Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:Q9NRP7 (fused)
 58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influenza A virus nucleoprotein (NP) has been examined with regard to its RNA-binding characteristics. NP, purified from virions and devoid of RNA, bound synthetic RNAs in vitro and interacted with the ribonucleotide homopolymers poly(A), poly(G), poly(U), and poly(C) in a salt-dependent manner, showing higher binding affinity for polypyrimidine homopolymers. To map the NP regions involved in RNA binding, a series of deleted forms of the NP were prepared, and these truncated polypeptides were tested for their ability to bind poly(U) and poly(C) homopolymers linked to agarose beads. Proteins containing deletions at the N terminus of the NP molecule showed reduced RNA-binding activity, indicating that this part of the protein was required to bind RNA. To identify the NP region or regions which directly interact with RNA, proteins having the maltose-binding protein fused with various NP fragments were obtained and tested for binding to radioactively labeled RNAs in three different assays: (i) nitrocellulose filter binding assays, (ii) gel shift assays, and (iii) UV light-induced cross-linking experiments. A maltose-binding protein fusion containing the N-terminal 180 amino acids of NP behaved as an RNA-binding protein in the three assays, demonstrating that the N terminus of NP can directly interact with RNA. This NP region could be further subdivided into two smaller regions (amino acids 1 to 77 and 79 to 180) that also retained RNA-binding activity.
 ...
PMID:Identification of an RNA binding region within the N-terminal third of the influenza A virus nucleoprotein. 774 27

We have constructed and transiently expressed in HeLa cells a series of hybrid proteins in which the cytoplasmic domain or both the transmembrane and the cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor were fused to the ectodomain of the hemagglutinin of the influenza virus (HA), a typical plasma membrane protein. In addition, we have expressed a hybrid protein containing the luminal domain of HA fused to the transmembrane and cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor. These hybrids were transported through and sorted from the secretory pathway as shown by acquisition of endo-H resistant oligosaccharides and their ability to recruit the Golgi assembly proteins AP-1 on the Golgi membrane. Like the mannose 6-phosphate receptors (MPRs), these hybrid proteins are also present in small amounts at the cell surface where they are likely to undergo endocytosis as disruption of the endocytosis signals contained in the MPR cytoplasmic domains induces their accumulation at the cell surface. Double immunofluorescence studies indicate that these chimeras codistribute with the endogenous MPRs at steady state. The results suggest that the cytoplasmic domains of the MPRs are sufficient to determine the steady-state distribution of the full-length proteins.
 ...
PMID:Chimeric proteins containing the cytoplasmic domains of the mannose 6-phosphate receptors codistribute with the endogenous receptors. 777 98

Previous data showed that six out of a group of nine cattle inoculated with NS1-p67, a recombinant form of a 67-kDa Theileria parva sporozoite surface protein, were immune to East Coast fever. This bacterially expressed antigen encoded all 709 amino acid residues of p67 fused to the C-terminal end of 87 residues derived from NS1, a structural protein of influenza virus, and a linker DNA sequence. NS1-p67 lacked reactivity with TpM 12, a monoclonal antibody to native p67, and had an estimated molecular mass of 110 kDa, as opposed to the calculated mass of 85,000 Da. We have used the baculovirus expression system in an attempt to express this parasite protein in a native form and thereby increase the protective capacity of the antigen. However, Spodoptera frugiperda SF21AE cells infected with recombinant virus expressed p67 as a 100-kDa molecule. The host cells exhibited a limited capacity to glycosylate this molecule to a 110-kDa form, and p67 was not exported to the surface membrane. TpM 12 did not bind to these recombinant forms but, at time points late during viral infection, reacted with a molecule of about 70 kDa. Since the bulk of insect cell-derived p67 was not expressed in an appropriate form, we tested the immunogenicity of these partially processed recombinant p67 forms in cattle. Two groups of three cattle were inoculated with antigen formulated either with saponin or Freund's adjuvant. As seen previously with NS1-p67, all animals developed high levels of anti-p67 antibodies that neutralized sporozoite infectivity in vitro, but antigen-specific T-cell proliferative responses were not detected in peripheral blood. Given the caveat of the small number of cattle analyzed, insect cell-derived p67 does not appear to be superior to NS1-p67 as an immunogen, and the latter remains the molecule of choice for the development of vaccines against East Coast fever.
 ...
PMID:Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle. 782 14

Influenza A/PR/8/34-derived chimeric (D) protein (SK&F 106160) composed of the first 81 amino acids (aa) of NS1 fused to the conserved 157 C-terminal aa of HA2 (NS1 1-81-HA2 65-222) was previously shown to induce H-2d-restricted protective cytotoxic T-lymphocyte (CTL) immunity in inbred mice. However, D protein, like other small peptides, exhibited haplotype dependence and was not immunogenic in H-2b and H-2K mice. A potential use of this antigen in humans and the role of T cells in any protection were evaluated in outbred Swiss and inbred CBF6F1 (H-2d/b) mice. Mice immunized with D protein and challenged by small-particle aerosol with a lethal dose of influenza virus were significantly protected against mortality from influenza A/H1N1 and A/H2N2 (p < 0.05-< 0.0000001), but not from A/H3N2 and influenza B viruses when compared with control mice. D protein did not induce serum virus-neutralizing antibody but caused virus to be cleared faster in immunized mice. Protection was long-lasting. In vivo depletion of either Lyt2 (CD8+) or L3T4 (CD4+) T cells with monoclonal antibodies led to abrogation of in vitro-generated CTL activity in CF6F1 mice and significant reduction in the protective efficacy of D protein against virus challenge in both Swiss and CF6F1 mice. These results suggest that protection was mediated by CD8+ and/or CD4+ cells and not antibody. Thus D protein, via a conserved sequence on the HA2 polypeptide, has the potential to induce partially cross-reactive CTL that may protect against influenza virus disease in humans.
 ...
PMID:Influenza A subtype cross-protection after immunization of outbred mice with a purified chimeric NS1/HA2 influenza virus protein. 785 2

Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fv construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the periplasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperonins resulted in complete proteolysis of the scFvNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions.
 ...
PMID:Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector. 785 83

Cytotoxic T lymphocytes (CTL) generally recognize peptides derived from endogenously expressed proteins in association with nascent major histocompatibility complex (MHC) class I molecules. In contrast, peptides derived from exogenous proteins associate with MHC class II following endocytosis to an endosomal compartment. However, we have recently demonstrated that exogenous fusion proteins consisting of the binding and translocating domains of Pseudomonas exotoxin (PE) fused with CTL epitopes derived from either influenza matrix protein (PEMa) or nucleoprotein are internalized, processed, targeted to and presented by MHC class I (Donnelly et al. 1993, Proc. Natl. Acad. Sci. USA 1993. 90: 3530). PE is known to be internalized, processed in endosomes, and translocated to the cytosol during intoxication of cells. However, our present studies demonstrate that, unlike PE, PEMa does not require translocation to the cytosol to exert its effect. First, two inhibitors of PE toxicity that exert their effects at steps subsequent to endosomal processing had no effect on the sensitization of target cells for CTL-mediated lysis by PEMa. NH4Cl, which inhibits PE by raising endosomal pH, and brefeldin A, which inhibits PE by disrupting the Golgi complex, did not inhibit sensitization of targets cells by PEMa. Second, PEMa was capable of sensitizing for lysis T2 mutant cells, which are defective in transport of peptides from the cytosol to the lumen of the endoplasmic reticulum for presentation by MHC class I. These results suggest that PEMa is proteolytically processed in endosomes, and association with MHC class I does not require nascent MHC molecules. Such a process may involve internalized MHC class I, and subsequent expression of the peptide-MHC complexes on the cell surface would then lead to recognition by CTL.
 ...
PMID:Presentation of an exogenous antigen by major histocompatibility complex class I molecules. 802 20

The trans-Golgi network (TGN) of MDCK cells is exquisitely sensitive to the fungal metabolite brefeldin A (BFA), in contrast to the refractory Golgi stack of these cells. At a concentration of 1 microgram/ml, BFA promoted extensive tubulation of the TGN while the medical Golgi marker alpha-mannosidase II was not affected. Tubules emerging minutes after addition of the drug contained both the apical marker influenza hemagglutinin (HA), previously accumulated at 20 degrees C, and the fusion protein interleukin receptor/TGN38 (TGG), a TGN marker that recycles basolaterally, indicating that, in contrast to TGN vesicles, TGN-derived tubules cannot sort apical and basolateral proteins. After 60 minutes treatment with BFA, HA and TGG tubules formed extensive networks widely spread throughout the cell, different from the focused centrosomal localization previously described in non-polarized cells. The TGG network partially codistributed with an early endosomal tubular network loaded with transferrin, suggesting that the TGG and endosomal networks had fused or that TGG had entered the endosomal network via surface recycling and endocytosis. The extensive structural alterations of the TGN were accompanied by functional disruptions, such as the extensive mis-sorting of influenza HA, and by the release of the TGN marker gamma-adaptin. Our results suggest the involvement of BFA-sensitive adaptor proteins in TGN-->surface transport.
 ...
PMID:Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells. 805 47

The host protective antigen gene VP2 of infectious bursal disease virus (IBDV) was genetically modified and expressed by recombinant fowlpox viruses (rFPV). To achieve cell surface localization, VP2 was expressed as a hybrid protein with signal sequence and membrane anchors of influenza virus hemagglutinin or neuraminidase. Native VP2 was expressed as VP2 alone or as self-processing VP2-VP4-VP3 polyprotein for coexpression of IBDV structural proteins. VP2 hybrid protein containing the carboxy-terminal membrane anchor sequence of influenza virus hemagglutinin was located on the cell surface and was N-glycosylated. The expression of VP2 fused to the N-terminal signal/anchor sequence of influenza virus neuraminidase led to cell lysis and the VP2 protein remained mainly unglycosylated. Cell surface localization of VP2 reduced immunogenicity (antibody induction) and abolished protection in poultry in comparison with the native VP2 expressed by FPV as VP2 alone or as the self-processing VP2-VP4-VP3. Vaccination of poultry with rFPV expressing native VP2 protein alone provided better protection from IBDV infection than VP2 derived from the VP2-VP4-VP3 polyprotein.
 ...
PMID:Modification of infectious bursal disease virus antigen VP2 for cell surface location fails to enhance immunogenicity. 807 13

We have monitored the fusion of intact A/PR/8/34 influenza virus with glycophorin-bearing liposomes and with ganglioside- (GD1a-) containing liposomes. The lipid bilayers of the glycophorin-bearing liposomes had several compositions, including pure dioleoylphosphatidylethanolamine (DOPE), pure egg phosphatidylethanolamine (EPE), and pure dioleoylphosphatidylcholine (DOPC). Examination of the temperature dependence of fusion for these and other compositions showed that even if the lipids are competent to form inverted hexagonal phases (HII), there is no enhancement of the fusion rate constant at the L alpha-HII phase transition temperature of the lipids, TH. Thus, the HII phase transition is not involved in the HA-mediated fusion mechanism. However, this mechanism is sensitive to lipid composition, in that PC bilayers fused more slowly than PE-containing bilayers above 20 degrees C. These results show that the HA-mediated fusion mechanism depends primarily upon specific lipid-protein interactions, although the fundamental parameters of lipid phase stability (interstice stabilization and monolayer spontaneous radius of curvature) may also be important. The fact that HII phase-component lipid bilayers in the glycophorin liposomes do not enhance the HA-mediated fusion rate strongly suggests that substantial bilayer-bilayer contact is not involved in HA-mediated fusion. Previously, we have shown that glycoprotein-bearing liposomes bind to HA-expressing cells specifically through HA-glycophorin interactions and that fusion is mediated by HAs not bound to glycophorin. Thus, with respect to the target membrane, the fusion site involves just the lipid bilayer. Our results with GD1a-containing liposomes strongly suggest that HAs bound to this sialic acid-bearing molecule are likewise incapable of participating in the fusion site. This could be due to a diminished lateral mobility of the HAs simultaneously bound to both closely apposed membranes. Finally, we find that the low-pH-induced viral inactivation is inhibited by binding to either glycophorin- or GD1a-containing target membranes.
 ...
PMID:Fusion of influenza virus with sialic acid-bearing target membranes. 811 54

Octadecylrhodamine (R18) has often been used to measure membrane fusion of enveloped viruses by fluorescence dequenching. In order to see whether non-specific R18 exchange between non-fused membranes occurs we have measured fusion of influenza virus with erythrocyte membranes by utilizing dequenching of the non-exchangeable lipid analogue N-(lissamine-rhodamine B-sulfonyl)diacylphosphatidylethanolamine (N-Rh-PE). Rather low concentration of N-Rh-PE (< 0.1 mol%) were required to assess fusion since self-quenching in the influenza virus membrane was more efficient in comparison to R18. For both markers we observed the same kinetics as well as the same extent of fluorescence dequenching upon triggering low pH-induced fusion. Non-specific marker transfer was not observed. Haemolysis was not affected by either type of fluorophore. Our results confirm that R18 is a valuable tool to investigate membrane fusion of enveloped viruses in a quantitative manner. Differences in the efficiency of self-quenching of both markers are discussed.
 ...
PMID:On the validity of lipid dequenching assays for estimating virus fusion kinetics. 814 37


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>