Boys IN et al. (2021), Functional-genomic analysis reveals intraspecie...

XB-ART-58124

PLoS Genet 2021 May 01;175:e1009578. doi: 10.1371/journal.pgen.1009578.

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Functional-genomic analysis reveals intraspecies diversification of antiviral receptor transporter proteins in Xenopus laevis.

Boys IN , Mar KB , Schoggins JW .

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The Receptor Transporter Protein (RTP) family is present in most, if not all jawed vertebrates. Most of our knowledge of this protein family comes from studies on mammalian RTPs, which are multi-function proteins that regulate cell-surface G-protein coupled receptor levels, influence olfactory system development, regulate immune signaling, and directly inhibit viral infection. However, mammals comprise less than one-tenth of extant vertebrate species, and our knowledge about the expression, function, and evolution of non-mammalian RTPs is limited. Here, we explore the evolutionary history of RTPs in vertebrates. We identify signatures of positive selection in many vertebrate RTP clades and characterize multiple, independent expansions of the RTP family outside of what has been described in mammals. We find a striking expansion of RTPs in the African clawed frog, Xenopus laevis, with 11 RTPs in this species as opposed to 1 to 4 in most other species. RNA sequencing revealed that most X. laevis RTPs are upregulated following immune stimulation. In functional assays, we demonstrate that at least three of these X. laevis RTPs inhibit infection by RNA viruses, suggesting that RTP homologs may serve as antiviral effectors outside of Mammalia.

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Species referenced: Xenopus laevis
Genes referenced: bcl6 dynlt2b masp1 ndrg1 rgmb rtp3 rtp3a.2 rtp3a.3 rtp3b rtp3d rtp3e.1 rtp3e.3 rtp3e.4

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	Fig 1. Evolutionary survey of vertebrate RTPs. A) A Maximum Likelihood tree for 303 vertebrate RTPs was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-99002.44) is shown. 500 bootstrap replicates were performed to test robustness of the ML tree, and branches are colored by percentage of bootstrap replicates which reflect this topology. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.8741)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Asterix (*) denotes the bearded dragon RTP that clusters with anamniote RTPs. Tree with species and gene names included is available in File B in S1 File. B) Left: A Maximum Likelihood tree for 24 representative fish RTPs was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-6168.52) is shown. 500 bootstrap replicates were performed to test robustness of the ML tree, and branches are colored by percentage of bootstrap replicates which reflect this topology. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 2.2558)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Right: A TimeTree describing the evolutionary relationships between the different fish species. Yellow squares denote inferred duplication events. Question marks denote ambiguous duplication events.
	Fig 2. RTP locus synteny and evolutionary analysis of X. laevis RTPs. A) Left: A TimeTree [51] of representative RTP-containing vertebrate species. Right: chromosomal arrangement of RTPs. Common proximal genes and chromosome numbers, when available, are indicated. Numbering of RTP genes, when indicated, matches the numbering used in Fig 1B, File A in S1 File, and S4 Table. Scaffold denotes contigs that are not mapped to chromosomes. B) Free-ratio analysis of X. laevis RTPs. Annotated values indicate the dN/dS of each branch. Branches with dN/dS > 1 are marked in red. C) A TimeTree for X. laevis RTPs inferred using the Reltime method [52,53] and the Tamura-Nei model [54]. Divergence times were estimated by calibration by designating mammalian RTP4 sequences as an outgroup. The estimated log likelihood value is -9335.49. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 2.9731)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 6.22% sites). White rectangles denote 95% CI of divergence times. Asterix (*) indicates estimated L and S genome divergence time. Dagger (†) indicates estimated L and S genome hybridization time.
	Fig 3. Identification of poly(I:C)-induced Xenopus laevis RTPs. A) qPCR was used to assess MX-family GTPase induction following transfection of A6 cells with poly(I:C) over a time course. As designed, primers are capable of detecting all X. laevis MX-family transcripts. Points represent the mean ± SD of n = 3 biological replicates. B) Volcano plots for A6 cells treated with poly(I:C) for 6, 12, or 24 hours, relative to mock transfection. Differentially-expressed genes (padj < 0.05, fold change > 4) are indicated. Data are derived from three biological replicates. C) PCA analysis of gene expression kinetics. Ten subclusters were identified. D) Kinetic profiles of top-induced, ISG-rich clusters (SC4 and SC5). Lines represent individual genes. Colored/bolded line indicates mean profile for the cluster. E) Annotation of genes in SC4 and SC5. ISGs annotated per inducibility in other datasets, as detailed in methods. Other: non-ISGs with clear annotation. Undetermined: insufficient homology to determine identify of transcript. F) Induction of RTP family members following poly(I:C) transfection. Plotted points indicate when significant enrichment over background was observed.
	Fig 4. Identification of antiviral Xenopus laevis RTPs. A) Huh7.5 cells ectopically expressing the indicated RTPs or a vector control were infected with a panel of RNA viruses at an MOI of 0.5 to 1. Cells were harvested after the completion of approximately one replication cycle and percent infection was assessed by flow cytometry. Cells represent the mean of n = 2 biological replicates, normalized to control. Raw data: S2B Fig. B) Huh7.5 cells ectopically expressing the indicated RTPs or a vector control were infected with a panel of flaviviruses at an MOI of 0.5 to 1. Cells were harvested after the completion of approximately one replication cycle and percent infection was assessed by flow cytometry. Bars represent the mean ± SD of n = 3 biological replicates. C) Data from Fig 4B, represented as percent of control. D) Huh7.5 cells ectopically expressing either RTPγS or a vector control were transfected with a YFV-RLuc replicon and protein production was assessed by luminometry. Bars represent the mean ± SD of n = 3 biological replicates. E) Huh7.5 cells ectopically expressing the indicated RTPs or a vector control were infected with a panel of picornaviruses at an MOI of 0.5 to 1. Cells were harvested after the completion of approximately one replication cycle and percent infection was assessed by flow cytometry. Bars represent the mean ± SD of n = 3 biological replicates. F) Data from Fig 4E, represented as percent of control. G) Huh7.5 cells expressing HA.RTPγS, HA.RTPγS.C151A, or a vector control were infected with YFV-17D for 24 hours. Cells were harvested after the completion of approximately one replication cycle and percent infection was assessed by flow cytometry. Bars represent the mean ± SD of n = 3 biological replicates. H) Huh7.5 cells expressing HA.RTPαL1, HA.RTPαL1.C154A, or a vector control were infected with MenV for 6 hours. Cells were harvested after the completion of approximately one replication cycle and percent infection was assessed by flow cytometry. Bars represent the mean ± SD of n = 3 biological replicates. I) Huh7.5 cells expressing HA.RTPγS or a vector control were infected with YFV (MOI of 2) for 24h. CLIP-qPCR identified RNA bound by HA.RTPγS. UV: UV crosslinked HA.RTPγS cells. NoUV: non-crosslinked HA.RTPγS cells. Vector: UV crosslinked vector control cells. Bars: mean ± SD of n = 3 biological replicates.
	S1 Fig. A)Collapsed maximum-likelihood trees for N-terminal and C-terminal fish RTP gene fragments, relative to a predicted recombination breakpoint (nt383). Numbers indicate the percentage of 50 bootstrap replicates for which the associated taxa clustered together. Nodes with <50% bootstrap confidence were collapsed. Trees were generated as in Fig 1B.
	S2 Fig. A) Huh7.5 cells ectopically expressing the indicate RTPs or a vector control were infected with a panel of viruses at an MOI of 0.5 to 1. Cells were harvested after the completion of approximately one replication cycle and percent infection was assessed by flow cytometry. Cells represent the mean of n = 2 (HCV) or n = 3 (all other viruses) biological replicates, normalized to control. B) Western blot of ectopically-expressed RTPs. For each screen replicate, an additional well of cells was harvested alongside infections for protein expression analysis. Blot is representative of n = 3 replicates. C) Representative micrograph of paRTP3-expressing Huh7.5 cells and vector control cells. Scale bar: 20μm. D) Huh7.5 cells expressing the indicated construct were seeded at 10,000 cells per well on a 96-well plate one day prior to assay. The day of WST assay, WST reagent (Takara) was added (10uL in 100uL media) and cells were incubated for two hours, after which visual absorbance was assayed. In parallel, cell density was measured using Cell Titer-Glo (Promega) and luminometry.
	S3 Fig. A) Western blot of ectopically-expressed RTPs. Vertical dotted line indicates splice site for two independently-run gels. For each screen replicate, an additional well of cells was harvested alongside infections for protein expression analysis. Blot is representative of n = 2 replicates. B) Raw data related to Fig 4A. Huh7.5 cells ectopically expressing the indicate RTPs or a vector control were infected with a panel of RNA viruses at an MOI of 0.5 to 1. Cells were harvested after the completion of approximately one viral life cycle and percent infection was assessed by flow cytometry. Bars represent the mean ± SD of n = 2 biological replicates. C) Cartoon representation of X. laevis RTPαL1 and RTPγS with paRTP4 as a reference. Yellow boxes denote N-terminal CXXC motifs, the third of which was targeted for disruption by site-directed mutagenesis to generate ZFD RTPs. D) Western blot of ectopically-expressed RTPγS and RTPγSZFD. Blot is representative of n = 2 replicates. E) Huh7.5 cells expressing HA.RTPαL1 or a vector control were infected with MenV (MOI of 2) for 6h. CLIP-qPCR identified RNA bound by HA.RTPαL1. UV: UV crosslinked HA.RTPαL1 cells. NoUV: non-crosslinked HA.RTPαL1 cells. Vector: UV crosslinked vector control cells. Bars: mean ± SD of n = 3 biological replicates.

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Behrens, Members of RTP and REEP gene families influence functional bitter taste receptor expression. 2006, Pubmed