Study On The Interaction Between Host Protein HNRNPA2B1 And Zika Virus NS5 Prontein And Its Effct On Viral Replication

Zika virus(ZIKV)is a mosquito-borne flavivirus that is associated with severe neurological complications such as Guillain-Barre syndrome(GBS)and microcephaly.Although the molecular mechanism of ZIKV infection in human cells is increasingly studied,the host factors that determine ZIKV replication and immune evasion are still to be deeplyexplored.NS5 protein is the largest and most conserved non-structural protein of ZIKV.It has a variety of enzyme activities and functions,and plays an important role in viral replication and immune escape.In the process of ZIKV replication,NS5 protein has the typical characteristics of localization in the nucleus,but the molecular details of its interaction with host proteins in the nucleus are still unclear.In this study,a combination of immunoprecipitation and mass spectrometry was used to screen the host proteins that interact with the ZIKV NS5 protein.The interaction and functional mechanism of the host protein HNRNPA2B1 and NS5 were further analyzed.The specific content and results are as follows:1.Screening and validation of ZIKV NS5 and host protein interactionIn HEK-293 T cells,host proteins that might interact with ZIKV NS5 were successfully screened out by immunoprecipitation combined with mass spectrometry analysis,and then nuclear heterogeneous ribonucleoprotein A2 /B1(HNRNPA2B1)was finally selected based on its function and localization in the cell.The results of co-immunoprecipitation(Co-IP),bimolecular fluorescence complementary systems(Bi FCs)and immunofluorescence analysis showed that ZIKV NS5 protein co-localized and interacted with the host HNRNPA2B1 protein in the nucleus.2.Identification of key areas of interaction between ZIKV NS5 and HNRNPA2B1Based on the domains of NS5 and HNRNPA2B1,truncated mutants of these two proteins were constructed and transfected into HEK-293 T cells.The specific regions of interaction were detected by Co-IP and Bi FCs.Western Blot andimmunofluorescence results showed that the NS5 MTase domain interacted with the RRM1,RRM2 and GRD domains of HNRNPA2B1.3.Effects of HNRNPA2B1 knockout and overexpression on ZIKV replicationUsing CRISPR-Cas9 technology,the HNRNPA2B1 gene was knocked out in HEK-293 T and JEG3 cell lines respectively and then corresponding cell lines were constructed.After infecting cells with Zika virus,samples at different time points were collected for q RT-PCR,Western Blot,and plaque assay to detect virus replication levels and proliferation titers.The results showed that knockout of HNRNPA2B1 significantly inhibited ZIKV RNA replication and NS5 protein production.The overexpression of HNRNPA2B1 could promote viral replication and protein production,indicating that HNRNPA2B1 had the function of promoting ZIKV replication.4.Molecular mechanism of the interaction between HNRNPA2B1 and NS5 to affect ZIKV replicationWestern Blot and IFA experiments verified that HNRNPA2B1 could up-regulate the expression of NS5 and promoted NS5 stability by preventing lysosome degradation of NS5,indicating that HNRNPA2B1 interacts with NS5 to promote the corresponding function of NS5 in the nucleus,thereby affecting virus replication.Overall,this study shows that host HNRNPA2B1 is a key factor that affects ZIKV replication,and the interaction between ZIKV NS5 and HNRNPA2B1 provides new clues for the development of anti-ZIKV therapy.