1R & D Department, Indian Immunologicals Limited, Gachibowli, Hyderabad - 500019.
2Department of Virology, Sri Venkateswara University, Tirupati-517502, A.P.
3Department of Biochemistry, Indian Institute of Science, Bangalore-526012.
*E-mail: pothursree@yahoo.com
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The ssRNA genome of Sugarcane streak mosaic virus (SCSMV-AP) naturally infecting sugarcane in India has been partially sequenced (4,475 nucleotides from 3′ end; GenBank, EMBL) and identified as a taxonomically distinct virus in the family Potyviridae. The coat protein gene (852 nucleotides) of SCSMV-AP isolate was amplified by PCR, cloned into pRSET-A vector, over expressed in E. coli pLys S (DE3) and purified the expressed coat protein either by Ni-NTA agarose affinity column chromatography or by differential centrifugation followed by linear sucrose density gradient (50–20%) centrifugation (26,000 rpm, 3 hrs). The authenticity of the expressed coat protein was confirmed by western blot analysis using polyclonal antiserum produced against purified virus. The recombinant coat protein (rCP) was used as an antigen to produce antibodies and for in vivo assembly studies. The electron microscopic studies of the top and bottom zones of purified rCP obtained by sucrose density gradient centrifugation revealed potyvirus-like particles (PVLPs) that are heterogeneous in length. This observation showed that the additional 36 amino acids at the N-terminus of rCP due to cloning strategy did not influence its assembly into PVLPs. The slot-blot analysis of RNA isolated from PVLPs using SCSMV-AP coat protein gene as a probe revealed that they encapsidated coat protein mRNAs. The potential applications of these studies shall be discussed.
