PLoS Pathogens最新文献

The RIG-I-like receptor (RLR) signaling pathway plays a critical role in the host defense against RNA virus infection. Among the RLR family members, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic sensors that initiate type I interferon (IFN-I) responses. Their controllable expression, activation, and degradation are essential for maintaining immune homeostasis. However, the precise regulatory mechanisms governing RIG-I and MDA5 function during viral infection remain unclear. Here, we uncover that the E3 ubiquitin ligase RNF20 exerts dual regulatory roles in RLR signaling by modulating the expression and promoting the degradation of RIG-I and MDA5 in a nucleocytoplasmic translocation-dependent manner during viral infection. Under resting conditions, RNF20 resides in the nucleus, where it maintains immune readiness by regulating the basal and inducible transcription of RIG-I and MDA5. Upon RNA virus infection, RNF20 translocates to the cytoplasm via the export receptor CRM1. There, it recognizes the degron motifs of RIG-I and MDA5 through its coiled-coil domain and catalyzes their K27-linked ubiquitination and degradation, thereby preventing excessive antiviral signaling. These findings shed light on the significant and dual regulatory roles of RNF20 in maintaining innate immune homeostasis.

Parvovirus B19 (B19V) is a prevalent human pathogen that can cross the placenta by a mechanism that remains unknown, posing a risk of severe fetal complications, particularly during the first trimester of pregnancy. We investigated the expression of B19V-specific receptors in the three trophoblast cell types, cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs), and assessed their susceptibility to infection. VP1uR, the receptor that mediates viral uptake and infection in erythroid progenitor cells, is expressed in CTBs and STBs, but not in EVTs. Globoside, a glycosphingolipid that is essential for the escape of the virus from endosomes, is also expressed in these cells, except for choriocarcinoma-derived CTBs. In the latter, the absence of globoside can be overcome by promoting endosomal leakage with polyethyleneimine. While erythropoietin receptor (EpoR) signaling is associated with the strict erythroid tropism of B19V, it is not required for infection in trophoblasts. Transfection experiments revealed that highly proliferative first-trimester CTBs are more susceptible to B19V infection than the low-proliferative CTBs from term placenta. These findings demonstrate that B19V targets specific trophoblast cells, where viral entry and replication are collectively mediated by VP1uR, globoside, and high cellular proliferative activity, but are independent of EpoR signaling.

Rift Valley fever virus (RVFV) poses a continued threat to human health and animal husbandry. Two neutralizing and protective human monoclonal antibodies (mAbs), RVFV-268 and RVFV-379, exhibit similar affinities and epitope footprints on the Gn glycoprotein component of the RVFV Gn-Gc capsomeric lattice. Here, we define fine details of the biophysical determinants of Gn recognition used by RVFV human monoclonal antibodies through studying an antibody encoded by a set of recombined genes not previously identified in RVFV antibodies. We find that RVFV-379 exhibits a larger footprint than that observed for RVFV-268 and other antibodies targeting the same region, which involves major contributions of both the light and heavy chains. RVFV-379 also uses an oblique angle of approach towards the virion surface that contrasts with the perpendicular angle of engagement observed for some other potently neutralizing human mAbs. Further, consistent with amino acid sequence variation within and proximal to the RVFV-379 epitope, in vitro neutralization screening reveals a limited degree of neutralization breadth across prevalent RVFV strains, suggesting that RVFV has fewer functional constraints at this region of the virus envelope. By dissecting the molecular determinants of mAb recognition of Gn, this integrated analysis refines strategies needed for the rational design of vaccines that can elicit a potent and species-wide protective antibody immune response to this important re-emerging pathogen.

Porcine astrovirus (PAstV) is globally prevalent in swine and is associated with diarrhea and encephalitis in piglets, posing a threat to porcine health. However, its pathogenic mechanisms remain poorly understood. This study used the PAstV1-GX1 strain to infect PK-15 cells, revealing that the virus induces significant apoptosis, with late apoptotic cells reaching 41.2% at 24 hours post-infection. The infection activates caspase-9 and caspase-3, but not caspase-8, and causes mitochondrial damage, indicating apoptosis via the mitochondrial pathway. The apoptosis inhibitor Z-VAD-FMK reduced viral replication, while apoptosis inducer ABT-263 enhanced it at later stages. The nsP1a/3 protein, which interacts with MAVS and localizes to mitochondria, was identified as key in inducing apoptosis. Its 3C-like serine protease domain likely mediates this interaction. Knocking down MAVS reduced apoptosis and increased early-stage replication but decreased it later. Overexpressing MAVS increased apoptosis and decreased replication. Furthermore, we observed that the expression of nsP1a/3 resulted in the cleavage of MAVS and the suppression of the type I interferon (IFN) response. Notably, treatment with Z-VAD-FMK did not influence nsP1a/3-mediated MAVS cleavage or type I IFN inhibition, suggesting that the induction of apoptosis and MAVS cleavage are distinct processes. By employing site-directed mutagenesis to substitute alanine for the catalytic triad residues (His459, Asp487, and Ser549) of the 3C-like serine protease, we significantly reduced the ability of nsP1a/3 to induce apoptosis, cleave MAVS, and suppress the type I IFN response, underscoring the essential role of protease activity in these functions. Furthermore, the use of a serine protease inhibitor markedly decreased PAstV replication. These findings provide significant insights into the pathogenesis of PAstV and establish a foundation for the development of novel antiviral therapies.

The Mab21/cGAS protein family has diversified across metazoans to regulate development and innate immunity. In vertebrates, cGAS detects cytosolic DNA and synthesizes 2'3'-cGAMP to activate STING-TBK1-IRF signaling, while invertebrate cGAS-like receptors (cGLRs) recognize RNA or DNA and generate non-canonical cyclic dinucleotides. However, whether shrimp Mab21 proteins function as canonical nucleic acid sensors remains unresolved. Here, we identified three Mab21 proteins from Litopenaeus vannamei-LvMab21-1, LvMab21-2, and LvMab21-3. Although they are phylogenetically related to cGAS-like proteins, none bound dsDNA or dsRNA or synthesized cGAMP in response to ISD or poly(I:C). Instead, all three interacted directly with the TBK1 homolog LvIKKε, promoted its phosphorylation at serine 175, and thereby activated the downstream IRF-Vago4 signaling axis. This mechanism defines a non-canonical nucleic acid sensing paradigm, whereby Mab21 proteins act as protein-based enhancers of kinase activation rather than as nucleic acid-dependent CDN synthases. We further show that these proteins display tissue-specific antiviral functions: all three act in hemocytes, LvMab21-1 predominates in hepatopancreas, LvMab21-2 and LvMab21-3 are most critical in gills, and LvMab21-1 and LvMab21-3 cooperate in intestine. Silencing any Mab21 paralog reduced survival and increased white spot syndrome virus (WSSV) burden, underscoring their physiological relevance. Together, our findings expand the known repertoire of innate immune strategies within the Mab21 family, highlight a previously unrecognized non-canonical mechanism of interferon-like activation, and reveal tissue-specific specialization that tailors antiviral responses across shrimp organs. These insights provide both evolutionary context and candidate targets for breeding disease-resistant shrimp.

Encephalomyocarditis virus (EMCV) is a rodent-borne picornavirus that has repeatedly caused severe outbreaks resulting in the deaths of zoo mammals - most notably elephants - for decades. However, within North America, little is known regarding the diversity of EMCV that exists in nature, the reservoir or amplifying hosts important for maintaining the virus, and the epidemiology of zoo-associated outbreaks. This lack of knowledge of the EMCV strains that circulate in North America has impeded a more thorough understanding of how genetic and antigenic variation may affect pathogenicity or potentially vaccine-induced protection from disease. Herein, following a zoological outbreak of EMCV in Florida, we conducted the most comprehensive comparative phylogenomic analysis of virus isolates from fatal zoo animal cases and local rodent species to date, identifying both non-native, invasive rodents and native mice and rat species as potentially important in precipitating and/or maintaining outbreaks, with multiple transmissions to zoo animals. After development of an autogenous vaccine, we investigated the duration and magnitude of neutralizing antibody responses in elephants monthly for multiple years, providing a unique fine-scale, long-term profile of responses to vaccination. To better understand how antigenic variation may affect vaccine-induced protection, we constructed a reverse genetics system to determine the level of cross-protection afforded by autogenous vaccination against capsids derived from various divergent EMCV strains and serotypes. These results provide new advancements in understanding EMCV transmission and antigenicity in North America, which can be used as a foundation to ultimately enable zoos to better protect animals from this important pathogen.

Dengue virus (DENV) is a mosquito-transmitted flavivirus that circulates globally as four distinct serotypes and poses a substantial threat to public health. There are an estimated ~96 million symptomatic infections yearly, including severe cases of dengue fever, underscoring the urgency of identifying effective therapeutics targeting all four serotypes. Nucleoside analogs, which mimic endogenous nucleosides to inhibit viral RNA replication, offer a promising strategy for broad-spectrum antiviral development. Here, we conducted a high-throughput screen of 1,101 nucleoside analogs against DENV serotype 2 (DENV2) in a panel of human cell models, including human epithelial cells, hepatocytes, and fibroblasts. Candidates that were active against DENV2 were screened against all four serotypes. Since flaviviruses including West Nile virus and Zika virus are also important human pathogens, we screened these compounds for activity and identified compounds that were broadly active in these cellular and viral models. We further evaluated antivirals in primary human keratinocytes and fibroblasts, which are early targets of mosquito-transmitted DENV infection. From this screen, we identified 23 nucleoside analogs with broad antiviral activity against DENV and focused on two purine analogs UPGNUC255 and UPGNUC558, that demonstrated potent pan-flaviviral activity achieving >10-fold viral load reduction across all four DENV serotypes and other flaviviruses across cell models. Mechanistic studies revealed that both compounds target the viral RNA-dependent RNA polymerase (RdRp) domain of NS5. Resistance to UPGNUC558 was associated with a conserved S604T substitution, conferring cross-resistance to other 2'C-substituted nucleoside analogs. Resistance to UPGNUC255 was linked to a previously unknown R355Q mutation, located near the catalytic GDD motif of RdRp. These findings highlight UPGNUC255 and UPGNUC558 as promising leads for the development of broad-spectrum antiviral agents against flaviviruses.

Translocation of Campylobacter concisus from the oral cavity to the intestinal tract is increasingly recognised as a contributor to inflammatory bowel disease (IBD). The C. concisus secreted protein Csep1 has emerged as a molecular marker of C. concisus strains associated with Crohn's disease, a form of IBD. However, its structure and role in inflammation remain unknown. Here, we report the X-ray crystal structure of plasmid-encoded Csep1P that reveals a unique α-helical fold with structural similarity to Helicobacter pylori cysteine-rich proteins HcpB and HcpC. Because HcpA, another Hcp family member, is known to affect monocyte differentiation, this structural similarity led us to hypothesise that Csep1P may modulate monocyte differentiation and macrophage function. Transcriptomic analysis revealed that Csep1P induced a chemokine-dominant inflammatory state in macrophages, M1-chem. Protein-level validation in both THP-1-derived and primary human macrophages confirmed this selective chemokine response. While Csep1P alone did not upregulate proinflammatory cytokines, THP-1-derived macrophages pre-incubated with Csep1P produced a higher level of proinflammatory cytokines in response to commensal Escherichia coli, which was validated on primary human macrophages. Furthermore, silencing the delta like canonical notch ligand 4 (DLL4) gene decreased the proinflammatory response of Csep1P-mediated macrophages to E. coli. Collectively, our data demonstrate that the structurally unique Csep1P reprograms macrophage response, which provides a mechanistic link between C. concisus infection and Crohn's disease pathogenesis, and identifies Csep1P as a potential target for therapeutic intervention.