result in tolerance and therefore drive persistence of the mosquito vector in the context of ZIKV infection. Additionally, the model simulations showed slower accumulation of infected cells under higher activation of the DVG associated processes, which indicates potential role of DVGs in virus attenuation. Importance. One of ideas on lessening Zika pathogenicity is addition of its natural or engineered defective virus genomes (DVG: have no pathogenicity) to the infection pool: DVG is redirecting the wild type (WT) associated virus development resources to its own maturation. The presented here mathematical model, attuned to the data from interplays between Zika WT viruses and their natural DVG in mammalian and mosquito cells, provides evidence that loss of uninfected cells is attenuated by the DVG development processes. This model enabled us to estimate rates of the virus development processes in the WT/DVG interplay, determine the key processes, and show that the key processes are faster in mosquito cells than in mammalian ones. In general, the presented model and its detailed study suggest in what important virus development processes the therapeutically efficient DVG might compete with WT: this may help in assembling engineered DVGs for ZIKV and other flaviviruses.

7.
Mol Cell Probes ; 59: 101749, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34214632

RESUMO

New vaccine platforms are crucial to address complex parasitic infections such as cutaneous leishmaniasis. Self-amplifying mRNA (SAM) based vaccines represent the next generation nucleic acid-based platform. In the present study, we compared the expression levels of PpSP15-LmSTI1 fusion gene in BHK-21 cells following transfection with Semliki Forest virus (SFV)-derived SAM, SFV-derived plasmid DNA (pSFV-PD) and conventional plasmid DNA (pcDNA3.1+). PpSP15-LmSTI1 fusion gene expression levels were evaluated at different time points, using quantitative Real-time PCR. All data were validated and normalized by two internal control genes. According to the results, mean values of relative expression were significantly higher for SFV-PD SAM/fusion than pcDNA/fusion and pSFV-PD/fusion at all concentrations and time points. Our results showed that higher levels of PpSp15-LmSTI1 antigen expression could be achieved using a SAM vector than pcDNA and pSFV-PD, making it a valuable and efficient alternative to conventional plasmid DNA-based vaccines against leishmaniasis.

8.
Science ; 373(6554): 541-547, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326236

RESUMO

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacos
9.
Science ; 373(6557): 931-936, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285133

RESUMO

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).


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Coronavirus Humano OC43/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Tiazóis/farmacologia , Células A549 , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , COVID-19/virologia , Domínio Catalítico , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Coronavirus Humano OC43/fisiologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
10.
PLoS One ; 16(6): e0252595, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086776

RESUMO

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.

11.
Sci Rep ; 11(1): 7893, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846459

RESUMO

APOBEC3 (A3) enzymes are best known for their role as antiviral restriction factors and as mutagens in cancer. Although four of them, A3A, A3B, A3F and A3G, are induced by type-1-interferon (IFN-I), their role in inflammatory conditions is unknown. We thus investigated the expression of A3, and particularly A3A and A3B because of their ability to edit cellular DNA, in Systemic Lupus Erythematosus (SLE), a chronic inflammatory disease characterized by high IFN-α serum levels. In a cohort of 57 SLE patients, A3A and A3B, but also A3C and A3G, were upregulated ~ 10 to 15-fold (> 1000-fold for A3B) compared to healthy controls, particularly in patients with flares and elevated serum IFN-α levels. Hydroxychloroquine, corticosteroids and immunosuppressive treatment did not reverse A3 levels. The A3AΔ3B polymorphism, which potentiates A3A, was detected in 14.9% of patients and in 10% of controls, and was associated with higher A3A mRNA expression. A3A and A3B mRNA levels, but not A3C or A3G, were correlated positively with dsDNA breaks and negatively with lymphopenia. Exposure of SLE PBMCs to IFN-α in culture induced massive and sustained A3A levels by 4 h and led to massive cell death. Furthermore, the rs2853669 A > G polymorphism in the telomerase reverse transcriptase (TERT) promoter, which disrupts an Ets-TCF-binding site and influences certain cancers, was highly prevalent in SLE patients, possibly contributing to lymphopenia. Taken together, these findings suggest that high baseline A3A and A3B levels may contribute to cell frailty, lymphopenia and to the generation of neoantigens in SLE patients. Targeting A3 expression could be a strategy to reverse cell death and the generation of neoantigens.

12.
FEBS J ; 288(17): 5148-5162, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33864728

RESUMO

Small linear motifs targeting protein interacting domains called PSD-95/Dlg/ZO-1 (PDZ) have been identified at the C terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A, and N showing significant interactions with dissociation constants values ranging from 3 to 82 µm. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Among the binders of the SARS-CoV-2 proteins E, 3a, or N, seven significantly affect viral replication under knock down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.


Assuntos
COVID-19/genética , Interações Hospedeiro-Patógeno/genética , Domínios PDZ/genética , SARS-CoV-2/genética , COVID-19/virologia , Proteínas de Transporte/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Cinesina/genética , Miosinas/genética , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 13/genética , SARS-CoV-2/patogenicidade , Proteínas do Envelope Viral/genética , Proteínas Viroporinas/genética , Internalização do Vírus , Replicação Viral/genética , Proteína da Zônula de Oclusão-1/genética
13.
bioRxiv ; 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33791693

RESUMO

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost. One Sentence Summary: Drug-induced phospholipidosis is a single mechanism that may explain the in vitro efficacy of a wide-variety of therapeutics repurposed for COVID-19.

14.
Nat Commun ; 12(1): 2290, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863888

RESUMO

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.


Assuntos
Antivirais/administração & dosagem , Vírus Defeituosos/genética , Mosquitos Vetores/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Zika virus/genética , Aedes/efeitos dos fármacos , Aedes/virologia , Animais , Chlorocebus aethiops , Biologia Computacional , Evolução Molecular Direcionada , Modelos Animais de Doenças , Feminino , Aptidão Genética , Genoma Viral/genética , Células HEK293 , Humanos , Camundongos , Controle de Mosquitos/métodos , Mosquitos Vetores/virologia , Fases de Leitura Aberta/genética , RNA Viral/genética , Células Vero , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
15.
PLoS Pathog ; 17(2): e1009110, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33556143

RESUMO

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.


Assuntos
Aedes/virologia , Antivirais/farmacologia , Febre de Chikungunya/transmissão , Vírus Chikungunya/genética , Vírus Defeituosos/genética , Genoma Viral , Replicação Viral , Animais , Febre de Chikungunya/imunologia , Febre de Chikungunya/virologia , Vírus Chikungunya/crescimento & desenvolvimento , Vírus Chikungunya/isolamento & purificação , Humanos , Mosquitos Vetores/virologia
16.
bioRxiv ; 2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33501440

RESUMO

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted an unbiased CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is an essential node in the cellular response to SARS-CoV-2 infection. BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells. BRD2 also controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates ACE2 levels. It is possible that the previously reported interaction between the viral E protein and BRD2 evolved to manipulate the transcriptional host response during SARS-CoV-2 infection. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.

17.
Emerg Infect Dis ; 27(2): 620-623, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33496226

RESUMO

Since May 2019, the Central African Republic has experienced a poliomyelitis outbreak caused by type 2 vaccine-derived polioviruses (VDPV-2s). The outbreak affected Bangui, the capital city, and 10 districts across the country. The outbreak resulted from several independent emergence events of VDPV-2s featuring recombinant genomes with complex mosaic genomes. The low number of mutations (<20) in the viral capsid protein 1-encoding region compared with the vaccine strain suggests that VDPV-2 had been circulating for a relatively short time (probably <3 years) before being isolated. Environmental surveillance, which relies on a limited number of sampling sites in the Central African Republic and does not cover the whole country, failed to detect the circulation of VDPV-2s before some had induced poliomyelitis in children.


Assuntos
Poliomielite , Poliovirus , República Centro-Africana/epidemiologia , Criança , Surtos de Doenças , Humanos , Poliomielite/epidemiologia , Poliomielite/prevenção & controle , Poliovirus/genética , Vacina Antipólio Oral/efeitos adversos
18.
Science ; 371(6532): 926-931, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33495306

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Depsipeptídeos/farmacologia , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Antivirais/uso terapêutico , COVID-19/prevenção & controle , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/biossíntese , Proteínas do Nucleocapsídeo de Coronavírus/genética , Depsipeptídeos/administração & dosagem , Depsipeptídeos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Humanos , Pulmão/virologia , Camundongos Endogâmicos C57BL , Mutação , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , RNA Viral/biossíntese , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
19.
PLoS One ; 15(11): e0241592, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33180795

RESUMO

Superinfection exclusion (SIE) is a process by which a virally infected cell is protected from subsequent infection by the same or a closely related virus. By preventing cell coinfection, SIE favors preservation of genome integrity of a viral strain and limits its recombination potential with other viral genomes, thereby impacting viral evolution. Although described in virtually all viral families, the precise step(s) impacted by SIE during the viral life cycle have not been systematically explored. Here, we describe for the first time SIE triggered by chikungunya virus (CHIKV), an alphavirus of public health importance. Using single-cell technologies, we demonstrate that CHIKV excludes subsequent infection with: CHIKV; Sindbis virus, a related alphavirus; and influenza A, an unrelated RNA virus. We further demonstrate that SIE does not depend on the action of type I interferon, nor does it rely on host cell transcription. Moreover, exclusion is not mediated by the action of a single CHIKV protein; in particular, we observed no role for non-structural protein 2 (nsP2), making CHIKV unique among characterized alphaviruses. By stepping through the viral life cycle, we show that CHIKV exclusion occurs at the level of replication, but does not directly influence virus binding, nor viral structural protein translation. In sum, we characterized co-infection during CHIKV replication, which likely influences the rate of viral diversification and evolution.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Superinfecção/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Vírus Chikungunya/genética , Vírus Chikungunya/patogenicidade , Chlorocebus aethiops , Genoma Viral , Vírus da Influenza A/patogenicidade , Camundongos , Vírus Sindbis/patogenicidade , Células Vero , Proteínas não Estruturais Virais/genética
20.
RNA ; 26(12): 1905-1918, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32929001

RESUMO

Like most RNA viruses, influenza viruses generate defective viral genomes (DVGs) with large internal deletions during replication. There is accumulating evidence supporting a biological relevance of such DVGs. However, further understanding of the molecular mechanisms that underlie the production and biological activity of DVGs is conditioned upon the sensitivity and accuracy of detection methods, that is, next-generation sequencing (NGS) technologies and related bioinformatics algorithms. Although many algorithms were developed, their sensitivity and reproducibility were mostly assessed on simulated data. Here, we introduce DG-seq, a time-efficient pipeline for DVG detection and quantification, and a set of biological controls to assess the performance of not only our bioinformatics algorithm but also the upstream NGS steps. Using these tools, we provide the first rigorous comparison of the two commonly used sample processing methods for RNA-seq, with or without a PCR preamplification step. Our data show that preamplification confers a limited advantage in terms of sensitivity and introduces size- but also sequence-dependent biases in DVG quantification, thereby providing a strong rationale to favor preamplification-free methods. We further examine the features of DVGs produced by wild-type and transcription-defective (PA-K635A or PA-R638A) influenza viruses, and show an increased diversity and frequency of DVGs produced by the PA mutants compared to the wild-type virus. Finally, we demonstrate a significant enrichment in DVGs showing direct, A/T-rich sequence repeats at the deletion breakpoint sites. Our findings provide novel insights into the mechanisms of influenza virus DVG production.


Assuntos
Vírus Defeituosos/genética , Genoma Viral , Influenza Humana/genética , Orthomyxoviridae/genética , RNA Viral/genética , RNA-Seq/métodos , Humanos , Influenza Humana/virologia , Replicação Viral
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