Els Wessels

A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport

ABSTRACT The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.

Effects of Picornavirus 3A Proteins on Protein Transport and GBF1-Dependent COP-I Recruitment

ABSTRACT The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence, to inhibit protein transport. These results show that the N terminus of the CVB3 3A protein is important for binding of GBF1 and its transport-inhibiting function. Taken together, our data demonstrate that the activity of the enterovirus 3A protein to inhibit GBF1-dependent COP-I recruitment is unique among the picornaviruses.

Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus, Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex real-time PCR assays

BACKGROUND Norovirus, Rotavirus group A, Astrovirus, Sapovirus and Adenovirus serotypes 40 and 41, are common causes of gastroenteritis. Conventional diagnosis of these causative agents is based on antigen detection and electron microscopy. OBJECTIVE To improve the diagnostic possibilities for viral gastroenteritis, two internally controlled multiplex real-time PCRs have been developed. STUDY DESIGN Individual real-time PCRs were developed and optimized for the specific detection of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, Astrovirus, Adenovirus group F and Sapovirus. Subsequently, the PCRs were combined to two multiplex PCR reactions. The multiplex assays were clinically evaluated using 239 fecal samples submitted to our laboratory over a 1-year period for the routine detection of Rotavirus and/or Adenovirus antigens using the Vikia(®) Rota/Adeno test (bioMérieux, Boxtel, The Netherlands). RESULTS In general, the multiplex real-time PCR assays showed comparable sensitivity and specificity to the individual assays. A retrospective clinical evaluation showed increased pathogen detection in samples from 14% using conventional methods to 45% using PCR. Subsequently, the assay was implemented as a routine diagnostic tool. From September 2007 up to December 2009, 486 positive results were obtained in 1570 samples (31%) analyzed. Norovirus genogroup II was found the most frequently (61.1%), followed by Adenovirus (9.9%), Rotavirus (9.3%), Astrovirus (6.0%), Norovirus genogroup I (3.3%) and Sapovirus (0.4%). CONCLUSIONS Two internally controlled multiplex real-time PCR assays for the simultaneous detection of Astrovirus, Adenovirus group F, Rotavirus, Norovirus genogroups I and II and Sapovirus have shown significant improvement in the diagnosis of viral gastroenteritis.

Added value of multiplex Luminex Gastrointestinal Pathogen Panel (xTAG® GPP) testing in the diagnosis of infectious gastroenteritis.

The Luminex Gastrointestinal Pathogen Panel (xTAG(®) GPP) detects in one assay the most common gastroenteritis-causing pathogens and toxins, namely adenovirus 40/41, norovirus genogroup (NG) I/II, rotavirus A, Clostridium difficile toxin A/B, Campylobacter sp., Escherichia coli O157, Enterotoxigenic E. coli heat-labile enterotoxin/heat-stable enterotoxin, Salmonella sp., Shiga-toxin producing E. coli, Shiga-like toxin (Stx)1/2, Shigella sp., Vibrio cholerae, Yersinia enterocolitica, Cryptosporidium sp., Entamoeba histolytica and Giardia sp. In this study, we compared the results that were obtained by testing 393 faecal samples, collected during November and December 2011 at our laboratory, using the xTAG(®) GPP assay with the results of the routine diagnostic procedure. This procedure includes culture for bacteria and real-time PCR for viruses and parasites, but only if the test was requested by the clinician. If the clinician did not request the test for an xTAG(®) GPP-positive target, real-time PCR assays were used to confirm xTAG(®) GPP positivity. Discrepant results were also tested with real-time PCR assays. A total of 83 targets were detected in 76 samples using xTAG(®) GPP. The xTAG(®) GPP assay detected 43 additional positives compared with the routine diagnostic procedure, of which 11 targets could not be confirmed by real-time PCR. The non-confirmed targets were Campylobacter (one sample), Salmonella (four samples), Shigella (one sample) and E. histolytica (five samples). The xTAG(®) GPP was shown to be a convenient and sensitive assay for detection of 15 major gastrointestinal pathogens in a single molecular test, but for detection of E. histolytica and Salmonella, a confirmatory assay is indicated.

Molecular Determinants of the Interaction between Coxsackievirus Protein 3A and Guanine Nucleotide Exchange Factor GBF1

ABSTRACT 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.

Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples

ABSTRACT The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO2 atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O2 atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated.

Structure-Function Analysis of the Coxsackievirus Protein 3A IDENTIFICATION OF RESIDUES IMPORTANT FOR DIMERIZATION, VIRAL RNA REPLICATION, AND TRANSPORT INHIBITION

The coxsackievirus B3 3A protein forms homodimers and plays important roles in both viral RNA (vRNA) replication and the viral inhibition of intracellular protein transport. The molecular determinants that are required for each of these functions are yet poorly understood. Based on the NMR structure of the closely related poliovirus 3A protein, a molecular model of the coxsackievirus B3 3A protein was constructed. Using this structural model, specific mutants were designed to study the structure-function relationship of 3A. The mutants were tested for their capacity to dimerize, support vRNA replication, and block protein transport. A hydrophobic interaction between the monomers and an intermolecular salt bridge were identified as major determinants required for dimerization. We show that dimerization is important for both efficient vRNA replication and inhibition of protein transport. In addition, determinants were identified that were not required for dimerization but that were essential for either one of the biological functions of 3A. The combination of the in silico and in vivo results obtained in this study provides important insights in both the structural and functional aspects of 3A.

Differential Membrane Association Properties and Regulation of Class I and Class II Arfs

ADP‐ribosylation factor (Arf) proteins are small guanosine triphosphatases (GTPases) that act as major regulators of intracellular vesicular trafficking and secretory organelle pathway integrity. Like all small monomeric GTPases, Arf proteins cycle between a GDP‐bound and a GTP‐bound state, and this cycling is catalysed by guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins. While the class I Arfs, especially Arf1, have been studied extensively, little is known as yet about the function and regulation of class II Arfs, Arf4 and Arf5. In this study, we show that Arf proteins show class‐specific dynamic behaviour. Moreover, unlike class I Arfs, membrane association of class II Arfs is resistant to inhibition of large Arf GEFs by Brefeldin A. Through the construction of Arf chimeric proteins, evidence is provided that the N‐terminal amphipathic helix and a class‐specific residue in the conserved interswitch domain determine the membrane‐binding properties of class I and class II Arf proteins. Our results show that fundamental differences exist in behaviour and regulation of these small GTPases.

First report of Atopobium vaginae bacteremia with fetal loss after chorionic villus sampling.

ABSTRACT Infectious complications after chorionic villus sampling (CVS) are rare (<0.1%) but can lead to maternal sepsis and spontaneous abortion. We report the first bacteremia with Atopobium vaginae and suggest A. vaginae to be a pathogenic microorganism that can lead to intrauterine infection and fetal death following CVS.

Rapid Genotyping of Cytomegalovirus in Dried Blood Spots by Multiplex Real-Time PCR Assays Targeting the Envelope Glycoprotein gB and gH Genes

ABSTRACT Genotyping of cytomegalovirus (CMV) is useful to examine potential differences in the pathogenicity of strains and to demonstrate coinfection with multiple strains involved in CMV disease in adults and congenitally infected newborns. Studies on genotyping of CMV in dried blood spots (DBS) are rare and have been hampered by the small amount of dried blood available. In this study, two multiplex real-time PCR assays for rapid gB and gH genotyping of CMV in DBS were developed. Validation of the assays with 39 CMV-positive plasma samples of transplant recipients and 21 urine specimens of congenitally infected newborns was successful in genotyping 100% of the samples, with gB1 and gB3 being the most prevalent genotypes. Multiple gB and gH genotypes were detected in 36% and 33% of the plasma samples, respectively. One urine sample from a newborn with symptomatic congenital CMV was positive for gB1 and gB2. DBS of congenitally infected newborns (n = 41) were tested using 9 μl of dried blood, and genotypes were detected in 81% (gB) and 73% (gH) of the samples, with gB3 being the most prevalent genotype. No clear association of specific genotypes with clinical outcome was observed. In conclusion, the CMV gB and gH PCR assays were found to be rapid, sensitive for detecting mixed infections, and suitable for direct usage on DBS. These assays are efficient tools for genotyping of CMV in DBS of congenitally infected newborns.