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. 2022 Jul 7:13:923256.
doi: 10.3389/fmicb.2022.923256. eCollection 2022.

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization

Elena Smertina  1   2   3 , Adam J Carroll  4 , Joseph Boileau  4 , Edward Emmott  5 , Maria Jenckel  1 , Harpreet Vohra  6 , Vivien Rolland  7 , Philip Hands  7 , Junna Hayashi  8 , Matthew J Neave  9 , Jian-Wei Liu  10 , Robyn N Hall  1   3 , Tanja Strive  1   3 , Michael Frese  1   2
Affiliations

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization

Elena Smertina et al. Front Microbiol. .

Abstract

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they 'chaperone' p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

Keywords: Caliciviridae; SILAC; heat shock protein; non-structural protein; viroporin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic representation of a typical lagovirus genome. Both genomic (Top) and subgenomic RNAs (Bottom) contain two ORFs, are covalently linked to the viral protein VPg at the 5′-end, and are polyadenylated at the 3′-end (An). The genomic RNA encodes non-structural (shown in white and pink) and structural proteins (shown in gray), whereas the subgenomic RNA encodes only structural proteins. The coding sequence for p23 (224 amino acid residues) is shown in pink; two copies are shown to indicate that p23 dimerizes; cleavage sites at the N-terminus (E, glutamine and G, glycine) and C-terminus (E, glutamine and D, aspartic acid) are indicated; HR, hydrophobic region.
FIGURE 2
FIGURE 2
Dimerization of p23 depends on disulfide bond formation. (A) Recombinant FLAG-tagged p23 (wt, wild-type) expressed in transiently transfected RK-13 cells was eluted from an anti-FLAG resin with reducing or non-reducing Laemmli buffer containing β-mercaptoethanol (+β-me) or without β-mercaptoethanol (–β-me), respectively. Affinity purified p23 was separated by SDS-PAGE and analyzed by Western blotting using anti-FLAG antibodies; bands corresponding to monomeric p23 and dimeric forms can be observed. Untransfected RK-13 cell lysate was subject to immunoprecipitation as a negative control. The position of molecular mass standards is shown at the right. (B) RHDV/GI.1 p23 (NC_001543.1) protein sequence; the three cysteine residues at positions 41, 207, and 212 are highlighted using blue circles; and amino acids truncated in variant FLAG:p23 Δ146–224 are indicated using blue square brackets. (C) A consensus sequence and identity plot were generated using 567 lagovirus p23 sequences; the positions of the cysteine residues at position 41, 207, and 212 are indicated above the plot. The percentage identity at each amino acid position is indicated by column height and color (green means completely conserved, olive means less than complete identity, and red refers to a low identity). (D) Variants with C-terminal cysteine substitutions. Dimer formation of p23 wild-type (wt) and variants with cysteine-to-serine substitutions at positions 207 (C207S) or 212 (C212S) or both, ‘double’ was not affected under non-reducing conditions; the possible formation of a trimer is suggested by a weak band. (E) Dimers did not form between p23 variants with a cysteine-to-serine substitution at position 41 or in variants that had all cysteines substituted to serine (‘triple’); higher-order oligomers, trimers, and tetramers are seen in non-reducing conditions. (F) Unboiled samples were analyzed in a gel that did not contain SDS. Under these conditions, the C41S variant did not form dimers and both the wt protein and the C41S variant failed to form higher-order oligomers. In panel D, 10-μl samples were loaded per lane, and in E and F, 20-μl samples were loaded per lane (to enhance the detection of higher- order oligomers). (G) The truncated p23 Δ146–224 variant without transmembrane helices was not detected in either monomeric or dimeric form.
FIGURE 3
FIGURE 3
Prediction of transmembrane helices and disordered regions using in silico sequence analyses. (A–D) p23 protein sequences from Rabbit haemorrhagic disease virus (RHDV/GI.1; NC_001543.1), (E–H) Rabbit calicivirus (RCV/GI.4; MF598302.1), and (I–L) European brown hare syndrome virus (EBHSV/GII.1; NC_002615.1). (A,C,E,G,I,K) PSIPRED secondary structure prediction with MEMSAT-SVM algorithm and Kyte-Doolittle hydropathy plot revealed the presence of two putative transmembrane helices in the p23 proteins of all lagoviruses examined. The helices (‘helix 1’ and ‘helix 2’) are shown as striped boxes. (D,H,L) PONDR plot shows predicted disordered and ordered regions; the strength of the prediction is indicated by the score on the y-axis (>0.5 are considered disordered). (B,F,J) HeliQuest was used to generate a wheel diagram for the amino acid sequence of ‘helix 2.’ Arrows represent direction and magnitude of the hydrophobic moment. Note that the orientation of p23 within the membrane is not clear.
FIGURE 4
FIGURE 4
Transiently expressed recombinant p23 localizes to the ER. RK-13 cells were transfected with FLAG-tagged p23 (RHDV), incubated overnight, fixed, immunostained, and analyzed by confocal microscopy. (A–C) The ER marker calnexin (green) and FLAG-tagged p23 (magenta) were detected by immunofluorescence using anti-calnexin and anti-FLAG antibodies, respectively. (E–H) The cytoskeleton protein α-tubulin (green) and FLAG-tagged p23 (magenta) were detected by immunofluorescence using anti-tubulin and anti-FLAG antibodies, respectively. (I–L) The cell periphery lacks p23 expression and therefore this area was used to estimate the threshold for Rr. (D,H,L) The cytofluorograms show the distribution of green and magenta pixels in the cytoplasm (areas under investigation are bounded by a green line); Rr, Pearson correlation coefficient and Col. rate (%), colocalization rate. (M,N) Boxplots represent the distribution of Rr and colocalization rates for seven individual cells (n = 7), respectively. Each cell is depicted as a black dot, the median for each group is shown as a black line inside the boxes, the position of the mean is shown as a red diamond, and the value of mean is given in red numbers. Significance was analyzed using one-way ANOVA followed by Tukey’s honest significant difference test; adjusted p-values are shown above the plots; plots were produced using ggplot2 (Gómez-Rubio, 2017).
FIGURE 5
FIGURE 5
Heat shock proteins interact with p23 but not the viral polymerase. (A,B) A quantitative proteomics approach (SILAC) was used to identify cellular interaction partners of p23 and the RNA-dependent RNA polymerase (RdRp) of RHDV, respectively. Proteins identified in at least two out of three biological replicates were included in the analysis. Cellular proteins significantly enriched in comparison to an internal GFP control are shown as blue dots; the viral bait proteins p23 and RdRp are highlighted in pink. Significance was analyzed using a one-sample t-test with an unadjusted p-value of <0.05 and a log2 fold-change > 1. Heat shock proteins Hsp70 and Hsp90 were identified as p23 interactors (A), and one or more BRO1 domain-containing proteins were identified as RdRp interactors (B). (C) Western blot analysis of immunoprecipitated complexes isolated from transfected cells expressing recombinant FLAG:p23 or FLAG:RdRp (p23-IP and RdRp-IP, respectively). Aliquots of affinity purified complexes were separated by SDS-PAGE and analyzed by Western blot using anti-FLAG and anti-Hsp70 antibodies. Hsp70 was detected in p23 but not in RdRp expressing cells (we did not attempt to detect Hsp110 or BRO1 domain-containing proteins).
FIGURE 6
FIGURE 6
Ca2+ transmembrane transport pathways are altered in the liver of RHDV2- infected rabbits. A gene ontology (GO) enrichment analysis was conducted using the list of differentially expressed genes from RNA sequencing. (A) The graph induced by the top 5 GO terms identified by the weight01 algorithm (Alexa et al., 2006); the square shape indicates the most significant GO term. (B) The graph induced by the top 5 GO terms identified by weight01 algorithm with Fisher exact test also identified pathways associated with viral infection and regulation of reactive oxygen species generation. Box colors represents the relative significance of GO terms, from white (least significant) to red (most significant).
FIGURE 7
FIGURE 7
Flow cytometry Ca2+ flux measurements. Mean fluorescence intensity was measured for 25,000 cells in each condition, with three replicates per condition. Boxplots represent the distribution of mean fluorescence intensity value. Median values are shown as a horizontal line and mean values as a red diamond. The results from four independent experiments were normalized and combined. Significance was analyzed using one-way Kruskal-Wallis test and pairwise Wilcoxon rank test with multiple testing correction; adjusted p-values are shown above the plot; the plot was produced using ggplot2 (Gómez-Rubio, 2017); n.s., not significant (p > 0.05).
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