|Hein, Gary - UNI OF NE|
Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 8, 2006
Publication Date: July 5, 2006
Citation: Stenger, D.C., Hein, G.L., French, R.C. 2006. Nested deletion analysis of Wheat streak mosaic virus HC-Pro: Mapping of domains affecting polyprotein processing and eriophyid mite transmission. Virology volume 350: 465-474. Interpretive Summary: Functions of the Wheat streak mosaic virus (WSMV) HC-Pro gene were evaluated using mutant WSMV genomes in which portions of the HC-Pro gene were deleted. WSMV mutants in which two-thirds of the HC-Pro gene was removed retained the ability to systemically infect wheat plants and were able to process viral proteins through proteinase activity. However, WSMV mutants in which three-fourths of the HC-Pro gene was removed were unable to systemically infect plants and also unable to process viral proteins. These results indicated that proteinase functions of WSMV HC-Pro mapped to the carboxy-terminal end of the HC-Pro protein and that HC-Pro mediated processing of viral proteins was necessary for infection. In contrast, small deletions in the amino-terminus of HC-Pro eliminated transmission of WSMV by the wheat curl mite. Collectively, these results indicate that vector transmission and protein processing functions of WSMV HC-Pro are distinct and located in separate, domains of the protein.
Technical Abstract: A series of in frame and nested deletion mutations which progressively removed 5’-proximal sequences of the Wheat streak mosaic virus (WSMV) HC-Pro coding region (1152 nucleotides) was constructed and evaluated for pathogenicity to wheat. WSMV HC-Pro mutants with 5’-proximal deletions of 12 to 720 nucleotides systemically infected wheat. Boundary sequences flanking the deletions were stable and unaltered by passage through plants for all deletion mutants except HCD12 (lacking HC-Pro codons 3-6) which exhibited strong bias for G to A substitution at nucleotide 1190 in HC-Pro codon 2 (aspartic acid to asparagine). HC-Pro mutants with 5’-proximal deletions of up to 720 nucleotides retained autoproteolytic activity in vitro. In contrast, 5’-proximal deletion of 852 nucleotides of the HC-Pro coding region (HCD852) abolished both infectivity and in vitro proteolytic activity. These results confirm that the proteolytic domain of WSMV HC-Pro resides within the carboxy-terminal third of the protein and includes the cysteine proteinase motif (GYCY) conserved among four genera of the family Potyviridae. Inoculation of wheat with HC-Pro deletion mutants also bearing the GUS reporter gene revealed that HCD852 was unable to establish primary infection foci in inoculated leaves, indicating that processing of the P3 amino-terminus was essential. Deletion of as few as 24 nts 5’-proximal nucleotides of HC-Pro (codons 3-10) eliminated transmission by the eriophyid mite vector Aceria tosichella Keifer. Collectively, these results demonstrated similar organization of proteinase and vector transmission functional domains among divergent HC-Pro homologues encoded by potyviruses and tritimoviruses.