Biological And Molecular Characterization Study Of Two Closely Related Arepaviruses

Potyviridae is the largest family of plant-infecting RNA viruses,which threaten a variety of important economic and food crops.Previously,our group characterized two novel closed-related viruses of the Potyviridae from Areca catechu L.in Hainan.The two viruses were named areca palm necrotic spindle-spot virus(ANSSV)and areca palm necrotic ringspot virus(ANRSV).Both are taxonomically classified into a new genus,Arepavirus.The two viruses are highly prevalent in the field and induce severe necrosis and tree vigor declining symptoms,thus severely restricting the healthy development of the areca palm industry.However,their biological and pathological properties,virus-virus interactions,key infection sites are yet unknown.Here,we newly constructed an infectious c DNA clone of ANRSV,which,together with our previously described ANSSV clone,were used to investigate the biology and pathological properties,functional compatibility of genome segments,virus-virus interactions,and key infection sites of ANSSV.The obtained results are mainly as follows:1.The full-length c DNA clone of ANRSV(p RS)was constructed and tested to be highly infectious in the experimental host N.benthamiana.Subsequently,a GFP sequence was inserted into the virus genome at the NIb and CP junction to generate a GFP-tagged virus clone(p RS-G),which is highly infectious in N.benthamiana as well.2.The pathological properties between ANSSV and ANRSV are investigated.In comparison with ANSSV,ANRSV induces more severe leaf chlorosis and rugosity symptoms in planta and the pathogenicity discrepancy is associated with virus multiplication velocity.3.The functional compatibility of viral genome segments was investigated.A series of hybrid viruses were constructed via substitution of multiple genome segments in the ANRSV infectious clone with the counterparts of ANSSV.The replacement of either 5’-UTRHCPro1–HCPro2 or CI effectively supported replication and systemic infection of ANRSV,whereas individual substitution of P3-7K,9K-NIa,and NIb-CP-3’-UTR abolished viral infectivity.4.The virus-virus interactions between ANSSV and ANRSV was studied.Both coinfection and super-infection assays were conducted.The two assays consistently confirmed the antagonistic interaction between them,and ANRSV confers efficient exclusion of ANSSV,providing a clue to understanding the molecular basis underlying the epidemiological discrepancy in the field.5.The key pathogenicity sites of ANSSV was identified.In this study,we identified three amino acid mutations in the viral genome of ANSSV progeny.Further,two amino acids I883 M and E2704 G,located in replication-related proteins P3 and NIb,respectively,were characterized to promote viral infectivity efficiently via testing a series of virus mutants(including single,double,and triple aa site mutants).These findings laid an important foundation for further dissecting the underlying molecular mechanism of viral infectivity.In summary,in this study,we developed the reverse genetic system of ANRSV.Subsequently,we investigated the pathological properties,functional compatibility of genomic segments,and virus-virus interactions of ANRSV and ANSSV.These findings advance our understanding of these two viruses’ biology and molecular properties.Besides,we identified two amino acids in the viral replication-related protein crucial for the ANSSV infectivity,which provided an important basis for dissecting the underlying molecular mechanism.