RESUMO
Adhesion G protein-coupled receptors (aGPCRs) play an important role in neurodevelopment, immune defence and cancer; however, their role throughout viral infections is mostly unexplored. We have been searching for specific aGPCRs involved in SARS-CoV-2 infection of mammalian cells. In the present study, we infected human epithelial cell lines derived from lung adenocarcinoma (Calu-3) and colorectal carcinoma (Caco-2) with SARS-CoV-2 in order to analyse changes in the level of mRNA encoding individual aGPCRs at 6 and 12 h post infection. Based on significantly altered mRNA levels, we identified four aGPCR candidates-ADGRB3/BAI3, ADGRD1/GPR133, ADGRG7/GPR128 and ADGRV1/GPR98. Of these receptors, ADGRD1/GPR133 and ADGRG7/GPR128 showed the largest increase in mRNA levels in SARS-CoV-2-infected Calu-3 cells, whereas no increase was observed with heat-inactivated SARS-CoV-2 and virus-cleared conditioned media. Next, using specific siRNA, we downregulated the aGPCR candidates and analysed SARS-CoV-2 entry, replication and infectivity in both cell lines. We observed a significant decrease in the amount of SARS-CoV-2 newly released into the culture media by cells with downregulated ADGRD1/GPR133 and ADGRG7/GPR128. In addition, using a plaque assay, we observed a reduction in SARS-CoV-2 infectivity in Calu-3 cells. In summary, our data suggest that selected aGPCRs might play a role during SARS-CoV-2 infection of mammalian cells.
Assuntos
Adenocarcinoma de Pulmão , COVID-19 , RNA Mensageiro , Receptores Acoplados a Proteínas G , SARS-CoV-2 , Regulação para Cima , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , SARS-CoV-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , COVID-19/genética , COVID-19/virologia , COVID-19/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/virologia , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Regulação para Cima/genética , Linhagem Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/virologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Células CACO-2RESUMO
The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m1A profiling and deep sequencing. We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.
Assuntos
Adenosina/análogos & derivados , HIV-1/genética , RNA Citoplasmático Pequeno/genética , RNA Viral/genética , Partícula de Reconhecimento de Sinal/genética , Vírion/genética , Adenosina/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Genoma Viral/genética , HIV-1/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Espectrometria de Massas/métodos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Vírion/metabolismo , Montagem de Vírus/genéticaRESUMO
HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.