Poly-Thymidine Oligonucleotides Mediate Activation of Murine Glial Cells Primarily Through TLR7, Not TLR8

The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS

The Ubiquitin-Like Protein PLIC-1 or Ubiquilin 1 Inhibits TLR3-Trif Signaling

Background: The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9) or cytoplasmic double-stranded RNA (dsRNA)-recognizing RNA helicases RIG-I and MDA5. To avoid causing injury to the host, these signaling pathways must be switched off in time by negative regulators. Methodology/Principal Findings: Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1) interacted with the Toll/interleukin-1 receptor (TIR) domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA) enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV) infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP) and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner. Conclusions: Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif. © 2011 Biswas et al

Genomic and biological characterization of chiltepin yellow mosaic virus, a new tymovirus infecting Capsicum annuum var. aviculare in Mexico.

The characterization of viruses infecting wild plants is a key step towards understanding the ecology of plant viruses. In this work, the complete genomic nucleotide sequence of a new tymovirus species infecting chiltepin, the wild ancestor of Capsicum annuum pepper crops, in Mexico was determined, and its host range has been explored. The genome of 6,517 nucleotides has the three open reading frames described for tymoviruses, putatively encoding an RNA-dependent RNA polymerase, a movement protein and a coat protein. The 5′ and 3′ untranslated regions have structures with typical signatures of the tymoviruses. Phylogenetic analyses revealed that this new virus is closely related to the other tymoviruses isolated from solanaceous plants. Its host range is mainly limited to solanaceous species, which notably include cultivated Capsicum species. In the latter, infection resulted in a severe reduction of growth, indicating the potential of this virus to be a significant crop pathogen. The name of chiltepin yellow mosaic virus (ChiYMV) is proposed for this new tymovirus

Engineering resistance against physalis mottle tymovirus by expression of the coat protein and 3' noncoding region

A 748 nucleotides cDNA fragment corresponding to the 3' terminal of physalis mottle virus, PhMV (formerly known as belladonna mottle virus) ($\#$Y16104) genomic RNA encompassing the tymobox, coat protein ORF and 3' noncoding region was cloned into the binary vector pKYLX 71 35 $S^2$ and introduced into N. tabacum cv. Havana plants using Agrobacterium-mediated transformation. The R0 transgenic plants showed accumulation of coat protein which self-assembled into capsids in vivo. The transgenic R1 and R2 plants showed delay in symptom expression and virus accumulation upon challenge with PhMV. 55 and 65% of the plants showed no detectable symptoms in the R1 and R2 transgenic plants respectively, when challenged with 10 $\mu$g/ml virus. Further, no detectable symptoms were observed in 75% and 25% of the R1 and R2 transgenic plants respectively, after 50 days of post infection when challenged with 10 $\mu$g/ml RNA. Thus the expression of PhMV coat protein and 3' noncoding sequence confers a high level of resistance against PhMV infection

Yeast Two Hybrid screening of host factors that directly interact with g-1 RdRp and ORF4.

<p>Yeast Two Hybrid screening of host factors that directly interact with g-1 RdRp and ORF4.</p

ORF4 is a target of host ubiquitin-proteasome machinery.

<p><b>(A)</b> Western blot using anti-Flag (top) and anti-GAPDH (bottom). <b>(B)</b> Western blot using anti-ubiquitin (top) and anti-Flag (bottom). <b>(C)</b> Anti-Flag western blot of Huh7 cells expressing WT and K51N mut ORF4, treated with cycloheximide (panel 1, 3). Same blots were reprobed with anti-GAPDH (panel 2, 4). <b>(D)</b> Immunofluorescence of ORF4 in Huh7 cells transfected with <i>in vitro</i> synthesized WT HEV or K51N HEV. Scale: 20μm. Shown are merged images of nuclei (blue) and ORF4 (green). “→”: positive staining, “►”: unstained. <b>(E)</b> QRT-PCR of HEV sense and anti-sense RNA from Huh7 cells transfected and treated as indicated. Veh: Vehicle, TUN:tunicamycin.</p

G-3 RdRp, X and Helicase associate with each other in Huh7 cells.

<p><b>(A)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 RdRp and X, immunoprecipitated and revealed using indicated antibodies. <b>(B)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 Helicase and X, immunoprecipitated and revealed using indicated antibodies. <b>(C)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 RdRp and Helicase, immunoprecipitated and revealed using indicated antibodies. <b>(D)</b> CoIP and western of Huh7 cell extract transiently expressing g-3 RdRp, Helicase and X, immunoprecipitated and revealed using indicated antibodies.</p