sesbania mosaic virus (semv) infectious clone possible mechanism of 3′ and 5′ end repair and role of polyprotein processing in viral replication田菁属花叶病毒(semv)感染性克隆可能3u2032,5u2032末端修复机制,多蛋白处理病毒复制的作用.pdf

Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 39 and 59 End Repair and Role of Polyprotein Processing in Viral Replication 1 ¨ 2 1 Kunduri Govind , Kristiina Makinen , Handanahal S. Savithri * 1 Indian Institute of Science, Bangalore, Karnataka, India, 2 University of Helsinki, Helsinki, Finland Abstract Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 39 and 59 terminal deletion mutants, we propose a possible mechanism for 39 and 59 end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic