Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family results in the activation of type I interferon (IFN) responses.To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural protein NSs of the newly described severe fever with thrombocytopenia syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the beta interferon (IFN-) promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of the Atg7 gene, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures colocalize with Rab5 but not with Golgi apparatus or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and point toward a novel mechanism for the suppression of IFN responses.
IMPORTANCEThe mechanism by which the newly described SFTSV inhibits host antiviral responses has not yet been fully characterized. In this study, we describe the redistribution of RIG-I signaling components into virus-induced cytoplasmic structures in cells infected with SFTSV. This redistribution correlates with the inhibition of host antiviral responses. Further characterization of the interplay between the viral protein and components of the IFN responses could potentially provide targets for the rational development of therapeutic interventions.
The nucleotide-binding site-leucine-rich repeat (NBS-LRR)-encoding gene family has attracted much research interest because approximately 75% of the plant disease resistance genes that have been cloned to date are from this gene family. We cloned the NBS-LRR-encoding genes from polyploid cotton by a polymerase chain reaction-based approach. A sample of 150 clones was selected from the NBS-LRR gene sequence library and was sequenced, and 61 resistance gene analogs (RGA) were identified. Sequence analysis revealed that RGA are abundant and highly diverged in the cotton genome and could be categorized into 10 distinct subfamilies based on the similarities of their nucleotide sequences. The numbers of members vary many fold among different subfamilies, and gene index analysis showed that each of the subfamilies is at a different stage of RGA family evolution. Genetic mapping of a selection of RGA indicates that the RGA reside on a limited number of the cotton chromosomes, with those from a single subfamily tending to cluster and two of the RGA loci being colocalized with the cotton bacterial blight resistance genes. The distribution of RGA between the two subgenomes A and D of cotton is uneven, with RGA being more abundant in the A subgenome than in the D subgenome. The data provide new insights into the organization and evolution of the NBS-LRR-encoding RGA family in polyploid plants.
Matrix metalloproteinase-9 (MMP-9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor-1 (FGF-1) on the expression of MMP-9 in ENU1564, an ethyl-N-nitrosourea-induced rat mammary adenocarcinoma cell line. We observed that FGF-1 induces a dose-dependent increase in MMP-9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP-9 regulation by FGF-1, we investigated the role of components of PI3K-Akt and MEK1/2-ERK signaling pathways in our system since NF-kappaB and AP-1 transcription factor binding sites have been characterized in the upstream region of the MMP-9 gene. We demonstrated that FGF-1 increases Akt phosphorylation, triggers nuclear translocation of NF-kappaBp65, and enhances degradation of cytoplasmic IkappaBalpha. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP-9 protein expression in FGF-1-treated cells. Conversely, our data show that FGF-1 increases ERK phosphorylation in ENU1564 cells, increases c-jun and c-fos mRNA expression in a time-dependent manner, and triggers nuclear translocation of c-jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP-9 protein expression in FGF-1 treated cells. Finally, we observed increased DNA binding of NF-kappaB and AP-1 in FGF-1-treated cells and that mutation of either NF-kappaB or AP-1 response elements prevented MMP-9 promoter activation by FGF-1. Taken together, these results demonstrated that FGF-1-induced MMP-9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF-kappaB and AP-1 and involve activation of a dual signaling pathway, PI3K-Akt and MEK1/2-ERK.
Physical mapping with large-insert clones is becoming an active area of genomics research, and capillary electrophoresis (CE) promises to revolutionize the physical mapping technology. Here, we demonstrate the utility of the CE technology for genome physical mapping with large-insert clones by constructing a robust, binary bacterial artificial chromosome (BIBAC)-based physical map of Penicillium chrysogenum. We fingerprinted 23.1× coverage BIBAC clones with five restriction enzymes and the SNaPshot kit containing four fluorescent-ddNTPs using the CE technology, and explored various strategies to construct quality physical maps. It was shown that the fingerprints labeled with one or two colors, resulting in 40–70 bands per clone, were assembled into much better quality maps than those labeled with three or four colors. The selection of fingerprinting enzymes was crucial to quality map construction. From the dataset labeled with ddTTP–dROX, we assembled a physical map for P.chrysogenum, with 2–3 contigs per chromosome and anchored the map to its chromosomes. This map represents the first physical map constructed using the CE technology, thus providing not only a platform for genomic studies of the penicillin-producing species, but also strategies for efficient use of the CE technology for genome physical mapping of plants, animals and microbes.
Equine infectious anemia virus (EIAV) infection is distinctive in that it causes a rapid onset of clinical disease relative to other retroviruses. In order to understand the interaction dynamics between EIAV and the host immune response, we explored the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine response in macrophages. EIAV infection markedly altered the expression pattern of a variety of pro-inflammatory cytokines and chemokines monitored in the study. Comparative studies in the cytokine response between EIAV17 and EIAV17ΔS2 infection revealed that S2 enhances the expression of IL-1α, IL-1β, IL-8, MCP-2, MIP-1β and IP-10. Moreover, S2 specifically induced the expression of the newly discovered cytokine, IL-34. Taken together, these results may help explain the effect of cytokine and chemokine dysregulation in EIAV pathogenesis and suggest a role of S2 in optimizing the host cell environment to promote viral dissemination and replication.
The spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (WTD) and its ability to transmit from deer to deer raised concerns about the role of WTD in the epidemiology and ecology of the virus. Here, we present a comprehensive cross-sectional study assessing the prevalence, genetic diversity, and evolution of SARS-CoV-2 in WTD in the State of New York (NY). A total of 5,462 retropharyngeal lymph node samples collected from free-ranging hunter-harvested WTD during the hunting seasons of 2020 (Season 1, September to December 2020, n = 2,700) and 2021 (Season 2, September to December 2021, n = 2,762) were tested by SARS-CoV-2 real-time RT–PCR (rRT-PCR). SARS-CoV-2 RNA was detected in 17 samples (0.6%) from Season 1 and in 583 samples (21.1%) from Season 2. Hotspots of infection were identified in multiple confined geographic areas of NY. Sequence analysis of SARS-CoV-2 genomes from 164 samples demonstrated the presence of multiple SARS-CoV-2 lineages and the cocirculation of three major variants of concern (VOCs) (Alpha, Gamma, and Delta) in WTD. Our analysis suggests the occurrence of multiple spillover events (human to deer) of the Alpha and Delta lineages with subsequent deer-to-deer transmission and adaptation of the viruses. Detection of Alpha and Gamma variants in WTD long after their broad circulation in humans in NY suggests that WTD may serve as a wildlife reservoir for VOCs no longer circulating in humans. Thus, implementation of continuous surveillance programs to monitor SARS-CoV-2 dynamics in WTD is warranted, and measures to minimize virus transmission between humans and animals are urgently needed.
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