Biological Function Study On The Unique Leader Cysteine Proteases In Tandem Encoded By Areca Palm Necrotic Spindle-spot Virus

Potyviridae is the largest family of plant-infecting RNA viruses,and the viruses in this family(potyvirids)threaten a variety of important economic and alimentary crops.The N-terminal regions of potyvirids encode the leader proteases,which vary greatly in sequence and play key roles during viral infection.Previously,we identified two novel virus species from Areca catechu L.in Hainan,China,i.e.,Areca palm necrotic spindle-spot virus(ANSSV)and Areca palm necrotic ringspot virus(ANRSV).The genomic sequence analysis revealed that both ANRSV and ANSSV are classified into a putative new genus(designated as Arepavirus)in the family Potyviridae.Bioinformatic analysis predicted that either ANSSV or ANRSV has a unique pattern of leader proteases formed,apparently,by two cysteine proteases(HCProl and HCPro2)in tandem,which is different from other viruses in this family.In order to explore the biological functions of HCProl-HCPro2 encoded by ANSSV during viral infection,we achieved the following main results:1.We demonstrated that the 5'-terminal region of the ANSSV genome encodes two cysteine proteases in tandem(HCProl-HCPro2).The wheat germ extract(WGE)-based in vitro translation system was employed in this assay.The results verified that the 5'-terminal region of the ANSSV genome contains two cysteine protease domains,and the catalytic active sites are C161 and C463,respectively.Subsequently,we constructed a series of alanine substitution mutants,in which the conserved amino acids 'Gly' in the predictable cis-cleavage sites of HCProl and HCPro2 were replaced by 'Ala'.The in vitro translation assay showed the cis-cleavage sites of HCProl and HCPro2 were located at position 273G/V274 and 574G/G575,respectively,and thus these two viral cistrons in ANSSV genome are clearly delineated.2.The functional differentiation of HCProl and HCPro2 in host RNA silencing suppression.For potyvirids,HCPro is one of the most variable proteins.In the present study,a comprehensive analysis of HCPro sequences in the family Potyviridae was performed.The results showed that potyvirid's HCPro factors comprise a variable N-terminal region and a conserved C-terminal cysteine protease domain.Usually,one of the leader proteases of potyvirids executes essential roles in RNA silencing suppression,such as HCPro factor from members of the Potyvirus genus.The Agrobacteriummediated transient co-expression assays revealed the functional divergence of HCProl and HCPro2 in RNA silencing suppression:HCPro2 is a viral suppressor of RNA silencing(VSR),whereas HCProl is not.Consistently,HCPro is the potyviral VSR factor,and its activity depends on its release from the preceding P1.On the contrary,the self-cleavage of HCProl to release free HCPro2 is not essential for HCPro2-mediated antisilencing activity.Both N2 and D2 domains of HCPro2 confer RNA silencing suppression activity independently.Intriguingly,the N-terminal region of HCProl also has RNA silencing suppression activity,which is,however,suppressed by its C-terminal protease domain,leading to abolish RNA silencing suppression activity of the whole HCPro1 protein.3.Development of ANSSV-derived full-length infectious cDNA clones.We generated the complete cDNA clone of ANSSV(named pSS-I),and infectivity test showed that that the pSS-I was infectious in experimental host,Nicotiana benthamiana.The GFP cistron was integrated into the NIb/CP intercistronic junction in pSS-I to create a GFP-tagged ANSSV clone(pSS-I-G).4.The biological functions of HCPro1 and HCPro2 during viral infection cycle.To dissect the biological significance of HCPro1 and HCPro2 during viral infection,we constructed either HCPro1-or HCPro2-deleted virus clone.Infectivity test showed that these clones are loss-of-infectivity in N.benthamina.Using a similar strategy,we domenstrated that neither of them(HCProl and HCPro2)can be functionally replaced,respectively,by the P1 and HCPro of a potyvirus(TuMV).All in all,HCPro1 and HCPro2 are indispensable during viral infection cycle,and they function in a coordinated manner to maintain viral infection.Here,we experimentally demonstrated the presence of a distinct pattern of leader proteases,HCPro1 and HCPro2 in tandem in ANSSV genome.They have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity.Altogether,the new knowledge provides the basis for further study on the interaction between virus and host,pathogenicity mechanism and the management of the vial agent.In addition,these novel findings advance our understanding of the functional diversification conveyed by the hypervariable N-terminal regions of potyvirids.