The complete nucleotide sequence and genomic organization of Citrus Leprosis associated Virus, Cytoplasmatic type (CiLV-C).

The Citrus leprosis disease (CiL) is associated to a virus (CiLV) transmitted by Brevipalpus spp. mites (Acari: Tenuipalpidae). CiL is endemic in Brazil and its recently spreading to Central America represents a threat to citrus industry in the USA. Electron microscopy images show two forms of CiLV: a rare nuclear form, characterized by rod-shaped naked particle (CiLV-N) and a common cytoplasmic form (CiLV-C) associated with bacilliform-enveloped particle and cytoplasmic viroplasm. Due to this morphological feature, CiLV-C has been treated as Rhabdovirus-like. In this paper we present the complete nucleotide sequence and genomic organization of CiLV-C. It is a bipartite virus with sequence similarity to ssRNA positive plant virus. RNA1 encodes a putative replicase polyprotein and an ORF with no known function. RNA2 encodes 4 ORFs. pl5, p24 and p61 have no significant similarity to any known proteins and p32 encodes a protein with similarity to a viral movement protein. The CiLV-C sequences are associated with typical symptoms of CiL by RT-PCR. Phylogenetic analysis suggests that CiLV-C is probably a member of a new family of plant virus evolutionarily related to Tobamovirus.

Parallel measurement of Ca2+ binding and fluorescence emission upon Ca2+ titration of recombinant skeletal muscle troponin C. Measurement of sequential calcium binding to the regulatory sites.

Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca(2+) affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca(2+) titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca(2+) binding to the two N-terminal Ca(2+) binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca(2+) binding data showed that the Asp --> Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp --> Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp --> Ala mutation at position 30 eliminates the differences in Ca(2+) affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca(2+) binding to site I is dependent on the previous binding of metal to site II.