Rare codons in uORFs of baculovirus p13 gene modulates downstream gene expression.

The p13 gene is a group II nucleopolyhedroviruses (NPVs) specific gene and featured by containing upstream mini ORFs (uORF) in its 5' UTR region. However, there are almost no reports published on the functions of the uORFs of p13 gene. In this study, the Luciferase Reporter Assay System was employed to investigate how the mini ORFs of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) p13 gene (Ha-p13) and its rare codons regulated the downstream gene expression. After the coding sequence of uORFs in the Ha-p13 gene was fused to the luciferase reporter gene in the expression vector pGL3 and the plasmid DNA was then transfected into the Hz-AM1 cells, the translation of the fusion protein could be initiated from the start codon of the uORFs. The uAUG and its context in uORF2 seemed to be more efficient for translation initiation than that in uORF1. Mutation of the start codons in one or both of uORFs (uORF1 or uORF2) could significantly increase the expression of the downstream reporter gene. The start codon mutation in uORF1 produced a higher reporter gene expression than that in uORF2, indicating that the uORF1 could be a stronger inhibitor than the uORF2, and the length of uORFs seemed not to be crucial for down-regulating translation. The expression of both uORFs could co-regulate the associated gene expression. Substituting the rare codons in uORF1, uORF2 or both with less rare codons dramatically increased the expression of the downstream reporter gene. Rare codon mutations in both uORFs were much more efficient in up-regulating the associate gene expression than mutations in either of the two uORFs alone.

Genome sequence of Leucania seperata nucleopolyhedrovirus.

The nucleotide sequence of the Leucania seperata (Ls) Nucleopolyhedrovirus (LsNPV) genome has been determined and analyzed. The circular dsDNA genome contains 168041 bp, making it the largest NPV sequenced to date. The genome has a G + C content of 48.6% and encodes 169 predicted open reading frames (ORFs), one unique repeat region, and eight homologous repeat regions that are divided into two groups. Of the genome, 82.8% encodes predicted ORFs including five dispersal ORFs that have a large overlaps (range in 149 approximately 390 bp) with their adjacent ORFs, respectively such as expression factor 10, 11, 5, 2 (lef-10, lef-11, lef-5, lef-2), and telokin-like protein-20 (tlp-20); 4.4% is in repeat regions; the remaining 12.8% of the genome comprises nonrepeat intergenic regions. LsNPV encodes homologues of 133 ORFs identified previously in other baculoviruses. Other than 10 'baculovirus repeat ORFs' (bro) and two 'inhibitor of apoptosis' (iap) genes, no duplicated ORFs were found. LsNPV lacks a homologue of the ubiquitin gene, which has been found in all fully sequenced baculoviruses. Iap3 and p49, two genes were proven to be inhibitors of apoptosis by experiment, and are found in the LsNPV genome. It is not found in other baculoviruses that two kinds of inhibitors of apoptosis present in a baculovirus genome.

Identification and functional analysis of LsMNPV anti-apoptosis genes.

Three anti-apoptosis genes, Ls-iap2, iap3 and p49 were found in Leucania separata multiple nuclear polyhedrovirus. Amino acid sequence homology of Ls-IAP2 and Ls-IAP3 with Op-IAP2 and Op-IAP3 from Orgyia pseddotsugata MNPV were 20% and 42%, while that of Ls-P49 is 28% with Sl-P49 from Spodoptera littorolis MNPV. Ls-IAP2 contains one baculoviral IAP repeat (BIR) domain followed by a RING domain, while Ls-IAP3 contains two BIRs and a RING. Ls-P49 contains a reactive site loop, predicted cleavage site (KKLD(74) downward arrow G) that is different from Sl-P49 (TVID(94) downward arrow G). Expressed Ls-iap3 or Ls-p49 under presence of actinomycin D in SF9 cells, DNA ladder assay revealed that Ls- IAP3 or Ls-P49 could block the apoptosis of SF9 cells induced by actinomycin D. Replication of p35 deficient-mutant Autographa californica MNPV in SF9 cells was also rescued when Ls-iap3 or Ls-p49 was expressed transiently. No anti-apoptotic activity was observed for Ls-IAP2. The results showed that both of Ls-IAP3 and Ls-P49 were functional apoptotic suppressors in SF9 cells.

The IRAK-1-BCL10-MALT1-TRAF6-TAK1 cascade mediates signaling to NF-kappaB from Toll-like receptor 4.

Our previous studies have revealed that the signaling protein BCL10 plays a major role in adaptive immunity by mediating NF-kappaB activation in the LPS/TLR4 pathway. In this study, we show that IRAK-1 acts as the essential upstream adaptor that recruits BCL10 to the TLR4 signaling complex and mediates signaling to NF-kappaB through the BCL10-MALT1-TRAF6-TAK1 cascade. Following dissociation from IRAK-1, BCL10 is translocated into the cytosol along with TRAF6 and TAK1, in a process bridged by a direct BCL10-Pellino2 interaction. RNA interference against MALT1 markedly reduced the level of NF-kappaB activation stimulated by lipopolysaccharide (LPS) in macrophages, which suggests that MALT1 plays a major role in the LPS/TLR4 pathway. MALT1 interacted with BCL10 and TRAF6 to facilitate TRAF6 self-ubiquitination in the cytosol, which was strictly dependent on the dissociation of BCL10 from IRAK-1. We show that BCL10 oligomerization is a prerequisite for BCL10 function in LPS signaling to NF-kappaB and that IRAK-1 dimerization is an important event in this process.

BCL10 mediates lipopolysaccharide/toll-like receptor-4 signaling through interaction with Pellino2.

Toll-like receptor (TLR) pathways signal through microbial components stimulation to induce innate immune responses. Herein, we demonstrate that BCL10, a critical molecule that signals between the T cell receptor and IkappaB kinase complexes, is involved in the innate immune system and is required for appropriate TLR4 pathway and nuclear factor-kappaB (NF-kappaB) activation. In response to lipopolysaccharide (LPS) stimulation, BCL10 was recruited to TLR4 signaling complexes and associated with Pellino2, an essential component down-stream of BCL10 in the TLR4 pathway. In a BCL10-deficient macrophage cell line, LPS-induced NF-kappaB activation was consistently defective, whereas activator protein-1 and Elk-1 signaling was intact. In addition, we found that BCL10 was targeted by SOCS3 for negative regulation in LPS signaling. The recruitment of BCL10 to TLR4 signaling complexes was attenuated by induced expression of SOCS3 in a feedback loop. Furthermore, ectopic SOCS3 expression blocked the interaction between BCL10 and Pellino2 together with BCL10-generated NF-kappaB activation and inducible nitric-oxide synthase expression. Together, these data define an important role of BCL10 in the innate immune system.