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Title: MUTAGENIC ANALYSIS AND LOCALIZATION OF A HIGHLY CONSERVED EPITOPE NEAR THE AMINO TERMINAL END OF THE CITRUS TRISTEZA CLOSTEROVIRUS CAPSID PROTEIN

Author
item PAPPU, H - UNIV. OF FLORIDA
item PAPPU, S - UNIV. OF FLORIDA
item KANO, T - MAFF, JAPAN
item KOIZUMI, M - MAFF, JAPAN
item CAMBRA, M - IVIA, VALENCIA, SPAIN
item MORENO, P - IVIA, VALENCIA, SPAIN
item SU, H - NATIONAL TAIWAN UNIV.
item Garnsey, Stephen
item LEE, R - UNIV. OF FLORIDA
item NIBLETT, C - UNIV. OF FLORIDA

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/8/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Citrus tristeza virus (CTV) is an economically important virus that affects citrus worldwide and is the object of intensive research. One goal has been to improve detection procedures for CTV through development of monoclonal antibodies. A number of these have now been produced and some react to only select isolates while others react to nearly all isolates. This indicates that some sites on the virus coat protein are common to nearly all isolates while others are not. Removing the amino end of the virus coat proteiny eliminatesy reactivity to certain monoclonals. To better understand the serological properties of CTV, sequencing studies and mutation analysis were done to locate they amino acids in the coat protein involved in the reaction to the monoclonal antibody 3DF1, a widely used monoclonal that is marketed commercially for detection of CTV. Comparisons with data from similar studies done with the strain-selective monoclonal MCA13 show that they sites on the coat protein that control reaction to each monoclonal are distinct. The reaction site for MCA13 is at mid molecule while the 3DF1 reaction site is at the amino end of the molecule. This explains why coat proteins that lose even a few amino acids at the amino end do not react to 3DF1. Changing a single amino acid in the reaction site of the coat protein can abolish activity. Use of intact virus coat protein is also important when using this monoclonal for virus testing.

Technical Abstract: The monoclonal antibody (MAb) 3DF1 is the first commercially available citrus tristeza closterovirus (CTV)-specific MAb. It detects a broad spectrum of CTV isolates from various parts of the world. To precisely map the antigenic determinant recognized by 3DF1, the capsid protein (CP) genes of four 3DF1-nonreactive isolates were cloned as complementary DNA and their nucleotide sequences determined. Comparison of the deduced CP sequences of the four nonreactive isolates with those of previously sequenced 3DF1-reactive isolates revealed differences at three positions near their amino terminal ends. The amino acids Asp-2, Lys-13, and Phe-28 conserved in all the 3DF1-reactive isolates, but they were replaced by Gly, Thr/Asp, and Tyr, respectively, in the CPs of the nonreactive isolates. Site-specific mutations were introduced into the cloned CP genes of the 3DF1-nonreactive isolate B215 and the 3DF1-reactive isolate T36. The serological reactivities of the wild-type and mutant CPs of B215 and T36 expressed as recombinant fusion proteins in Escherichia coli were evaluated by Western blot analysis. A point mutation (A > G) resulting in an Asp > Gly change at amino acid position 2 of the CP of isolate T36 abolished the reactivity with the Mab. Reverse mutation resulting in a Gly > Asp change at the same position conferred reactivity on the CP of the nonreactive B215 isolate. The implications of the observed antigenic diversity on virus detection are discussed.