RESUMO
SARS-CoV-2 entry into host cells is facilitated by the interaction between the receptor-binding domain of its spike protein (CoV2-RBD) and host cell receptor, ACE2, promoting viral membrane fusion. The virus also uses endocytic pathways for entry, but the mediating host factors remain largely unknown. It is also unknown whether mutations in the RBD of SARS-CoV-2 variants promote interactions with additional host factors to promote viral entry. Here, we used the GST pull-down approach to identify novel surface-located host factors that bind to CoV2-RBD. One of these factors, SH3BP4, regulates internalization of CoV2-RBD in an ACE2-independent but integrin- and clathrin-dependent manner and mediates SARS-CoV-2 pseudovirus entry, suggesting that SH3BP4 promotes viral entry via the endocytic route. Many of the identified factors, including SH3BP4, ADAM9, and TMEM2, show stronger affinity to CoV2-RBD than to RBD of the less infective SARS-CoV, suggesting SARS-CoV-2-specific utilization. We also found factors preferentially binding to the RBD of the SARS-CoV-2 Delta variant, potentially enhancing its entry. These data identify the repertoire of host cell surface factors that function in the events leading to the entry of SARS-CoV-2.
Assuntos
Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Domínios Proteicos , Células HEK293 , COVID-19/metabolismo , COVID-19/virologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Interações Hospedeiro-PatógenoRESUMO
Isoaspartate (isoAsp) is a damaging amino acid residue formed in proteins mostly as a result of spontaneous deamidation of asparaginyl residues. An association has been found between isoAsp in human serum albumin (HSA) and Alzheimer's disease (AD). Here we report on a novel monoclonal antibody (mAb) 1A3 with excellent specificity to isoAsp in the functionally important domain of HSA. Based on 1A3 mAb, an indirect enzyme-linked immunosorbent assay (ELISA) was developed, and the isoAsp occupancy in 100 healthy plasma samples was quantified for the first time, providing the average value of (0.74 ± 0.13)%. These results suggest potential of isoAsp measurements for supplementary AD diagnostics as well as for assessing the freshness of stored donor blood and its suitability for transfusion.