tRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes

Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972–4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA^Ser(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA^Ser(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA^Ser(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2α in the tRNA^Ser(CGA) transfected L1 cell lines. The tRNA^Ser(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.     

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-kappaB-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.     

Common ancestry of herpesviruses and tailed DNA bacteriophages

Comparative analysis of capsid protein structures in the eukaryote-infecting herpesviruses (Herpesviridae) and the prokaryote-infecting tailed DNA bacteriophages (Caudovirales) revealed a characteristic fold that is restricted to these two virus lineages and is indicative of common ancestry. This fold not only serves as a major architectural element in capsid stability but also enables the conformational flexibility observed during viral assembly and maturation. On the basis of this and other emerging relationships, it seems increasingly likely that the very diverse collection of extant viruses may have arisen from a relatively small number of primordial progenitors.     

Variation in conserved non-coding sequences on chromosome 5q and susceptibility to asthma and atopy

Background

Evolutionarily conserved sequences likely have biological function.

Methods

To determine whether variation in conserved sequences in non-coding DNA contributes to risk for human disease, we studied six conserved non-coding elements in the Th2 cytokine cluster on human chromosome 5q31 in a large Hutterite pedigree and in samples of outbred European American and African American asthma cases and controls.

Results

Among six conserved non-coding elements (>100 bp, >70% identity; human-mouse comparison), we identified one single nucleotide polymorphism (SNP) in each of two conserved elements and six SNPs in the flanking regions of three conserved elements. We genotyped our samples for four of these SNPs and an additional three SNPs each in the IL13 and IL4 genes. While there was only modest evidence for association with single SNPs in the Hutterite and European American samples (P < 0.05), there were highly significant associations in European Americans between asthma and haplotypes comprised of SNPs in the IL4 gene (P < 0.001), including a SNP in a conserved non-coding element. Furthermore, variation in the IL13 gene was strongly associated with total IgE (P = 0.00022) and allergic sensitization to mold allergens (P = 0.00076) in the Hutterites, and more modestly associated with sensitization to molds in the European Americans and African Americans (P < 0.01).

Conclusion

These results indicate that there is overall little variation in the conserved non-coding elements on 5q31, but variation in IL4 and IL13, including possibly one SNP in a conserved element, influence asthma and atopic phenotypes in diverse populations.     

Nuclear RelB+ cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions

Differentiated dendritic cells (DC) have been identified by the presence of nuclear RelB (nRelB) and HLA-DR, and the absence of CD20 or high levels of CD68, in lymph nodes and active rheumatoid arthritis synovial tissue. The current studies aimed to identify conditions in which nRelB is expressed in human tissues, by single and double immunohistochemistry of formalin-fixed peripheral and lymphoid tissue. Normal peripheral tissue did not contain nRelB+ cells. nRelB+ DC were located only in T- or B-cell areas of lymphoid tissue associated with normal organs or peripheral tissues, including tonsil, colon, spleen and thymus, or in association with T cells in inflamed peripheral tissue. Inflamed sites included skin delayed-type hypersensitivity reaction, and a wide range of tissues affected by autoimmune disease. Nuclear RelB+-HLA-DR- follicular DC were located in B-cell follicles in lymphoid organs and in lymphoid-like follicles of some tissues affected by autoimmune disease. Lymphoid tissue T-cell areas also contained nRelB(-)-HLA-DR+ cells,some of which expressed CD123 and/or CD68. Nuclear RelB+ cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions.     

A Novel DNA Vaccine Technology Conveying Protection against a Lethal Herpes Simplex Viral Challenge in Mice

While there are a number of licensed veterinary DNA vaccines, to date, none have been licensed for use in humans. Here, we demonstrate that a novel technology designed to enhance the immunogenicity of DNA vaccines protects against lethal herpes simplex virus 2 (HSV-2) challenge in a murine model. Polynucleotides were modified by use of a codon optimization algorithm designed to enhance immune responses, and the addition of an ubiquitin-encoding sequence to target the antigen to the proteasome for processing and to enhance cytotoxic T cell responses. We show that a mixture of these codon-optimized ubiquitinated and non-ubiquitinated constructs encoding the same viral envelope protein, glycoprotein D, induced both B and T cell responses, and could protect against lethal viral challenge and reduce ganglionic latency. The optimized vaccines, subcloned into a vector suitable for use in humans, also provided a high level of protection against the establishment of ganglionic latency, an important correlate of HSV reactivation and candidate endpoint for vaccines to proceed to clinical trials.