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, 10 (1), 359

A Viral Expression Factor Behaves as a Prion

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A Viral Expression Factor Behaves as a Prion

Hao Nan et al. Nat Commun.

Abstract

Prions are proteins that can fold into multiple conformations some of which are self-propagating. Such prion-forming proteins have been found in animal, plant, fungal and bacterial species, but have not yet been identified in viruses. Here we report that LEF-10, a baculovirus-encoded protein, behaves as a prion. Full-length LEF-10 or its candidate prion-forming domain (cPrD) can functionally replace the PrD of Sup35, a widely studied prion-forming protein from yeast, displaying a [PSI+]-like phenotype. Furthermore, we observe that high multiplicity of infection can induce the conversion of LEF-10 into an aggregated state in virus-infected cells, resulting in the inhibition of viral late gene expression. Our findings extend the knowledge of current prion proteins from cellular organisms to non-cellular life forms and provide evidence to support the hypothesis that prion-forming proteins are a widespread phenomenon in nature.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
LEF-10-EGFP forms aggregates in infected insect cells. a Diagram of the recombinant baculovirus sensor system functions by transmitting the aggregation state of LEF-10-EGFP to downstream gene reporter mCherry outputs. The cassette fragment was integrated into the polh locus of a lef-10 knock-out Bacmid via site-specific homologous recombination (Supplementary Fig. 1a). b LEF-10-EGFP exists in two distinct forms: as aggregated (Cell 1) or as diffuse (Cell 2) forms in infected insect cells (MOI = 3) at 60 hpi (left). The mCherry reporter protein representing late gene expression can only be detected in cells harboring diffuse LEF-10-EGFP (right). c The distribution of LEF-10-EGFP in the two cells (b, left) was analyzed by ImageJ software. The proportion of the pixels of a certain brightness to all the pixels harbored by one cell is defined as “percentage of pixel”. Higher fluorescence intensity, which leads to the curve shifting to the right in Cell 1, indicates the aggregation of LEF-10-EGFP and exhausts the pool of non-aggregated LEF-10-EGFP which occurs in the lower fluorescence intensity areas
Fig. 2
Fig. 2
LEF-10 functionally replaces the prion domain of the yeast Sup35 protein. a Yeast ade1-14 cells expressing LEF-10-Sup35MC were spread on complete (1/4 YPD) medium. [LEF+] strains formed white colonies which were distinguishable from [lef] strains by the red pigment accumulated through the adenine biosynthetic pathway (top). Both phenotypes could be sustained during cell propagation (middle). [lef] strains spontaneously generated [LEF+] colonies at a very low frequency (bottom, a colony with the [LEF+] phenotype is indicated by an arrow) and these [LEF+] colonies displayed Ade+ (suppression of ade1-14) phenotype when tested directly on SD-Ade medium. b Comparable characteristics of LEF-10-Sup35MC and full-length Sup35 in yeast. Serial dilutions of [LEF+] and [lef] strains were spotted on the 1/4 YPD plate (the top panel), or medium lacking adenine (SD-Ade) on which only prion-containing i.e., [PRION+] strains suppressing the stop codon in the yeast ade1-14 allele could grow (the second panel). SDS-resistant aggregates in cell lysates of yeast strains expressing LEF-10-Sup35MC were examined by SDD-AGE (the third panel). The expression levels of full-length Sup35 and LEF-10-Sup35MC were examined by Western blot, probing with a Sup35C-specific antibody (the fourth panel). Endogenous phosphoglycerate kinase 1 (PGK1) was detected with a PGK1-specific antibody and served as a loading control (the bottom panel). [PSI+] and [psi-] strains were used as positive and negative controls
Fig. 3
Fig. 3
The [LEF+] phenotype in yeast is Hsp104-dependent. a Phenotypic curing of [LEF+] by guanidine hydrochloride (GdnHCl), an inhibitor of molecular chaperone Hsp104. Phenotypes of [PSI+], [psi-], [LEF+], and [lef-] colonies were, respectively, re-streaked on 1/4 YPD medium with or without 5 mM GdnHCl. b The [LEF+] strain phenotype is curable by deletion of the HSP104 gene. The [PSI+] strain was used as control
Fig. 4
Fig. 4
Identification of a candidate prion-forming domain (cPrD) in LEF-10. a Cartoon of LEF-10 and its truncations. The location of three conserved regions (C1, C2 and C3) and the amino acid sequence of the predicted cPrD are presented above. The ten highly conserved amino acids in C1 are colored in red. L21 is indicated by the inverted triangle. b Characterization of LEF-10 truncations in yeast. LEF-10, LEF-101-41 and LEF-1012-34 display similar characters to Sup35 on 1/4 YPD and SD-Ade medium, and by SDD-AGE. Western blot detected all the truncated LEF-10-Sup35MC expressing at similar levels as full-length Sup35. PGK1 was measured as the loading control. Strains with Sup35 and Sup35MC were used as positive and negative controls. c The [LEF+] phenotype of LEF-101-41 and LEF-1012-34 is curable by deletion of the HSP104 gene
Fig. 5
Fig. 5
Characterization of the aggregates of LEF-10 and LEF-10L21A in virus-infected Sf9 cells. a Fluorescence microscope images displayed that wild-type LEF-10 and LEF-10L21A fused with EGFP were driven by a tandem actin/p10 promoter (Supplementary Fig. 1b) and they could rescue the BacmidΔlef-10 (more mutants in Supplementary Fig. 4a). b Flow cytometry analysis revealed that the expression level of LEF-10L21A-EGFP was higher than that of LEF-10-EGFP. c Laser confocal microscopy images of over-expressed LEF-10-EGFP and LEF-10L21A-EGFP in infected Sf9 cells at 48 hpi (Related to Supplementary Fig. 4c). Both diffuse fluorescence and non-diffuse fluorescence could be observed in Sf9 cells expressing virus-encoded LEF-10-EGFP and LEF-10L21A-EGFP. Scale bar, 50 μm. d Percentage of cells which harbored puncta fluorescence formed by over-expressed LEF-10-EGFP or LEF-10L21A-EGFP were examined from 30 individual fields under laser confocal microscopy images. Bars represented mean ± SD. Two-tailed unpaired Student’s t-test was performed (****P ≤ 0.0001). Source data are provided as a Source Data file. e Laser confocal microscopy images of LEF-10-EGFP and LEF-10L21A-EGFP under the control of native lef-10 promoter (Supplementary Fig. 1e) in infected Sf9 cells at 24, 36 and 48 hpi, MOI = 10. Scale bar, 50 μm. f Percentage of cells which harbored puncta fluorescence formed by LEF-10-EGFP or LEF-10L21A-EGFP under the control of native lef-10 promoter (Supplementary Fig. 1e) were examined from 30 individual fields of laser confocal microscopy images at the indicated hours post-infection. Bars represented mean ± SD. Two-tailed unpaired Student’s t-test was performed (****P ≤ 0.0001). Source data are provided as a Source Data file. g SDD-AGE analysis of aggregates formed by LEF-10-EGFP-His and LEF-10L21A-EGFP-His fusion proteins in infected Sf9 cells at 48 hpi. Both chimeras were able to form such aggregates, albeit with different polymer size distribution. h Western blot analysis of aggregates formed by LEF-10-EGFP-His and LEF-10L21A-EGFP-His fusion proteins in infected Sf9 cells at 48 hpi. The high molecular-weight fraction remained in the stacking gel, and the low molecular-weight fraction and monomers were detected in the resolving gel. The LEF-10-EGFP-His and LEF-10L21A-EGFP-His proteins in g and h were detected using α-His antibody
Fig. 6
Fig. 6
The viral late gene regulation mediated by LEF-10. a Sf9 cells were infected in parallel with serial dilutions of recombinant baculoviruses encoding wild-type LEF-10 or the LEF-10L21A mutant at the indicated MOI. EGFP served as the reporter of late gene expression (Supplementary Fig. 2d). Infected Sf9 cells expressing EGFP were gated and counted at 36 hpi. Source data are provided as a Source Data file. b Laser confocal microscopy images of infected (MOI = 10) Sf9 cells containing virus-encoded LEF-10 and LEF-10L21A at 24, 36 and 48 hpi. The recombinant baculoviruses produced LEF-10-EGFP or LEF-10L21A- EGFP under the control of native lef-10 promoter and harbored a p10 promoter driven mCherry as a reporter for baculovirus late gene expression (Supplementary Fig. 1e). Hoechst stains the nuclei. Scale bar, 50 μm. c Model of how the LEF-10 endows prion-mediated regulation on late genes, resulting in the limitation of viral replication

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