Expression of a synthetic rust fungal virus cDNA in yeast

Authors: Cooper, Bret; Campbell, Kimberly; Garrett, Wesley

Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2015
Publication Date: 10/23/2015
Publication URL: https://handle.nal.usda.gov/10113/62559
Citation: Cooper, B., Campbell, K., Garrett, W.M. 2015. Expression of a synthetic rust fungal virus cDNA in yeast. Archives of Virology. 161:111-123.

Interpretive Summary: Mycoviruses are viruses that infect fungi. Some mycoviruses slow the growth of fungi, and scientists have exploited mycoviruses to slow the growth of fungi to reduce the effects of fungal disease in plants. Recently, a mycovirus was found in the fungus that causes rust disease in soybeans. We sequenced the genome of this virus and found that it was similar to a yeast virus. Because the soybean rust fungus cannot be cultured outside of a plant, we tested a clone of the soybean rust virus in yeast. Rust virus RNA was expressed in yeast and viral coat protein and polymerase proteins were made in yeast, but the rust virus did not replicate independently in yeast. Nevertheless, the proof of RNA transcription and protein translation from the clone in yeast are steps toward validating the effects of the rust mycovirus on cell growth. Further development of a surrogate biological system for the study of rust mycoviruses is necessary, and derivative data may help government, university and other scientists develop biological control methods against rust diseases.

Technical Abstract: 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 Totiviridae family, 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 validating PpV2383 as 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.