[KIL-d] Protein Element Confers Antiviral Activity via Catastrophic Viral Mutagenesis  Genjiro Suzuki, Jonathan S. Weissman, Motomasa Tanaka  Molecular Cell  Volume 60, Issue 4, Pages 651-660 (November 2015) DOI: 10.1016/j.molcel.2015.10.020 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 60, 651-660DOI: (10.1016/j.molcel.2015.10.020) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 A Mutation in Killer Toxin Gene in M1 Viral Genome Causes Defective Killer Phenotypes in [KIL-d] Yeast (A) A proteinaceous aggregate is an infectious agent in [KIL-d] yeast. Infectivity of WT yeast with pellets of [KIL-d] (dark gray) and [PSI+] (light gray) yeast extracts after various treatments, including proteinase K, nucleases and UV, or boil. (B) RT-PCR analysis shows that M1 (upper) and L-A dsRNA virus (middle) as well as rRNA as a control (lower) were retained in three [KIL-d] yeast strains (1-2B, 2-1D, 1144). Results are also shown for WT (non-[KIL-d]) K+R+ and K−R− strains as controls. (C) RT-PCR analysis shows that [KIL-d] yeast strains contain similar levels of preprotoxin mRNA to WT K+R+ strains (upper). mRNA levels of TUB1 were also shown as a control (lower). (D) Killer activity and expression of killer toxin in three [KIL-d] strains. Concentrated killer toxin secreted from WT or [KIL-d] yeast is spotted on sterilized filter discs on the K−R− yeast lawn (upper). Secreted killer toxin is detected by SYPRO ruby staining (middle) or western blotting with a polyclonal anti-α killer toxin antibody (lower). (E) A subunit structure of preprotoxin and mutation sites in three [KIL-d] yeast strains. (F) The mutations in three [KIL-d] strains eliminate killer activity. K-R- yeast cells carrying empty or pRS426 episomal plasmids encoding a WT or mutant preprotoxin gene under the CUP1 promoter are tested for killer (K) (upper) and resistant (R) phenotypes (lower). See also Figure S1 and Tables S1 and S3. Molecular Cell 2015 60, 651-660DOI: (10.1016/j.molcel.2015.10.020) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 Both the Relative Ratio of Mutant to WT Virus and the Mutation Site in the Killer Viral Genome Determine Defective Killer Phenotypes in [KIL-d] Yeast (A–D) Killer phenotypes of four meiotic spores (right, A–D) derived from K+R+ diploids (center) formed by the mating between K+R+ WT (left, top) and K−R+ [KIL-d] (left, bottom). The A146C mutation confers K− or Kw phenotypes, depending on the relative ratio of the mutant to WT virus. (B) Kw phenotypes of two examples of four meiotic spores from two tetrad sets (center) and phenotypes of K+ and K− yeasts as controls (right). The T787C mutation confers Kw phenotypes even though only this mutant virus exists in yeast. (C) Yeast cells expressing A146C mutant killer toxin show no killer activity, whereas those expressing T787C mutant exhibit weak killer activity. (D) A schematic model of the regulation of defective killer phenotypes in [KIL-d] yeast. Upon sporulation, mutant viruses are randomly segregated into meiotic spores, and the relative ratio of the mutant to WT virus in yeast determines the extent of defective killer phenotypes. A mutation site in the killer toxin gene is another critical determinant; the A146C and T787C nucleotide mutation intrinsically cause K− and Kw phenotypes, respectively. In the figures, halo diameters relative to K+ control are indicated under the corresponding phenotypic designations. See also Tables S1 and S3. Molecular Cell 2015 60, 651-660DOI: (10.1016/j.molcel.2015.10.020) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 Virus Exchange Assay Reveals the [KIL-d] Element Selectively Accumulates Mutant Killer Virus and Shows Non-Mendelian Cytoplasmic Inheritance (A) A scheme of “virus exchange” assay. After several passages, non-[KIL-d] WT yeast (top) always show K+ phenotypes (top, right), whereas the yeast cells harboring the [KIL-d] element (bottom) display K− or Kw phenotypes (bottom, right). The following sequence analysis revealed de novo mutations in the viral genome in K− and Kw [KIL-d] yeast. (B) A selective growth advantage of [KIL-d] K−R+ yeast over WT K+R+ yeast. The round area caused by cell killing was decreased after passages, suggesting a selective growth advantage of [KIL-d] K−R+ yeast over WT K+R+ yeast. (C) A scheme for examination of non-Mendelian inheritance of the [KIL-d] element. K−R− [KIL-d] diploids lacking M1 virus are sporulated, and each meiotic spore is crossed with non-[KIL-d] K+R+ WT yeast. The resulting K+R+ diploids are passaged on YPAD plates, and their killer phenotypes and viral genome sequences were analyzed. (D) A representation of the data achieved using the scheme illustrated in (C) for examination of non-Mendelian inheritance of the [KIL-d] element. All of the four meiotic spores showed Kw phenotypes after passages (center). Subsequent sequence analysis revealed a de novo mutation in the killer toxin gene in the four meiotic spores (right). Note that the mutation in each spore is distinct each other and also different from the mutation (C295T) in the parental [KIL-d] diploid. (E) Non-Mendelian inheritance of the [KIL-d] element. Thirteen diploids were examined for appearance of viral mutation in their meiotic spores, and 11 diploids showed mutations in viral genome. (F) A schematic diagram of cytoduction experiments. Cytoductants are passaged on YPAD plates and their killer phenotypes and sequences of the killer toxin gene are analyzed. (G) A summary of the cytoduction experiments. In the figures, halo diameters relative to K+ control are indicated under the corresponding phenotypic designations. See also Figures S2 and S3 and Tables S1–S3. Molecular Cell 2015 60, 651-660DOI: (10.1016/j.molcel.2015.10.020) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 Deep Sequence Analysis Reveals the [KIL-d] Element Increases the Rate of De Novo Mutations in the Killer Viral Genome (A) G10 and G11 yeast show Kw and K− phenotypes, respectively, due to the introduction of T229C mutation into the killer toxin gene and selective accumulation of the mutant virus. Halo diameters relative to K+ control are indicated under the corresponding phenotypic designations. (B) A gradual increase in the frequency of mutant (T229C) virus relative to WT virus in [KIL-d] yeast over passages. A population (%) of WT (T229) killer toxin sequence in M1 viral genome was analyzed in different generations of WT and [KIL-d] yeast by deep sequencing. (C) [KIL-d] yeast show an increase in the rate of de novo mutation in the killer toxin gene in M1 viral genome. A frequency of WT killer toxin sequence in the M1 viral genome in WT and [KIL-d] yeast was determined by deep-sequence analysis. Error bars represent mean + SEM (n = 5). ∗∗∗p < 0.001. (D) The mutation rate of yeast chromosomal TUB1 gene is similar between WT and [KIL-d] yeast by deep-sequence analysis. Error bars represent mean + SEM (n = 2), p = 0.32. (E) The induction frequency of cycloheximide-resistant colonies is similar between WT and [KIL-d] yeast. Error bars represent mean + SEM (n = 3), p = 0.42. n means the number of independent colonies examined. See also Figure S4 and Tables S1, S3, and S4. Molecular Cell 2015 60, 651-660DOI: (10.1016/j.molcel.2015.10.020) Copyright © 2015 Elsevier Inc. Terms and Conditions