Integrin ?1 Promotes Peripheral Entry By Rabies Virus Integrin ?1 Promotes Peripheral Entry By Rabies Virus

Rabies,a serious zoonotic disease caused by rabies virus(RABV),is responsible for?59,000 human deaths and heavy economic burden annually worldwide.Human rabies is mostly spread by RABV direct exposure from infected dog bites or scratches,and the fatality rate is almost 100%once clinical symptoms appear.Currently,no therapy for rabies has been shown to prevent death.Further explication of RABV invasion into host cells is urgently needed for the elucidation of the pathogenesis of RABV and the development of rabies therapy.Now,several RABV receptors affecting RABV invasion have been identified,but some other key host factors remain unknown.Therefore,it is of great significance to further explore the key host factors affecting RABV invasion.Previous work has screened the host factors associated with the RABV life cycle using RNA interference(RNAi),and identified the integrin ?1(ITGB1)affects RABV infection in the Human Embryonic Kidney 293 cells(HEK-293).Here,we found that RABV infection was drastically decreased after ITGB1 siRNA knockdown in HEK-293 and mouse neuroblastoma(N2a)cells,and increased after ITGB1 overexpression in HEK-293 cells,suggesting that ITGB 1 is a key host factor for RABV infection.RABV glycoprotein(G)is thought to be of prime importance in early entry and virus-host interaction.In this study,we found that ITGB1 directly interacts with RABV G derived from several rabies virus strains.Integrins on the surface of the cell membrane can be endocytosed to enter cells using a variety pathways,including the clathrin-mediated endocytosis pathway,which is consistent with the classic endocytosis pathway of RABV.We found that ITGB1 is internalized into cells and transported to late endosomes together with RABV upon infection in N2s cells,and the interaction between ITGB1 and RABV G under acidic conditions was also confirmed,indicating that RABV could continue to bind to ITGB1 after co-endotherptosis.ITGB1 is proposed to facilitate early infection as a receptor for a variety of viruses.We found that ITGB1 soluble protein and ITGB1 monoclonal antibody(MAb)can neutralize or block RABV infection in a dose-dependent manner in vitro,which further indicates that ITGB1 plays an important role in early invasion of RABV infection.ITGB1 is a motif-dependent receptor of fibronectin(FN)through Arg-Gly-Asp(RGD)motifs.RGD peptide can competitively inhibit the interaction between ITGB1 and FN.In this study,FN interacts with ITGB1 and RABVG respectively,and a RGD peptide(GRGDSP)can block RABV infection in a dose-dependent manner in vitro,indicating that ITGB1 mediates RABV infection through FN.ITGB1,a type ? transmembrane glycoprotein,is ubiquitously expressed in all metazoan cell types,and is essential for normal morphological development of the peripheral and central nervous system.Howerver,ITGB1 is differentially expressed in the muscle and mature nerves of animals.Mouse studies shows that ITGB1 was distributed throughout the cells of thigh muscle,and co-existed with RABVstreet strain(GX/09)in infected muscle cells,suggesting that ITGB1 may affect peripheral infection of RABV.Nicotinic acetylcholine receptor ?1(nAChR?1)is the only known peripheral receptor for RABV,and we found that ITGB1 also interacts with nAChRal,showed that ITGB1 may be an important host factor for peripheral infection of RABV.In addition,ITGB1 soluble protein can neutralize GX/09 infection with a lethal dosage in mice,and ITGB1 MAb and GRGDSP blocked GX/09 infection via intramuscular but not intracerebral inoculation in mice,further suggesting that ITGB1 plays a key role in RABV peripheral entry.This study on the mechanism of peripheral invasion of RABV enriched the theoretical system of the mechanism of RABV invasion into the host,provided a reference for the study of RABV antiviral drugs,and laid a foundation for the in-depth study of the pathogenesis of RABV.