RESUMO
SARS-CoV-2 is the causative agent of the COVID-19 pandemic, the acute respiratory disease which, so far, has led to over 7 million deaths. There are several symptoms associated with SARS-CoV-2 infections which include neurological and psychiatric disorders, at least in the case of pre-Omicron variants. SARS-CoV-2 infection can also promote the onset of glioblastoma in patients without prior malignancies. In this study, we focused on the Envelope protein codified by the virus genome, which acts as viroporin and that is reported to be central for virus propagation. In particular, we characterized the electrophysiological profile of E-protein transfected U251 and HEK293 cells through the patch-clamp technique and FURA-2 measurements. Specifically, we observed an increase in the voltage-dependent (Kv) and calcium-dependent (KCa) potassium currents in HEK293 and U251 cell lines, respectively. Interestingly, in both cellular models, we observed a depolarization of the mitochondrial membrane potential in accordance with an alteration of U251 cell growth. We, therefore, investigated the transcriptional effect of E protein on the signaling pathways and found several gene alterations associated with apoptosis, cytokines and WNT pathways. The electrophysiological and transcriptional changes observed after E protein expression could explain the impact of SARS-CoV-2 infection on gliomagenesis.
Assuntos
COVID-19 , Glioblastoma , Potencial da Membrana Mitocondrial , SARS-CoV-2 , Humanos , Glioblastoma/metabolismo , Glioblastoma/virologia , Glioblastoma/patologia , Glioblastoma/genética , Células HEK293 , SARS-CoV-2/fisiologia , COVID-19/virologia , COVID-19/metabolismo , Linhagem Celular Tumoral , Proteínas do Envelope de Coronavírus/metabolismo , Proteínas do Envelope de Coronavírus/genética , Apoptose , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genéticaRESUMO
Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission.
Assuntos
COVID-19/epidemiologia , Regulação Viral da Expressão Gênica , Genoma Viral , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Animais , COVID-19/patologia , COVID-19/transmissão , COVID-19/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteínas do Envelope de Coronavírus/genética , Proteínas do Envelope de Coronavírus/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , RNA Viral/metabolismo , SARS-CoV-2/classificação , SARS-CoV-2/metabolismo , Células Vero , Carga Viral , VirulênciaRESUMO
Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.
Assuntos
Proteínas do Envelope de Coronavírus/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , SARS-CoV-2/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , COVID-19/patologia , COVID-19/virologia , Proteínas do Envelope de Coronavírus/genética , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Humanos , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Janus Quinases/genética , Janus Quinases/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Poli I-C/farmacologia , RNA Viral/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/isolamento & purificaçãoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of the coronavirus disease (COVID-19), is a part of the $\beta $-Coronaviridae family. The virus contains five major protein classes viz., four structural proteins [nucleocapsid (N), membrane (M), envelop (E) and spike glycoprotein (S)] and replicase polyproteins (R), synthesized as two polyproteins (ORF1a and ORF1ab). Due to the severity of the pandemic, most of the SARS-CoV-2-related research are focused on finding therapeutic solutions. However, studies on the sequences and structure space throughout the evolutionary time frame of viral proteins are limited. Besides, the structural malleability of viral proteins can be directly or indirectly associated with the dysfunctionality of the host cell proteins. This dysfunctionality may lead to comorbidities during the infection and may continue at the post-infection stage. In this regard, we conduct the evolutionary sequence-structure analysis of the viral proteins to evaluate their malleability. Subsequently, intrinsic disorder propensities of these viral proteins have been studied to confirm that the short intrinsically disordered regions play an important role in enhancing the likelihood of the host proteins interacting with the viral proteins. These interactions may result in molecular dysfunctionality, finally leading to different diseases. Based on the host cell proteins, the diseases are divided in two distinct classes: (i) proteins, directly associated with the set of diseases while showing similar activities, and (ii) cytokine storm-mediated pro-inflammation (e.g. acute respiratory distress syndrome, malignancies) and neuroinflammation (e.g. neurodegenerative and neuropsychiatric diseases). Finally, the study unveils that males and postmenopausal females can be more vulnerable to SARS-CoV-2 infection due to the androgen-mediated protein transmembrane serine protease 2.
Assuntos
COVID-19/genética , Genoma Viral/genética , Conformação Proteica , SARS-CoV-2/ultraestrutura , COVID-19/virologia , Proteínas do Envelope de Coronavírus/genética , Proteínas do Envelope de Coronavírus/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/ultraestrutura , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Proteínas do Complexo da Replicase Viral/genética , Proteínas do Complexo da Replicase Viral/ultraestrutura , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/ultraestruturaRESUMO
The human ß-coronavirus SARS-CoV-2 epidemic started in late December 2019 in Wuhan, China. It causes Covid-19 disease which has become pandemic. Each of the five-known human ß-coronaviruses has four major structural proteins (E, M, N and S) and 16 non-structural proteins encoded by ORF1a and ORF1b together (ORF1ab) that are involved in virus pathogenicity and infectivity. Here, we performed detailed positive selection analyses for those six genes among the four previously known human ß-coronaviruses and within 38 SARS-CoV-2 genomes to assess signatures of adaptive evolution using maximum likelihood approaches. Our results suggest that three genes (E, S and ORF1ab genes) are under strong signatures of positive selection among human ß-coronavirus, influencing codons that are located in functional important protein domains. The E protein-coding gene showed signatures of positive selection in two sites, Asp 66 and Ser 68, located inside a putative transmembrane α-helical domain C-terminal part, which is preferentially composed by hydrophilic residues. Such Asp and Ser sites substitutions (hydrophilic residues) increase the stability of the transmembrane domain in SARS-CoV-2. Moreover, substitutions in the spike (S) protein S1 N-terminal domain have been found, all of them were located on the S protein surface, suggesting their importance in viral transmissibility and survival. Furthermore, evidence of strong positive selection was detected in three of the SARS-CoV-2 nonstructural proteins (NSP1, NSP3, NSP16), which are encoded by ORF1ab and play vital roles in suppressing host translation machinery, viral replication and transcription and inhibiting the host immune response. These results are insightful to assess the role of positive selection in the SARS-CoV-2 encoded proteins, which will allow to better understand the virulent pathogenicity of the virus and potentially identifying targets for drug or vaccine strategy design.
Assuntos
COVID-19/virologia , Proteínas do Envelope de Coronavírus/genética , Pandemias , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/genética , Substituição de Aminoácidos , COVID-19/epidemiologia , Humanos , Poliproteínas/genética , Domínios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Virulência/genética , Replicação ViralRESUMO
BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Proteínas do Envelope de Coronavírus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Limite de Detecção , Poliproteínas/genética , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
SARS-CoV-2 is mutating and creating divergent variants across the world. An in-depth investigation of the amino acid substitutions in the genomic signature of SARS-CoV-2 proteins is highly essential for understanding its host adaptation and infection biology. A total of 9587 SARS-CoV-2 structural protein sequences collected from 49 different countries are used to characterize protein-wise variants, substitution patterns (type and location), and major substitution changes. The majority of the substitutions are distinct, mostly in a particular location, and lead to a change in an amino acid's biochemical properties. In terms of mutational changes, envelope (E) and membrane (M) proteins are relatively more stable than nucleocapsid (N) and spike (S) proteins. Several co-occurrence substitutions are observed, particularly in S and N proteins. Substitution specific to active sub-domains reveals that heptapeptide repeat, fusion peptides, transmembrane in S protein, and N-terminal and C-terminal domains in the N protein are remarkably mutated. We also observe a few deleterious mutations in the above domains. The overall study on non-synonymous mutation in structural proteins of SARS-CoV-2 at the start of the pandemic indicates a diversity amongst virus sequences.
Assuntos
SARS-CoV-2/química , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Substituição de Aminoácidos , Aminoácidos/química , Proteínas do Envelope de Coronavírus/química , Proteínas do Envelope de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Mutação , Taxa de Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93%, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Proteínas do Envelope de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Faringe/virologia , Fosfoproteínas/genética , Poliproteínas/genética , Proteínas Virais/genéticaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.