Replication of poliovirus requires binding of the poly(rC) binding protein to the cloverleaf as well as to the adjacent C-rich spacer sequence between the cloverleaf and the internal ribosomal entry site

The 5' nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CD(pro). The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C(93-95) and C(98-100)) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C(93-95)) resulted in slower viral growth, whereas the C(98-100)A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C(94-95)) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5'-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CD(pro) to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.     

Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.     

Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus

The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.     

Diversity Arrays Technology (DArT) Marker Platforms for Diversity Analysis and Linkage Mapping in a Complex Crop,the Octoploid Cultivated Strawberry (Fragaria × ananassa)

Cultivated strawberry (Fragaria × ananassa) is a genetically complex allo-octoploid crop with 28 pairs of chromosomes (2n = 8x = 56) for which a genome sequence is not yet available. The diploid Fragaria vesca is considered the donor species of one of the octoploid sub-genomes and its available genome sequence can be used as a reference for genomic studies. A wide number of strawberry cultivars are stored in ex situ germplasm collections world-wide but a number of previous studies have addressed the genetic diversity present within a limited number of these collections. Here, we report the development and application of two platforms based on the implementation of Diversity Array Technology (DArT) markers for high-throughput genotyping in strawberry. The first DArT microarray was used to evaluate the genetic diversity of 62 strawberry cultivars that represent a wide range of variation based on phenotype, geographical and temporal origin and pedigrees. A total of 603 DArT markers were used to evaluate the diversity and structure of the population and their cluster analyses revealed that these markers were highly efficient in classifying the accessions in groups based on historical, geographical and pedigree-based cues. The second DArTseq platform took benefit of the complexity reduction method optimized for strawberry and the development of next generation sequencing technologies. The strawberry DArTseq was used to generate a total of 9,386 SNP markers in the previously developed ‘232’ × ‘1392’ mapping population, of which, 4,242 high quality markers were further selected to saturate this map after several filtering steps. The high-throughput platforms here developed for genotyping strawberry will facilitate genome-wide characterizations of large accessions sets and complement other available options.     

Detection of subclinical infection in significant breast implant capsules

The pathogenesis of fibrous capsular contracture after augmentation mammaplasty is still debated. One hypothesis implicates low-grade bacterial infections as a cause. The presence of a staphylococcal biofilm in a patient with recurrent capsular contracture was previously reported. A comparative, prospective, blinded, clinical study of implants and capsules removed from patients with or without significant capsular contracture was conducted to investigate the association of biofilm contamination, breast implants, and capsular contracture.Capsule and implant samples obtained during explantation were tested by routine microbiological culture, sensitive broth culture (after maceration and sonication), and scanning electron microscopy. Clinical parameters were correlated with microbiological findings. A total of 48 implant and/or capsule samples were obtained from 27 breasts during a 22-month period. Of the 27 breasts, 19 exhibited significant contracture (Baker grade III/IV). The mean duration of implantation was 9.2 years (range, 0.4 to 26.0 years). Routine swab cultures obtained at the time of explantation were negative for bacterial growth for all samples. The sensitive broth culture technique yielded 24 positive samples (50 percent, n = 48). An analysis of capsules demonstrated that 17 of 19 samples obtained from patients with significant contracture were positive, compared with only one of eight samples obtained from patients with minimal or no contracture (p = 0.0006). Fourteen of the 17 positive cultures from significantly contracted breasts yielded coagulase-negative staphylococci, mainly, species of the Staphylococcus epidermidis group. The presence of coagulase-negative staphylococci was also significantly associated with capsular contracture (p = 0.01). There was no significant difference in the frequency of culture positivity for saline versus silicone implants (p = 0.885). Scanning electron microscopy confirmed the presence of extensive biofilm on implants and within capsules.Biofilm, in particular, S. epidermidis biofilm, was detected for a significant proportion of patients with capsular contracture. This implicates biofilm disease in the pathogenesis of contracture, and strategies for its prevention should be explored.     

Serum- and glucocorticoid-induced kinase 3 in recycling endosomes mediates acute activation of Na+/H+ exchanger NHE3 by glucocorticoids

Na(+)/H(+) exchanger 3 (NHE3) is the major Na(+) transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 or SGK2, SGK3 rapidly activated NHE3 within 15 min of Dex treatment in both PS120 and Caco-2bbe cells. Immunofluorescence analysis showed that SGK3 colocalized with NHE3 in recycling endosomes, whereas SGK1 and SGK2 were diffusely distributed. Mutation of Arg-90 of SGK3 disrupted the endosomal localization of SGK3 and delayed NHE3 activation. Activation of SGK3 and NHE3 by Dex was dependent on phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase 1 (PDK1), and Dex induced translocation of PDK1 to endosomes. Our study identifies SGK3 as a novel endosomal kinase that acutely regulates NHE3 in a PI3K-dependent mechanism.     

Development of lipid-core-peptide (LCP) based vaccines for the prevention of group A streptococcal (GAS) infection

Traditional vaccines consisting of whole attenuated micro-organisms, or microbial components administered with adjuvant, have been demonstrated as one of the most cost-effective and successful public health interventions. Their use in large scale immunisation programs has lead to the eradication of smallpox, reduced morbidity and mortality from many once common diseases, and reduced strain on health services. However, problems associated with these vaccines including risk of infection, adverse effects, and the requirement for refrigerated transport and storage have led to the investigation of alternative vaccine technologies. Peptide vaccines, consisting of either whole proteins or individual peptide epitopes, have attracted much interest, as they may be synthesised to high purity and induce highly specific immune responses. However, problems including difficulties stimulating long lasting immunity, and population MHC diversity necessitating multiepitopic vaccines and/or HLA tissue typing of patients complicate their development. Furthermore, toxic adjuvants are necessary to render them immunogenic, and as such non-toxic human-compatible adjuvants need to be developed. Lipidation has been demonstrated as a human compatible adjuvant for peptide vaccines. The lipid-core-peptide (LCP) system, incorporating lipid adjuvant, carrier, and peptide epitopes, exhibits promise as a lipid-based peptide vaccine adjuvant. The studies reviewed herein investigate the use of the LCP system for developing vaccines to protect against group A streptococcal (GAS) infection. The studies demonstrate that LCP-based GAS vaccines are capable of inducing high-titres of antigen specific IgG antibodies. Furthermore, mice immunised with an LCP-based GAS vaccine were protected against challenge with 8830 strain GAS.     

In differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment then reduced in post-mitotic cells

Previously we have shown that during in vivo muscle regeneration differentiating rat primary myoblasts transiently upregulate connexin43 (Cx43) gap junctions and leave cell cycle synchronously. Here, we studied the temporal regulation of Cx expression in relation to functional dye coupling in allogenic primary myoblast cultures using western blotting, immuno-confocal microscopy and dye transfer assays. As in vivo, Cx43 was the only Cx isotype out of Cx26, 32, 37, 40, 43 and 45 found in cultured rat myoblasts by immunostaining. Cultured myoblasts showed similar temporal regulation of Cx43 expression and phenotypic maturation to those regenerating in vivo. Cx43 protein was progressively upregulated in prefusion myoblasts, first by the cytoplasmic assembly in sparse myoblast meshworks and then in cell membrane particles in aligned cells. Dye injection using either Lucifer Yellow alone, Cascade Blue with a non-junction permeant FITC-dextran revealed an extensive gap junction coupling between the sparse interacting myoblasts and a reduced communication between the aligned, but still prefused cells. The aligned myoblasts, uniformly upregulate p21^waf1/cip1 and p27^kip1 cell cycle control proteins. Taken together, in prefusion myoblasts less membrane-bound Cx43 was found to mediate substantially more efficient dye coupling in the growing cell fraction than those in the aligned post-mitotic myoblasts. These and our in vivo results in early muscle differentiation are consistent with the role of Cx43 gap junctions in synchronizing cell cycle control of myoblasts to make them competent for a coordinated syncytial fusion.