Mengovirus leader is involved in the inhibition of host cell protein synthesis.

The presence of a leader peptide in picornaviruses is restricted to the Cardiovirus and Aphthovirus genera. However, the leader peptides of these two genera are structurally and functionally unrelated. The aphthovirus leader is a protease involved in viral polyprotein processing and host cell translation shutoff. The function of the cardiovirus leader peptide is still unknown. To gain an insight into the function of the cardiovirus leader peptide, a mengovirus leader peptide deletion mutant was constructed. The deletion mutant was able to grow at a reduced rate in baby hamster kidney cells (BHK-21). Mutant virus production in mouse fibroblasts (L929 cells), however, could be demonstrated only after inoculation of BHK-21 cells with the transfected L929 cells. Analysis of cellular and viral protein synthesis in mutant virus-infected cells showed a delayed inhibition of host cell protein synthesis and a reduced production of viral proteins. In a single-cycle infection, mutant virus produced only 1% of wild-type virus yield at 8 h postinfection. Host cell translation shutoff in L929 cells infected with mutant virus was restored by the addition of the kinase inhibitor 2-aminopurine. Mutant virus production in 2-aminopurine-treated L929 cells was increased to 60% of wild-type virus yield at 8 h postinfection. Our results suggest that the cardiovirus leader peptide is involved in the inhibition of host cell protein synthesis.     

Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication.

The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.     

Acid,protons and Helicobacter pylori.

The anti-ulcer drugs that act as covalent inhibitors of the gastric acid pump are targeted to the gastric H+/K+ ATPase by virtue of accumulation in acid and conversion to the active sulfenamide. This results in extremely effective inhibition of acid secretion. Appropriate dosage is able to optimize acid control therapy for reflux and peptic ulcer disease as compared to H2 receptor antagonists. However, clinical data on recurrence show that Helicobacter pylori eradication should accompany treatment of the lesion. These drugs have been found to synergize with many antibiotics for eradication. The survival of aerobes depends on their ability to maintain a driving force for protons across their inner membrane, the sum of a pH and potential difference gradient, the protonmotive force (pmf). The transmembrane flux of protons across the F1F0 ATPase, driven by the pmf, is coupled to the synthesis of ATP. The internal pH of H. pylori was measured using the fluorescent dye probe, BCECF, and the membrane potential defined by the uptake of the carbocyanine dye, DiSC3 [5] at different pHs to mimic the gastric environment. The protonmotive force at pH 7.0 was composed of a delta pH of 1.4 (-84mV) and a delta potential difference of -131mV, to give a pmf of -215 mV. The effect of variations in external pH on survival of the bacteria in the absence of urea correlated with the effect of external pH on the ability of the bacteria to maintain a pmf. The effect of the addition of 5 mM urea on the pmf was measured at different medium pH values. Urea restored the pmf at pH 3.0 or 3.5, but abolished the pmf at pH 7.0 or higher, due the production of the alkalinizing cation, NH3. Hence H. pylori is an acid-tolerant neutrophile due to urease activity, but urease activity also limits its survival to an acidic environment. These data help explain the occupation of the stomach by the organism and its distribution between fundus and antrum. This distribution and its alteration by proton pump inhibitors also explains the synergism of proton pump inhibition and antibiotics such as amoxicillin and clarithromycin in H. pylori eradication.     

Breaking pseudo-twofold symmetry in the poliovirus 3'-UTR Y-stem by restoring Watson-Crick base pairs

The previously described NMR structure of a 5'-CU-3'/5'-UU-3' motif, which is highly conserved within the 3'-UTR Y-stem of poliovirus-like enteroviruses, revealed striking regularities of the local helix geometry, thus retaining the pseudo-twofold symmetry of the RNA helix. A mutant virus with both pyrimidine base pairs changed into Watson-Crick replicated as wild type, indicating the functional importance of this symmetry relation in viral RNA replication. Here we investigated the effect of changing only one of the two pyrimidine base pairs to Watson-Crick. We determined the NMR structures of two Y-stem variants: one containing the 5'-CU-3'/5'-AU-3' motif, which has been found in wild-type virus isolates as well, and the other containing a 5'-CU-3'/5'-UG-3' motif, which is not present in any enterovirus sequenced to date. Both structures show single pyrimidine mismatches with intercalated bases. In the 5'-CU-3'/5'-AU-3' motif a C-U Watson-Crick-type base pair is formed that retains the pseudo-twofold symmetry, while in the 5'-CU-3'/5'-UG-3' motif a single asymmetric U-U mismatch breaks the twofold symmetry. Surprisingly, for the nonnatural variant no effect of the single base-pair replacement was observed on polioviral RNA replication using an in vitro replicon assay.     

Molecular determinants of the interaction between coxsackievirus protein 3A and guanine nucleotide exchange factor GBF1

The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum-to-Golgi complex transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (Arf1), thereby interfering with Arf1-mediated COP-I recruitment. This study was undertaken to gain more insight into the molecular determinants underlying the interaction between 3A and GBF1. Here we show that 3A mutants that have lost the ability to dimerize are no longer able to bind to GBF1 and trap it on membranes. Moreover, we identify a conserved region in the N terminus of 3A that is crucial for GBF1 binding but not for 3A dimerization. Analysis of the binding domain in GBF1 showed that the extreme N terminus, the dimerization/cyclophilin binding domain, and the homology upstream of Sec7 domain are required for the interaction with 3A. In contrast to that of full-length GBF1, overexpression of a GBF1 mutant lacking its extreme N terminus failed to rescue the effects of 3A. Together, these data provide insight into the molecular requirements of the interaction between 3A and GBF1.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.