Desenvolvimento de modelo in vitro de superexpressão de ace2 e tmprss2 para estudo de infecção por Sars-Cov-2
Data
2023-01-27
Autores
Bartolomeo, Cynthia Silva 
Orientadores
Prado, Carla Máximo
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), agente etiológico da COVID-19 (Coronavirus disease 2019), tornou-se uma grande preocupação mundial, principalmente devido aos impactos nos sistemas de saúde na maioria dos países e na vida das pessoas. A entrada do vírus na célula hospedeira ocorre após a ligação da proteína spike (S), presente na espícula viral, ao receptor celular ACE2 (ECA2 - enzima conversora de angiotensina 2). Após a ligação, o vírus precisa ganhar acesso ao citosol, processo esse dependente da clivagem proteolítica da proteína S pela serino protease tipo II (TMPRSS2). No presente projeto, após avaliação da cinética viral nas linhagens Vero-E6, HuH7, BEAS-2B, A549, HEK-293T e HUVEC verificamos a existência de dois perfis de replicação viral. Nomeamos como “high-viral profile” as linhagens Vero-E6 e HuH7 por terem demonstrado uma capacidade de replicação viral ao longo do tempo muito superior às linhagens BEAS-2B, A549, HEK-293T e HUVEC que foram classificadas como sendo “low-viral profile”. Determinamos a expressão gênica e proteica dos receptores ACE2 e TMPRSS2 nas linhagens acima mencionadas, bem como a atividade de ACE2, de maneira a estabelecer uma relação aos achados da cinética viral. O desenvolvimento de um modelo que permita estudos relacionados à COVID-19 é de extrema relevância clínica, acadêmica e terapêutica. Assim sendo, a partir da criação de modelos celulares in vitro de superexpressão de ACE2 e TMPRSS2, em linhagens reconhecidamente suscetíveis ao SARS-CoV-2, visamos elucidar a participação desses receptores nos mecanismos fisiopatológicos da COVID-19. Foram desenvolvidos dois modelos de estudo in vitro do SARS-CoV-2: 1) modelo de superexpressão de ACE2 via plasmideal e 2) modelo de superexpressão de ACE2, TMPRSS2 e co-expressão de ACE2 e TMPRSS2 através de um vetor lentiviral em linhagens BEAS-2B, A549, HUVEC, HEK-293T e HuH7. Após criação do modelo de superexpressão via plasmideal das linhagens BEAS-2B-ACE2 e A549-ACE2 verificamos uma maior cinética de replicação viral quando comparadas aos seus respectivos controles, demonstrando que uma maior expressão do receptor ACE2 é capaz de promover uma potencialização na infecção por SARS-CoV-2. Constamos uma maior atividade de ACE2 nas linhagens acima mencionadas, como também na HUVEC-ACE2. Sendo a proliferação diminuída em todas as linhagens criadas (BEAS-2B-ACE2, A549-ACE2, HEK-293T e HUVEC-ACE2). Em células BEAS-2B e HUVEC, a
superexpressão de ACE2 aumentou a cinética de replicação viral, em contrapartida a superexpressão de TMPRSS2 não promoveu maior entrada viral. Em relação à variante Ômicron, observamos uma cinética de replicação viral superior ao vírus selvagem em linhagem BEAS-2B-ACE2, uma cinética intermediária em linhagem BEAS-2B-ACE2-TMPRSS2 e inferior em BEAS-TMPRSS2. Ademais, foi possível verificar que a Ang II aumenta a replicação do SARS-CoV-2 WT em células HUVEC, sendo esse aumento suprimido quando o receptor ACE2 é superexpresso. Modelos in vitro que mimetizem a superexpressão de ACE2 e TMPRSS2, em diferentes tipos celulares, são de grande utilidade para o estudo dos mecanismos de infecção viral e desenvolvimento de terapias, podendo auxiliar em futuros estudos para verificação de uma possível potencialização da infecção, simulando a sensibilização das células após a entrada viral. Desta forma, um maior entendimento do papel da ACE2 e TMPRSS2 na COVID-19, poderá agregar conhecimentos de novos alvos terapêuticos, uma melhor compreensão dos mecanismos de entrada do vírus e o comportamento das células frente à infecção.
SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), the etiological agent of COVID-19 (Coronavirus disease 2019), has become a major concern worldwide mainly due to the devastating effects not only on the healthcare system but also on human lives. Regarding the infection, the entry of the virus into a host cell occurs upon binding of the spike protein (S), located in the viral spike, to the cellular receptor ACE2 (angiotensin-converting enzyme 2). After binding, the virus needs to gain access to the cytosol, a process that depends on the proteolytic cleavage of protein S by serine protease type II (TMPRSS2). In this project, after analyzing the viral kinetics in the cell lines Vero-E6, HuH7, BEAS-2B, A549, HEK293T and HUVEC, we verified the existence of two viral replication profiles. We named Vero-E6 and HuH7 as “high-viral profile” because they showed a much higher capacity for viral replication over time than the strains BEAS-2B, A549, HEK293T and HUVEC, which were classified as “low-viral profile". We determined the gene and protein expression of ACE2 and TMPRSS2 receptors in the aforementioned cell lines, as well as the activity of ACE2 in order to correlate it with the findings of viral kinetics. The development of a model that allows studies related to COVID-19 has clinical, academic and therapeutic relevance. Therefore, our creation of in vitro cell models of ACE2 and TMPRSS2 overexpression in cells known to be susceptible to SARS-CoV-2 aims to elucidate the participation of these receptors in the pathophysiology of COVID-19. Two in vitro models of SARS-CoV2 were developed: 1) model of overexpression of ACE2 via plasmid and 2) model of overexpression of ACE2, TMPRSS2 and co-expression of ACE2 and TMPRSS2 through a lentiviral vector in BEAS-2B, A549, HUVEC, HEK-293T and HuH7 strains. After creating the overexpression model, via plasmid, of the BEAS-2B-ACE2 and A549-ACE2 strains, we verified a greater kinetics of viral replication when compared to their respective controls, demonstrating that a greater expression of the ACE2 receptor is capable of promoting an increase in the infection by SARS-CoV-2. We found a greater activity of ACE2 in the strains mentioned above, as well as in HUVEC-ACE2. The proliferation was decreased in all strains created (BEAS-2BACE2, A549-ACE2, HEK-293T and HUVEC-ACE2). In BEAS-2B and HUVEC cells, ACE2 overexpression increased viral replication kinetics, on the other hand, TMPRSS2 overexpression did not promote greater viral entry. Regarding the Omicron variant, we observed a viral replication kinetics superior to the wild virus in the BEAS-2B-ACE2 lineage, an intermediate kinetics in the BEAS-2B-ACE2- TMPRSS2 lineage and inferior in BEAS-TMPRSS2. Furthermore, it was possible to verify that Ang II increases the replication of SARS-CoV-2 WT in HUVEC cells, this increase being suppressed when the ACE2 receptor is overexpressed. In vitro models that mimic the overexpression of ACE2 and TMPRSS2 in different cell types are very useful to the study of viral infection mechanisms and development of therapies. Thus, these models may contribute to studying SARS-CoV-2 infection enhancement, which could be of extreme importance to future COVID-19 studies. Therefore, a better understanding of the role of ACE2 and TMPRSS2 in COVID-19 may contribute not only to the development of new therapeutic targets but also to a deeper understanding of virus entry mechanisms and behavior of cells upon infection.
SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), the etiological agent of COVID-19 (Coronavirus disease 2019), has become a major concern worldwide mainly due to the devastating effects not only on the healthcare system but also on human lives. Regarding the infection, the entry of the virus into a host cell occurs upon binding of the spike protein (S), located in the viral spike, to the cellular receptor ACE2 (angiotensin-converting enzyme 2). After binding, the virus needs to gain access to the cytosol, a process that depends on the proteolytic cleavage of protein S by serine protease type II (TMPRSS2). In this project, after analyzing the viral kinetics in the cell lines Vero-E6, HuH7, BEAS-2B, A549, HEK293T and HUVEC, we verified the existence of two viral replication profiles. We named Vero-E6 and HuH7 as “high-viral profile” because they showed a much higher capacity for viral replication over time than the strains BEAS-2B, A549, HEK293T and HUVEC, which were classified as “low-viral profile". We determined the gene and protein expression of ACE2 and TMPRSS2 receptors in the aforementioned cell lines, as well as the activity of ACE2 in order to correlate it with the findings of viral kinetics. The development of a model that allows studies related to COVID-19 has clinical, academic and therapeutic relevance. Therefore, our creation of in vitro cell models of ACE2 and TMPRSS2 overexpression in cells known to be susceptible to SARS-CoV-2 aims to elucidate the participation of these receptors in the pathophysiology of COVID-19. Two in vitro models of SARS-CoV2 were developed: 1) model of overexpression of ACE2 via plasmid and 2) model of overexpression of ACE2, TMPRSS2 and co-expression of ACE2 and TMPRSS2 through a lentiviral vector in BEAS-2B, A549, HUVEC, HEK-293T and HuH7 strains. After creating the overexpression model, via plasmid, of the BEAS-2B-ACE2 and A549-ACE2 strains, we verified a greater kinetics of viral replication when compared to their respective controls, demonstrating that a greater expression of the ACE2 receptor is capable of promoting an increase in the infection by SARS-CoV-2. We found a greater activity of ACE2 in the strains mentioned above, as well as in HUVEC-ACE2. The proliferation was decreased in all strains created (BEAS-2BACE2, A549-ACE2, HEK-293T and HUVEC-ACE2). In BEAS-2B and HUVEC cells, ACE2 overexpression increased viral replication kinetics, on the other hand, TMPRSS2 overexpression did not promote greater viral entry. Regarding the Omicron variant, we observed a viral replication kinetics superior to the wild virus in the BEAS-2B-ACE2 lineage, an intermediate kinetics in the BEAS-2B-ACE2- TMPRSS2 lineage and inferior in BEAS-TMPRSS2. Furthermore, it was possible to verify that Ang II increases the replication of SARS-CoV-2 WT in HUVEC cells, this increase being suppressed when the ACE2 receptor is overexpressed. In vitro models that mimic the overexpression of ACE2 and TMPRSS2 in different cell types are very useful to the study of viral infection mechanisms and development of therapies. Thus, these models may contribute to studying SARS-CoV-2 infection enhancement, which could be of extreme importance to future COVID-19 studies. Therefore, a better understanding of the role of ACE2 and TMPRSS2 in COVID-19 may contribute not only to the development of new therapeutic targets but also to a deeper understanding of virus entry mechanisms and behavior of cells upon infection.
Descrição
Citação
BARTOLOMEO, Cynthia Silva. Desenvolvimento de modelo in vitro de superexpressão de ace2 e tmprss2 para estudo de infecção por Sars-Cov-2. 2023. 178 f. Dissertação (Mestrado Interdisciplinar em Ciências da Saúde) - Instituto de Saúde e Sociedade, Universidade Federal de São Paulo, Santos, 2023.