We have studied the role of protein turnover in the induction of NF-kappa B DNA binding activity. Treatment of cells with tumour necrosis factor (TNF), double-stranded RNA (dsRNA), or phorbol esters is shown to be associated with an increase in the rate of p105 to p50 processing, and the loss of immunologically detectable MAD3/I kappa B alpha. Phosphate-labelling experiments indicate that these events are preceded by the phosphorylation of MAD3 and p105. The protease inhibitors TLCK (N alpha-p-Tosyl-L-Lysine Chloromethyl Ketone) and TPCK (N alpha-p-Tosyl-L-Phenylalanine Chloromethyl Ketone) inhibit both p105 to p50 processing and MAD3 degradation, and also cause a complete block to NF-kappa B activation. These data suggest a model for NF-kappa B activation in which phosphorylation destabilises the NF-kappa B/MAD3 complex but that, in vivo, this is insufficient to lead to activation in the absence of an obligatory mechanism that degrades MAD3.

No abstract

Adenovirus virus‐associated (VA) RNAI is required for efficient protein synthesis at late times of adenoviral infection, and in some other situations where double‐stranded RNA (dsRNA) is present. It prevents inhibition of protein synthesis by a dsRNA‐activated protein kinase and the secondary structure of VA RNAI is though to be important for its activity. To test this idea and to define structures and sequences responsible for VA RNAI activity, we constructed several mutant VA RNA genes and tested them in a transient expression assay. Activity is unaffected by deletions within a small region near the center of the gene, nt 72‐85, but it is greatly diminished by deletion or substitution of sequences on the 3′ side of this region. The structures of wild‐type and mutant RNAs were examined by nuclease‐sensitivity analysis. We propose a model for wild‐type VA RNAI which differs from that predicted to be the most stable structure. Surprisingly disruption of the longest duplex region in the molecule is tolerated, provided that adjacent structural elements are not rearranged. However, perturbations of elements located in the center of the structure correlate well with loss of function.

An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 RVA segment-specific (+)ssRNAs, a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Secondly, a genetically modified MA104 cell line was used in which several compounds of the innate immune were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low replicating attenuated vaccine candidates or low cell culture passage clinical isolates from humans or animals. IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies and expression vectors.

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.

Double‐stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM‐1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM‐1 is also induced by the cytokine interleukin 1β (IL‐1β), activation of the dsRNA‐activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL‐1β. Incubation of HUVE cells with the synthetic dsRNA, poly (I) · poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2α, and increased activation of the transcription factor NF‐кB. Promoter analysis in HUVE cells using a VCAM‐1 promoter linked to CAT reporter gene demonstrates that poly (I) · poly (C) responsiveness resides in the minimal VCAM‐1 promoter that contains two NF‐кB sites, and deletion of the NF‐кB sites eliminates basal and poly (I) · poly (C)‐induced CAT activity, supporting the importance of NF‐кB in the poly (I) · poly (C)‐mediated induction of VCAM‐1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating IкBα (the inhibitory subunit of NF‐кB) in a dsRNA‐dependent manner. This suggests that phosphorylation of IкBα by PKR could be an initial step in the activation of NF‐кB by dsRNA. NF‐кB is also activated by IL‐1β in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2α phosphorylation. Poly (I) · poly (C) induces VCAM‐1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL‐1β. Although phosphorylation of eIF2α interferes with protein translation, sufficient VCAM‐1 mRNA translation occurs in response to poly (I) · poly (C) to yield VCAM‐1 protein levels that are similar to levels that are induced by IL‐1bT. This suggests that the higher, sustained VCAM‐1 mRNA levels that occur in response to incubation with poly (I) · poly (C) compensate for the partial translational block resulting from increased eIF2α phosphorylation. These studies indicate that transcrip‐tional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM‐1 gene expression in HUVE cells.

Campylobacter jejuni is a food-borne pathogen responsible for infectious enterocolitis. The early-response transcription factor NF-κB triggers the expression of genes associated with cellular immune and inflammatory responses. Co-incubation of HeLa cells with viable C. jejuni leads to the activation of the transcription factor NF-κB as determined by specific induction of a cellular luciferase-based reporter. Boiled cell-free extracts of C. jejuni are also potent dose-dependent stimulators of NF-κB-dependent transcription, the levels of which can reach up to 1000-fold as compared with independent controls. Using both cultured HeLa cells and human colonic epithelial (HCA-7) cells, the activation of NF-κB by C. jejuni boiled extract has been monitored through the degradation of IKBα and DNA binding of the nuclear translocated p50/p65 heterodimer of NF-κB. These events are co-ordinated with elaboration of the pro-inflammatory cytokine interleukin-8. Fractionation of the boiled C. jejuni extract suggests that the majority of the bioactive component has a molecular mass of 3 kDa or less, which is insensitive to proteinase K treatment.

Six restriction fragment length polymorphisms (RFLPs) detected in the human growth hormone-human chorionic somatomammotropin (hGH-hCS) gene duster were studied in Mediterraneans, Northern Europeans, and American Blacks; the polymorphisms showed that, on the average, one of 500 bases in this cluster is variant. Haplotypes constructed for four of these RFLPs display strong nonrandom associations. However, the strongest associations were between RFLPs that are in homologous DNAs rather than between the physically closest RFLPs. From this and other evidence we argue that duplication of an ancestral hCS gene occurred at least twice, the second event being relatively recent.