Evolution of a Novel Antiviral Immune-Signaling Interaction by Partial-Gene Duplication

<div><p>The RIG-like receptors (RLRs) are related proteins that identify viral RNA in the cytoplasm and activate cellular immune responses, primarily through direct protein-protein interactions with the signal transducer, IPS1. Although it has been well established that the RLRs, RIG-I and MDA5, activate IPS1 through binding between the twin caspase activation and recruitment domains (CARDs) on the RLR and a homologous CARD on IPS1, it is less clear which specific RLR CARD(s) are required for this interaction, and almost nothing is known about how the RLR-IPS1 interaction evolved. In contrast to what has been observed in the presence of immune-modulating K63-linked polyubiquitin, here we show that—in the absence of ubiquitin—it is the first CARD domain of human RIG-I and MDA5 (CARD1) that binds directly to IPS1 CARD, and not the second (CARD2). Although the RLRs originated in the earliest animals, both the IPS1 gene and the twin-CARD domain architecture of RIG-I and MDA5 arose much later in the deuterostome lineage, probably through a series of tandem partial-gene duplication events facilitated by tight clustering of RLRs and IPS1 in the ancestral deuterostome genome. Functional differentiation of RIG-I CARD1 and CARD2 appears to have occurred early during this proliferation of RLR and related CARDs, potentially driven by adaptive coevolution between RIG-I CARD domains and IPS1 CARD. However, functional differentiation of MDA5 CARD1 and CARD2 occurred later. These results fit a general model in which duplications of protein-protein interaction domains into novel gene contexts could facilitate the expansion of signaling networks and suggest a potentially important role for functionally-linked gene clusters in generating novel immune-signaling pathways.</p></div

RIG-like receptor (RLR) and IPS1 CARD domains diversified in early deuterostomes.

<p>(A) We inferred the consensus phylogeny of RLR and IPS1 CARD domains using maximum likelihood and Bayesian inference (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). We report statistical confidence supporting key nodes on the phylogeny using maximum-likelihood SH-like aLRT (top) and Bayesian posterior probability (bottom); branch lengths are scaled to substitutions/site. Inferred gene and CARD duplication events are indicated by circles and stars, respectively. (B) We calculated BLAST e-values between all pairs of RLR and IPS1 CARD protein sequences and visualized sequence similarity using a force-directed network layout implemented in Cytoscape v2.8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.ref023" target="_blank">23</a>]. Nodes in the network represent individual sequences and are colored by CARD type. Edges indicate pairwise e-value calculations; e-values > 10⁻³ are not shown, to improve clarity. Nodes close to one another are more similar, based on e-value calculations, than nodes far away from one another. The central cluster of each RLR and IPS1 CARD domain is indicated by a dotted oval.</p

Association between Knowledge of Zika Transmission and Preventative Measures among Latinas of Childbearing Age in Farm-Working Communities in South Florida

Zika infection, an otherwise usually mild disease, is of serious public health concern due to the potential teratogenic effects of the virus. The incidence of Zika infection is difficult to document since it is mostly asymptomatic and detection of those carrying Zika is usually not possible. Currently, there is no vaccine for Zika; therefore, use of personal preventative measures is the only method of avoiding transmission. The aim of this study was to evaluate the association between knowledge of Zika transmission and the use of preventive measures among Latinas of childbearing age who lived in or near farm-working communities in South Florida. A secondary data analysis was performed on a cross-sectional study, sampling 100 Latina women aged 18&#8211;50 years. Sixty-nine percent demonstrated a high degree of knowledge of Zika transmission, and 68% were categorized as taking good preventative measures. Women with high knowledge were 5.86 times more likely to take good preventative measures than those with no knowledge (p-value = 0.05). Knowledge was associated with more preventative measures. Therefore, it is essential to further investigate this relationship in order to develop effective public health interventions for this population

Structure-based protein-protein docking suggests RIG-I CARD1 interacts with IPS1 CARD through a novel antiparallel α1-α3-α4 interface.

<p>We used ClusPro (A) and Dot2 (B) to predict the most likely structural orientation of human RIG-I CARD1 bound to IPS1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). We show the best-scoring complex (top panels) as well as the 20 top-scoring orientations (bottom panels) from each analysis. (C) We calculated the surface electrostatic charge (kT/e) of human RIG-I CARD1 and IPS1 CARD (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). Top panel displays IPS1 (left) and RIG-I CARD1 (right) in the same antiparallel α1-α3-α4 orientation as A and B. Arrows indicate regions of inferred shape and electrostatic complementarity. Gray Xs indicate locations of mutations examined in D. In the bottom panel, IPS1 is shown in mirror image and made 35% transparent; regions of electrostatic complementarity appear purple in the overlay (also highlighted by dotted ovals). (D) We created mutant RIG-I CARD1 (Glu66,67—Arg) and IPS1 (Arg66,67—Glu) and measured the affinities with which mutant proteins bind their wild-type partners. We plot the mean and standard error–log steady-state dissociation (pKd) and initial binding rate (pKm) of each interaction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s006" target="_blank">S6 Fig</a>). Wild-type RIG-I CARD1 and CASP9 interacting with wild-type IPS1 were used as positive and negative controls, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g001" target="_blank">Fig 1B</a>).</p

RIG-like receptor (RLR) and IPS1 CARD domains structurally diverged in early deuterostomes, and this structural divergence was associated with protein-coding adaptation.

<p>We inferred the 3-dimensional structures of ancestral RLR and IPS1 CARD domains at key points on the RLR-IPS1 CARD phylogeny (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g003" target="_blank">Fig 3</a>). We plot the electrostatic surface potential (kT/e) across the α1-α3-α4 surface of each CARD domain, with large acidic and basic patches indicated by dotted red and blue ovals, respectively. We additionally used phylogenetic techniques to infer the presence of adaptive protein-coding substitutions on each branch of the phylogeny (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). Stars indicate significant support for protein-coding adaptation on the indicated branch (<i>p</i><0.05 after correcting for multiple tests). Large dotted vertical line indicates the approximate time of RLR-IPS1 CARD proliferation in early deuterostomes.</p

Ancestral RIG-like receptor (RLR) CARDs bound the IPS1 CARD progenitor before the proliferation of RLR-IPS1 CARD domains in early deuterostomes, but did not bind the ancestral IPS1 CARD after it diverged from RLR CARDs.

<p>We resurrected ancestral RLR and IPS1 CARD domains and measured their binding kinetics in vitro (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s001" target="_blank">S1 Fig</a>). The simplified phylogeny at left indicates the ancestral proteins examined in this set of experiments, with the dotted vertical line indicating the approximate time of the RLR-IPS1 CARD proliferation in deuterostomes (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g005" target="_blank">5</a>). Colored circles indicate ancestral RLR CARDs, while gray circles indicate the ancestral IPS1 CARD progenitor before (A) and after (B) the deuterostome proliferation. We show the mean and standard error in–log-transformed steady-state dissociation (pKd) and initial binding rate (pKm) of in vitro kinetics assays, with dotted vertical line indicating approximate nonspecific binding (indicated by CASP9 negative control) and longer bars indicating tighter binding (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s016" target="_blank">S16 Fig</a>).</p

Deuterostome RIG-like receptors (RLRs) and IPS1 cluster with other potential CARD-signaling immune receptors in the <i>Branchiostoma floridae</i> genome.

<p>(A) We plot the physical location of each identified RLR and IPS1 gene along the assembled <i>B</i>. <i>floridae</i> chromosome (bottom). Other genes in the cluster are shown along the top. Functional domains are indicated by colors. Genes with significant BLAST hits to the <i>B</i>. <i>floridae</i> expressed sequence tag (EST) database are shaded according to e-value. (B) We measured the kinetics of <i>B</i>. <i>floridae</i> RIG-I CARD1+2 and MDA5 CARD1+2 domains binding to <i>B</i>. <i>floridae</i> IPS1 CARD in vitro (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s001" target="_blank">S1 Fig</a>). We plot the mean and standard error of–log-transformed steady-state dissociation (pKd) and initial binding rate (pKm), with human CASP9 used as a negative control. Dotted vertical line indicates approximate level of non-specific binding, with longer bars indicating tighter binding (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s012" target="_blank">S12 Fig</a>).</p

In the absence of ubiquitin, the RIG-like receptors (RLRs), RIG-I and MDA5, bind their signaling partner, IPS1, via a direct interaction between the first RLR CARD domain (CARD1) and the CARD domain of IPS1.

<p>(A) After binding viral RNA, RIG-I and MDA5 interact directly with IPS1 via CARD-CARD interactions. K63 Polyubiquitin chains—either covalently linked or noncovalently bound to RLR CARDs—can potentiate RLR-IPS1 signaling. In the case of noncovalent polyubiquitin binding, studies suggest RIG-I CARD2 interacts with IPS1 (bottom middle). Our results suggest that, in the absence of ubiquitin, RLR CARD1 interacts with IPS1 (bottom right). (B) We measured the kinetics of human RIG-I and MDA5 CARD1, CARD2 and CARD1+2 domains bound to IPS1 CARD in vitro (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s001" target="_blank">S1 Fig</a>). We plot the mean and standard error in–log-transformed steady-state dissociation (pKd) and initial binding rate (pKm), with longer bars indicating tighter binding (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s002" target="_blank">S2 Fig</a>). Human CASP9 CARD was used as a negative control to indicate an approximate level of nonspecific binding (dotted line); CASP9 is the CARD domain most closely related to RLR/IPS1 CARDs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g003" target="_blank">Fig 3</a>), and direct interactions between RLRs and CASP9 have not been reported.</p

Functional differentiation of ancestral RIG-I CARD1 and CARD2 domains occurred after the proliferation of RLR-IPS1 CARDs in deuterostomes, due to coevolution between RIG-I and IPS1 CARDs.

<p>Ancestral RLR and IPS1 CARD domains were resurrected, and we measured the kinetics of RIG-I and MDA5 CARDs bound to IPS1 CARD before (A) and after (B) the RLR-IPS1 CARD proliferation in early deuterostomes (dotted vertical line on simplified CARD phylogeny) (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g005" target="_blank">5</a>). We plot the mean and standard error of–log-transformed steady-state dissociation (pKd) and initial binding rate (pKm) when ancestral RLR CARD domains bind to IPS1 CARD before (A) and after (B) the deuterostome proliferation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). The approximate level of nonspecific binding is indicated by a vertical dotted line (inferred using human CASP9), and longer bars indicate tighter binding (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s017" target="_blank">S17</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s018" target="_blank">S18</a> Figs).</p