Cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences

We investigated the requirements of the carboxyterminal sequence for surface expression of the influenza viral hemagglutinin (HA). Deletions in the cloned hemagglutinin gene were introduced at locations upstream from and spanning into the region that codes for the hydrophobic carboxyl terminus. Primate cells infected with recombinants of the deleted HA gene and an SV40 vector were negative for surface immunofluorescence and failed to adsorb erythrocytes. Polypeptide analysis showed that the mutant hemagglutinins lacking the normal hydrophobic carboxy-terminal sequences were secreted into the medium. These data provide evidence that these sequences of the influenza hemagglutinin are responsible for accumulation at the cell surface. During infection with each deletion mutant, a truncated HA polypeptide was found intracellularly. Both intracellular and extracellular HAs were glycosylated, since a third species representing the unglycosylated mutant hemagglutinin was detected in the presence of tunicamycin. Interestingly, the secreted and intracellular mutant HA polypeptides differ from the surface HA in their sensitivity to endoglycosidase H, indicating that an alteration of glycosylation has occurred.     

In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci

BACKGROUND: Monoclonal (mAbKT) and recombinant single-chain (scFvKT) anti-idiotypic antibodies were produced to represent the internal image of a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity, including gram-positive cocci. Pathogenic eukaryotic and prokaryotic microorganisms, such as Candida albicans, Pneumocystis carinii, and a multidrug-resistant strain of Mycobacterium tuberculosis, presenting specific, although yet undefined, KT-cell wall receptors (KTR), have proven to be killed in vitro by mAbKT and scFvKT. mAbKT and scFvKT exert a therapeutic effect in vivo in experimental models of candidiasis and pneumocystosis by mimicking the functional activity of protective antibodies naturally produced in humans against KTR of infecting microorganisms. The swelling tide of concern over increasing bacterial resistance to antibiotic drugs gives the impetus to develop new therapeutic compounds against microbial threat. Thus, the in vitro bactericidal activity of mAbKT and scFvKT against gram-positive, drug-resistant cocci of major epidemiological interest was investigated. MATERIALS AND METHODS: mAbKT and scFvKT generated by hybridoma and DNA recombinant technology from the spleen lymphocytes of mice immunized with a KT-neutralizing monoclonal antibody (mAb KT4) were used in a conventional colony forming unit (CFU) assay to determine, from a qualitative point of view, their bactericidal activity against Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae strains. These bacterial strains are characterized by different patterns of resistance to antibiotics, including methicillin, vancomycin, and penicillin. RESULTS: According to the experimental conditions adopted, no bacterial isolate proved to be resistant to the activity of mAbKT and scFvKT. CONCLUSIONS: scFvKT exerted a microbicidal activity against multidrug resistant bacteria, which may represent the basis for the drug modeling of new antibiotics with broad antibacterial spectra to tackle the emergence of microbial resistance.     

The topology of bulges in the long stem of the flavivirus 3' stem-loop is a major determinant of RNA replication competence

All flavivirus genomes contain a 3'terminal stem-loop secondary structure (3'SL) formed by the most downstream approximately 100 nucleotides (nt) of the viral RNA. The 3'SL is required for virus replication and has been shown to bind both virus-coded and cellular proteins. Results of the present study using an infectious DNA for WN virus strain 956 initially demonstrated that the dengue virus serotype 2 (DEN2) 3'SL nucleotide sequence could not substitute for that of the WN 3'SL to support WN genome replication. To determine what WN virus-specific 3'SL nucleotide sequences were required for WN virus replication, WN virus 3'SL nucleotide sequences were selectively deleted and replaced by analogous segments of the DEN2 3'SL nucleotide sequence such that the overall 3'SL secondary structure was not disrupted. Top and bottom portions of the WN virus 3'SL were defined according to previous studies (J. L. Blackwell and M. A. Brinton, J. Virol. 71:6433-6444, 1997; L. Zeng, L., B. Falgout, and L. Markoff, J. Virol. 72:7510-7522, 1998). A bulge in the top portion of the long stem of the WN 3'SL was essential for replication of mutant WN RNAs, and replication-defective RNAs failed to produce negative strands in transfected cells. Introduction of a second bulge into the bottom portion of the long stem of the wild-type WN 3'SL markedly enhanced the replication competence of WN virus in mosquito cells but had no effect on replication in mammalian cells. This second bulge was identified as a host cell-specific enhancer of flavivirus replication. Results suggested that bulges and their topological location within the long stem of the 3'SL are primary determinants of replication competence for flavivirus genomes.     

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.     

Characterization of the murine polycystic kidney disease (Pkd2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent genetically transmitted disorders among Europeans with an attributed frequency of 0.1%. The two most common genetic determinants for ADPKD are the PKD1 and PKD2 genes. In this study we report the genomic structure and pattern of expression of the Pkd2 gene, the murine homolog of the human PKD2 gene. Pkd2 is localized on mouse Chromosome (Chr) 5 proximal to anchor marker D5Mit175, spans at least 35 kb of the mouse genome, and consists of 15 exons. Its translation product consists of 966 amino acids, and the peptide shows a 95% homology to human polycystin2. Functional domains are particularly well conserved in the mouse homolog. The expression of mouse polycystin2 in the developing embryo at day 12.5 post conception is localized in mesenchymally derived structures. In the adult mouse, the protein is mostly expressed in kidney, which suggests its functional relevance for this organ. Received: 13 March 1998 / Accepted: 11 May 1998     

Requirements for West Nile virus (-)- and (+)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (-)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (-)- and (+)-strand template RNAs containing 5'- and 3'-terminal regions (TR) with the conserved sequence elements. Our results show that both 5'- and 3'-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3'-TR of the (-)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5'-CYC motif is critical for (-)-strand RNA synthesis but neither the (-)-strand 5'- nor 3'-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5'-cap inhibits the (-)-strand RNA synthesis from the 3' fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that "cyclization" of the viral RNA play a role for (-)-strand RNA synthesis but not for (+)-strand RNA synthesis.     

In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV cross-linking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5'-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'-UTR, however, requires long range interactions involving the 5'-UTR, CYC motifs, and the 3'-stem-loop region that includes the tertiary pseudoknot structure.