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Expression of a synthetic rust fungal virus cDNA in yeast

Bret Cooper, Kimberly B. Campbell, Wesley M. Garrett
Archives of Virology 2016 v.161 no.1 pp. 111-123
Phakopsora pachyrhizi, RNA-directed RNA polymerase, Saccharomyces cerevisiae virus L-A, biological control, coat proteins, complementary DNA, double-stranded RNA, genome, mass spectrometry, molecular cloning, mycoviruses, open reading frames, pathogenicity, phylogeny, soybean rust, transcription (genetics), translation (genetics), virus replication, yeasts
Mycoviruses are viruses that infect fungi. Recently, mycovirus-like RNAs were sequenced from the fungus Phakopsora pachyrhizi, the causal agent of soybean rust. One of the RNAs appeared to represent a novel mycovirus and was designated Phakopsora pachyrhizi virus 2383 (PpV2383). The genome of PpV2383 resembles Saccharomyces cerevisiae virus L-A, a double-stranded (ds) RNA mycovirus of yeast. PpV2383 encodes two major, overlapping open reading frames with similarity to gag (capsid protein) and pol (RNA-dependent RNA polymerase), and a -1 ribosomal frameshift is necessary for the translation of a gag-pol fusion protein. Phylogenetic analysis of pol relates PpV2383 to members of the family Totiviridae, including L-A. Because the obligate biotrophic nature of P. pachyrhizi makes it genetically intractable for in vivo analysis and because PpV2383 is similar to L-A, we synthesized a DNA clone of PpV2383 and tested its infectivity in yeast cells. PpV2383 RNA was successfully expressed in yeast, and mass spectrometry confirmed the translation of gag and gag-pol fusion proteins. There was, however, no production of PpV2383 dsRNA, the evidence of viral replication. Neither the presence of endogenous L-A nor the substitution of the 5’ and 3’ untranslated regions with those from L-A was sufficient to rescue replication of PpV2383. Nevertheless, the proof of transcription and translation from the clone in vivo are steps toward confirming that PpV2383 is a mycovirus. Further development of a surrogate biological system for the study of rust mycoviruses is necessary, and such research may facilitate biological control of rust diseases.