CITRUS PSOROSIS VIRUS - NUCLEOTIDE SEQUENCING OF THE COAT PROTEIN GENE AND DETECTION BY HYBRIDIZATION AND RT-PCR

Citrus psorosis virus (CPV) is a multicomponent ssRNA virus with a coa t protein of approximately 48 kDa. The viral genome is encapsidated in short anti long particles that are readily separated by sucrose densi ty-gradient centrifugation. CPV particles are spiral filaments that ar e referred to as spiroviruses (SV). A cDNA library of purified short p articles from isolate CPV-4 was prepared in a Lambda vector and screen ed for expression of the colt protein gene (CPG) with a monoclonal ant ibody to the coat protein. Sequencing of immunopositive clones indicat ed a single ORF encoding a 49 kDa protein. This ORF, when expressed in E. coli, gave a protein identical in size and immunoreactivity to the CPV coat protein. A full-length clone of the CPG was transcribed and used in Northern hybridization assays to establish that short particle RNA of CPV is negative sense and contains the CPG, Moreover, the CPG was not found on RNA extracted from long particles or on the sedimenta ble dsRNA from CPV infected tissue. RT-PCR assays were developed for t he amplification of a 600 bp fragment of CPG and for the complete CPG (1317 bp). The 600 bp fragment from a biologically and serologically d ifferent isolate, CPV-6, was cloned, sequenced and found to share 86% (nucleotide) and 96% (amino acid) identity with CPV-4. BLAST analysis of sequences from CPV-4 and CPV-6 detected no significant nucleic acid or protein similarity with any known viral sequences.