Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR

Abstract

Citrus psorosis virus (CPV) is a multicomponent ssRNA virus with a coat protein of approximately 48 kDa. The viral genome is encapsidated in short and long particles that are readily separated by sucrose density-gradient centrifugation. CPV particles are spiral filaments that are referred to as spiroviruses (SV). A cDNA library of purified short particles from isolate CPV-4 was prepared in a Lambda vector and screened for expression of the coat protein gene (CPG) with a monoclonal antibody to the coat protein. Sequencing of immunopositive clones indicated 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 sedimentable dsRNA from CPV infected tissue. RT-PCR assays were developed for the amplification of a 600 bp fragment of CPG and for the complete CPG (1317 bp). The 600 bp fragment from a biologically and serologically different 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.