High-throughput Screening Of Anti-arenavirus Drugs And Quantitative Proteomic Analysis Of Herpes Simplex Virus Type 1-infected HEK 293T Cells

Arenaviruses is an enveloped virus with negative stranded and bi-segmented RNA genome,members including Guanarito virus,Junin virus,Lassa virus,Lujo virus,Machupo virus,Sabia virus and Whitewater Arroyo virus spread in many areas and caused severe hemorrhagic fever syndromes with high mortality in infected humans,seriously threat to public health.However,there are no FDA-licensed clinical treatments,drugs,and vaccines except the off-labeled use of ribavirin with many side effects and limited therapeutic efficacy.So,there are urgent needs to develop new antiviral drugs for arenavirus treatment.In this study,Old World arenavirus LCMV（Lymphocytic choriomeningitis virus）was used to screen anti-arenavirus drugs from an FDA-approved drug library by a high content image-based screening method.5 drugs out of all 1018 compounds were choosed including mycophenolic acid,benidipine hydrochloride,clofazimine,dabrafenib,and apatinib for stronger antiviral effect than other drugs.The dihydropyridine derived calcium channel blocker and cardioprotective drug benidipine hydrochloride which could be used clinically for hypertension and ischemic heart diseases,was confirmed to block LCMV and other hemorrhagic fever arenavirus infection,with IC₅₀=0.548μM,CC₅₀=113.8μM and SI=207.66.The antiviral activity was also confirmed in a LCMV infection mouse model.Through the analysis of different stages of LCMV life cycle,benidipine hydrochloride was confirmed to block LCMV GPC mediated membrane fusion stage of virus entry.And after incubated in drug containing medium for ten generation,an inhibitor-active adaptive mutant virus LCMV _GPC-D414A was identified to antagonize the antiviral efficacy of benidipine hydrochloride,and the membrane fusion mediated by GPC-D414A was also unaffected through benidipine hydrochloride treatment.So,we proposed that benidipine hydrochloride might interact with the D414 site on LCMV GPC directly or indirectly,blocked the structural changes of GPC when exposed to low p H environment,and the membrane fusion was inhibited;When the D414 site on LCMV GPC was mutated,the interaction was affected,the structural changes and membraned fusion could happen normally.Another dihydropyridine derived calcium channel blockers were also proved to inhibit LCMV,and their antiviral efficacy on LCMV _GPC-D414A was also blocked.We can conclude that the antiviral mechanism of these dihydropyridine derived calcium channel blockers was similar to benidipine hydrochloride,and the D414 mutation site on GPC provided us more insights into virus fusion mechanism and intersubunit interactions between inhibitors and virus.We also explored that the identified anti-arenavirus drugs mycophenolic acid,clofazimine,dabrafenib,and apatinib could also inhibit the replication of SARS-Co V-2（Severe acute respiratory syndrome coronavirus 2）,and mycophenolic acid showed strongest anti-SARS-Co V-2 activity,with IC₅₀=0.101μM,CC₅₀>100μM and SI>1000.The anti-viral mechanism of mycophenolic acid was investigated,and the results indicated that mycophenolic acid inhibited LCMV and SARS-Co V-2 by depleting GTP production.Since their FDA-approved status,these drug candidates have the potential to be rapidly clinical used for arenavirus and SARS-Co V-2 treatment.Herpes simplex virus type 1（HSV-1）is a double-stranded DNA virus widely carried by human hosts.HSV-1 could establish latency infection in infected people with periodically episodes,and caused severe symptoms in immunodeficiency patients.In order to study the interaction between virus and host cells,the regulation of host cell proteomics after HSV-1 infection was analyzed by LC-MS/MS.A total of6607 host proteins were identified,of which 498 proteins were differentially regulated.And further functional study indicated that three host proteins IFITM3（IFN-inducible transmembrane protein 3）,CHCHD2（Coiled-coil-helix-coiled-coil-helix domain containing 2）and TRIM27（Tripartite motif-containing protein 27） could inhibit viral genome DNA replication.Through bioinformatics analysis,multiple host cell signaling pathways involved in HSV-1 infection were identified,and provided a global perspective of the HSV-1 infection effect on host cells.