Functional analysis of the human cytomegalovirus immune evasion protein, pUS3(22kDa)

Human cytomegalovirus (HCMV) is an important opportunistic pathogen that infrequently causes disease in individuals with mature immune systems. The HCMV US3 gene encodes a 22-kDa protein that interferes with immune recognition of virally infected cells. The 22-kDa US3 protein binds to major histocompatibility complex (MHC) class I complexes, retaining them in the endoplasmic reticulum (ER), thereby decreasing the presentation of viral antigen to cytotoxic T cells. Our studies demonstrate that correct folding of the ER lumenal domain of the US3 protein is essential, but insufficient for interactions with MHC class I complexes. We demonstrate a requirement for the transmembrane domain of the 22-kDa US3 protein, confirming the results of others, and also show that the cytosolic carboxyl-terminal tail influences the function of the protein. Anchoring of the ER-lumenal immunoglobulin-like fold of the US3 protein to the membrane of the endoplasmic reticulum is critical for the binding and retention of MHC class I complexes.     

Expression of L-histidine decarboxylase in granules of elicited mouse polymorphonuclear leukocytes

Infiltrating polymorphonuclear leukocytes (PMN) in the peritoneal cavity were found to express L-histidine decarboxylase (HDC), the rate-limiting enzyme of histamine synthesis, in a csein-induced peritonitis model. Expression of HDC was detected in the elicited PMN, but not in the peripheral blood leukocytes. The peritoneal lavage fluids in this model were found to augment histamine synthesis in PMN isolated from the bone marrow. Rapid post-translational processing of HDC was observed in PMN, and the dominant form of HDC was the mature 53-kDa form, which was found to co-localize with a granule enzyme, matrix metalloproteinase-9 (MMP-9). Treatment of PMN with the phorbol ester PMA, which stimulates the release of MMP-9, did not liberate the granular HDC. Immunofluorescence studies using an anti-HDC antibody strongly suggested that HDC is bound to the cytosolic side of the granule membranes. These observations suggest that HDC is induced upon infiltration of PMN into the mouse peritoneal cavity and that histamine is synthesized by HDC attached to the granule membranes of PMN.     

A nuclear localization signal and a membrane association domain contribute to the cellular localization of the Tobacco mosaic virus 126-kDa replicase protein

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.     

Purification of novel calcium binding proteins from Trypanosoma brucei: properties of 22-, 24- and 38-kilodalton proteins.

The present study was undertaken to systematically purify calcium binding proteins (CaBPs) from homogenates of Trypanosoma brucei. This work is important since CaBPs either serve as intracellular calcium buffers or mediate cellular response to calcium signals. Disruption of either process should be lethal to trypanosomes. We report that the 45Ca-gel overlay assay can be used to detect CaBPs following fractionation on DE-52, phenyl-Sepharose, Mono-Q, and Superose 12. Specific CaBPs of 22, 24, and 38 kDa were purified. Each of these proteins associated with 45Ca under denaturing and non-denaturing conditions. An approximate Kd for calcium of 8 microM was calculated for 22-kDa CaBP. None of the trypanosome CaBPs were related to known calcium binding protein families. They did not associate with hydrophobic interaction columns or cellular membranes in a calcium-dependent way, nor cross-react with 2 separate antibodies against annexin consensus sequences. A synthetic peptide corresponding to amino terminal residues 16-30 of 22-kDa CaBP was used to generate polyclonal antibodies. Immunoblots identified 22-kDa CaBP in African trypanosomes but not in other Kinetoplastidae or mammalian cells. Nonetheless, significant homology (58%) was observed between the amino terminal 37 residues of 22-kDa CaBP and the amino terminus of translationally controlled p21 from mammalian tumor cells. The present study is the first to apply systemic fractionation techniques to identify the complement of CaBPs in T. brucei. We conclude that novel CaBPs other than calmodulin and annexin family members contribute towards calcium pathways in these organisms.     

Domain structure of herpes simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous and discontinuous residues

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is a multifunctional glycoprotein required for infectivity; it is thought to promote fusion of the viral envelope with the cell membrane and entry of virions into cells. To map the antigenic and functional domains on gB, we constructed amino terminal derivatives lacking the entire carboxyl terminus and internal deletion mutants lacking defined regions of the extracellular and transmembrane domains. Transient expression of the mutants in COS-1 cells revealed that the amino terminal derivatives were released into the medium whereas those with deletions in the extracellular domain were mostly retained within the transfected cells. Analysis of intact gB and the amino terminal derivatives showed that the intact molecule formed dimers whereas the mutant derivatives did not. Reactions of the derivatives with a panel of well-characterized monoclonal antibodies to gB showed that the neutralizing epitopes cluster in two domains. The first maps in the amino terminal 190 residues and contains seven continuous epitopes, five of which are HSV-1-specific. Reactions of antibodies with a set of oligopeptides fine-mapped the epitopes between residues 1 and 47. The second domain is composed of discontinuous epitopes and was expressed by amino terminal derivatives that were at least 457 residues in length or longer. Eleven epitopes map in this region, including those of four potent neutralizing antibodies whose cognitive sites mapped between residues 273 and 298 in mapping studies using antibody-resistant mutants. Results of the present study indicate that the cognitive sites of these antibodies are assembled into the discontinuous domain by juxtaposing residues from the amino-terminal half of gB monomers.     

Analysis of nucleotide sequence of the rightmost 43 kbp of herpesvirus saimiri (HVS) L-DNA: general conservation of genetic organization between HVS and Epstein-Barr virus.

We present an analysis of 43,658 bp of contiguous nucleotide sequence comprising the right terminal region (conventional orientation) of the unique protein-coding component (L-DNA) of the herpesvirus saimiri (HVS) genome. Within this region lie the genes encoding the 160-kDa virion protein, which is homologous to the 140-kDa membrane antigen of Epstein-Barr virus (EBV), thymidylate synthase (TS), and the immediate-early (IE) 52-kDa protein which is homologous to the EBV BMLF1 product. The 160-kDa gene of HVS lies at the right terminus of HVS L-DNA, its homologue in EBV occurring at the left terminus of the EBV genome (conventional orientation). The TS gene of HVS occurs within a group of 5 genes that have no homologues in EBV. The translation product of one of these genes, ECRF3, shows amino acid sequence and hydrophobicity pattern similarities to the HCMV and cellular G-protein-coupled receptor family of proteins. Another, ECLF2, is homologous to the cyclin family of cellular proteins. The 5 nonconserved genes lie adjacent to the 160-kDa gene. In EBV, the region to the right of the 140-kDa gene (BNRF1) contains the latent replication origin (OriP) and the open reading frames BCRF1, BWRF1 (repeated 12 times), BYRF1, BHLF1, and BHRF1, counterparts of which are not present in this position in HVS. The subsequent 18 genes in EBV (BFLF2 to BLRF2, approximate positions 56,000-89,500) are represented in HVS, and the relative positions and orientations of these genes are directly comparable between the two viruses. There then occurs a nonhomologous gene in HVS, and genes BLLF2 to BZLF1 (positions 89,500 to 103,200) in EBV which are not present in this region of HVS, before collinearity resumes. Thus, the HVS sequence presented here shows general collinearity between conserved genes in the right terminal region of HVS and the left terminal region of EBV and reveals the presence of two sets of unique genes which occur in exactly analogous positions in HVS and EBV.     

Identification of beta-subunit of GTP-binding regulatory protein in mitotic spindle.

BACKGROUND: The heterotrimeric GTP-binding regulatory protein (G protein) is important in membrane signal transduction. Since the function of the beta-subunit of G protein (G beta) in tumor cells is not well-documented, identification of G beta in tumor cells was performed. EXPERIMENTAL DESIGN: Immunolocalization, Western blotting, and immunoprecipitation of G beta in mammalian tumor and normal cells were investigated using rabbit antisera against amino and carboxyl terminal peptide fragments of G beta. A human nasopharyngeal carcinoma cell line (NPC-TW039) was used as the cell model because of its short doubling time. RESULTS: Anti-G beta immunoreactivity was found to be associated with the plasma membrane and the mitotic spindle throughout the mitotic phase of cell replication. Colcemid pretreatment resulted in random distribution of the anti-G beta reaction product in the mitotic cells. The same phenomenon was also seen in various other tumor and normal cell lines. When solubilized membranous, cytosolic, and mitotic spindle fractions were analyzed by Western blotting, G beta (35 +/- 1 kDa) was found to be associated with the plasma membrane and mitotic spindle fractions. Immunoprecipitation of isolated mitotic spindles with anti-G beta further confirmed these results. CONCLUSIONS: These findings indicate that G beta is closely associated with the mitotic spindle as well as the plasma membrane and may be important in regulation of cell mitosis in addition to transmembrane signal transduction.     

Neurotensin — Was ist gesichert über seine Rolle als Hormon im Magen-Darmtrakt?

Summary Neurotensin is a tridecapeptide originally isolated and characterized from bovine hypothalamus and later, in identical form, from bovine and human intestine. In the rat about 85% of immunoreactive neurotensin is found in the gut and about 10% in the brain. When an antibody specific for the amino terminal region of neurotensin was used the highest concentrations were found in the mucosa of the ileum, while an antibody specific for the biologically active region, the carboxyl terminus, also detected large amounts in the mucosa of the upper gastrointestinal tract. After a meal neurotensin — as measured by carboxyl terminal antibodies — rises after 5 min, a time in which the chymus has not yet reached the ileum, the main source of whole neurotensin. It is therefore possible that the carboxyl terminal molecules of neurotensin, found in the upper gastrointestinal tract, play an important physiological role. In plasma, neurotensin is rapidly degraded into smaller amino terminal and therefore biologically inactive molecules. Increases of carboxyl terminal neurotensin have been found in plasma in only a very few studies. The nature of this immunoreactive material has not yet been established. Therefore, the physiological role of neurotensin as a circulating hormone is unknown. Potential actions of neurotensin include thermoregulation, regulation of hormone release from brain (pituitary hormones) and gut (glucagon, insulin, somatostatin, pancreatic polypeptide), increase of vascular permeability, vasodilatation, inhibition of gastric acid secretion, stimulation of pancreatic secretion and changes of gut motility from the fasting to the fed type.

     

Antibodies to distal carboxyl terminal epitopes in the v-fms-coded glycoprotein do not cross-react with the c-fms gene product

The product of the v-fms oncogene is an integral transmembrane glycoprotein that is closely related to the cell surface receptor for the macrophage colony stimulating factor, CSF-1. A fragment of the v-fms gene encoding a major portion of the extracellular amino terminal domain, the membrane-spanning segment, and the entire carboxyl terminal tyrosine kinase domain of the glycoprotein was molecularly cloned into an inducible prokaryotic expression plasmid. Polypeptide products consisting only of v-fms-coded amino acids were produced in bacteria and were used to prepare immune reagents that precipitated the v-fms-coded glycoproteins expressed in transformed cells. Whereas rabbit antisera to recombinant polypeptides detected antigenic determinants of the c-fms proto-oncogene product, seven mouse monoclonal antibodies to these same antigens reacted only with v-fms-specific epitopes. Proteolytic mapping experiments and studies with a mutant v-fms-coded glycoprotein lacking the 37 carboxyl terminal amino acids of the wild-type product showed that the monoclonal antibodies were restricted in their reactivity to epitopes at the extreme carboxyl terminus of the glycoprotein. The v-fms and c-fms gene products must differ significantly in this region.     

Functional interactions of the domains of the adenovirus DNA binding protein

The 34-kDa fragment of the carboxyl end of the adenovirus (Ad) DNA binding protein (DBP) binds to single-stranded (ss) DNA and is able to replace the intact 72-kDa DBP needed for Ad DNA replication in vitro. A similar fragment prepared from the temperature-sensitive (ts) mutant, H5ts107, which has a single amino acid change in the carboxyl end of the DBP, is temperature sensitive for DNA replication and defective in binding to ssDNA. However, in 20 mM NaCl which is the salt concentration during Ad DNA replication in vitro, the intact 72-kDa H5ts107 DBP is defective only in replication but not binding to DNA at nonpermissive temperatures. These observations indicate that the amino domain of the H5ts107 DBP can stabilize the binding of its carboxyl end to DNA.     

Recombinant α-chains of type IV collagen demonstrate that the amino terminal of the Goodpasture autoantigen is crucial for antibody recognition

Goodpasture's disease, an autoimmune disorder causing severe glomerulonephritis and pulmonary haemorrhage, is characterized by antibodies to the glomerular basement membrane (GBM). The principal target antigen has been identified as the carboxyl terminal non-collagenous (NC1) domain of the α3-chain of type IV collagen. Anti-GBM antibodies appear to recognize one major epitope that is common to all patients, and is largely conformational. We have analysed antibody binding to recombinant α(IV)NC1 domains using a construct and expression system shown to produce correctly folded antigen that is strongly recognized by autoantibodies. In this system, as with the native antigen, α3(IV)NC1 was bound strongly by antibodies from all patients, whereas the closely related α1(IV) and α5(IV)NC1 domains, similarly expressed, showed no such binding. A series of chimeric NC1 domains, between human α3(IV) and α1(IV), and between human and rat α3(IV), were expressed as recombinant molecules, and were recognized by autoantibodies to varying degrees. Strong binding required the presence of human α3(IV) sequence in the amino terminal region of both sets of chimeric molecules. This work strongly suggests that the amino terminal of α3(IV)NC1 is critical for antibody recognition, whereas the carboxyl terminal end of α3(IV)NC1 has a less important role.     

Influence of antigen structure on the activation and induction of unresponsiveness in cloned human T lymphocytes.

Using cloned human helper T lymphocytes reactive with a 24 amino acid peptide (p20) of the carboxyl terminal of the HA-1 molecule of influenza haemagglutinin we have investigated the influence of antigen structure on the activation and in the induction of antigen specific unresponsiveness of T cells. For this analysis stereoisomers and structural isomers of p20 have been constructed. P20 in the form of a single loop created by a disulphide based between residue 306 and an additional cysteine at position 330 was able to activate the helper T cells in the presence of accessory cells but unable to induce tolerance. This result suggested that critical residues were prevented from direct interaction with the T-cell receptor and/or the MHC Class II determinants and required processing to expose them. The enantiomer (D-p20) and the inverted sequence (retro-L-p20) which have non complementary side chain topography as compared to the parent peptide neither activated nor tolerized the T cells. Furthermore the retro-D-p20 isomer which has the same side chain topography as L-p20 but with a reversal of amino and carboxyl acid groups also failed to stimulate or tolerize. Therefore T-cell antigen recognition is not determined by side chains alone. The results presented suggest that structure of extrinsic antigen influences T-cell antigen recognition.     

Mutations of the Hepatitis C virus protein NS4B on either side of the ER membrane affect the efficiency of subgenomic replicons

The non-structural protein NS4B of the Hepatitis C virus (HCV) is an integral membrane protein located in the endoplasmic reticulum (ER). Although the function of the NS4B in the viral life cycle is unknown a critical role in replication has been indicated. In order to investigate which components are involved we initially introduced restriction sites near the extremities of the NS4B in a subgenomic replicon that resulted in the alterations of six amino acid residues. This totally abolished replication. We subsequently introduced 14 single point mutations into different regions of NS4B based on the current topology model. One mutation abolished replication, while most conferred reduced replicon establishment and one mutation resulted in improved efficiency. Neither the protein processing nor the membrane altering capacity of NS4B was affected. Surprisingly, mutations situated in the ER lumen also conferred strong effects, despite the fact that replication occurs on the cytosolic side of the ER membrane. We conclude that the molecular integrity of NS4B is vital for HCV replication. Our results suggest that NS4B interacts with itself and with other viral and cellular factors, and may carry intrinsic capacities in order to allow replication.     

Infection of macrophage-like THP-1 cells with Mycobacterium avium results in a decrease in their ability to phosphorylate nucleolin

This study of the phosphorylation ability of macrophage-like cells upon infection with Mycobacterium avium was undertaken to establish potential targets of the interference with host response mechanisms. Cytosolic and membrane fractions from noninfected and infected cells were incubated with [gamma-(32)P]ATP, in the presence of Mg(2+) and the absence of Ca(2+), and the patterns of phosphoproteins synthesized were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lower levels of a 110-kDa phosphoprotein were observed in association with cytosolic fractions from mycobacterium-infected cells compared to noninfected cells or cells treated with lipopolysaccharide or having ingested Escherichia coli or killed M. avium. The 110-kDa phosphoprotein was present in the soluble fraction (230,000 x g supernatant) after the kinase incubation, from where it was partially purified and identified as phosphonucleolin by amino acid sequencing. The decrease in nucleolin phosphorylation observed was not related to changes in the cytosolic or membrane levels of this protein, and was detected also in the cytosolic fraction of (32)P-labeled intact cells.     

Molecular cloning of human RP105

RP105 is a 105-kDa type I membrane protein of the leucine-rich repeat (LRR) family. Anti-RP105 sensitizes B cells to antigen-receptor-mediated apoptosis, but protects B cells from radiation-induced apoptosis and stimulates B cell proliferation. The sequence of the mouse RP105 has been reported. Here, we report the characterization of the human RP105. The 2.6-kb cDNA encodes a protein of 661 amino acids which displays 78% homology with mouse RP105. The 22 LRR and the 9 potential N-linked glycosylation sites within the extracellular region are conserved. While previous studies have shown that RP105 is expressed on surface IgM⁺IgD⁺⁺ B cells in mice, human RP105 was shown to be expressed on all subsets of mature B cells and dendritic cells. Human RP105 gene was mapped to the long arm of chromosome 5, where numerous cytokines and receptors have been localized.     

Components and Mechanisms of Import,Modification, Folding,and Assembly of Immunoglobulins in the Endoplasmic Reticulum

In mammalian cells, the endoplasmic reticulum (ER) plays a central role in biogenesis of secretory- and plasma membrane proteins as well as in cellular calcium (Ca²⁺) homeostasis. The protein biogenesis function involves an aqueous polypeptide conducting channel in the ER membrane, which is formed by the heterotrimeric Sec61 complex; the store- and receptor-controlled Ca²⁺- release function requires a steep ER to cytosol gradient, with more than 500 μM free Ca²⁺ in the ER and 50 nM Ca²⁺ in the cytosol. Recent work demonstrated that the Sec61 complex can transiently allow passive ER Ca²⁺ efflux. Therefore, gating of the Sec61 channel has to be tightly regulated by substrates as well as allosteric effectors. The ER lumenal Hsp70-type molecular chaperone, immunoglobulin heavy-chain binding protein (BiP), together with its membrane resident co-chaperone Sec63 facilitates channel opening in a precursor specific manner. In addition, BiP, together with its lumenal co-chaperones, ERj3 and ERj6, as well as cytosolic Ca²⁺-calmodulin (CaM) in collaboration with the membrane resident Sec62 protein represent allosteric effectors for channel closure. In the course of the last couple of years several human diseases were linked to the Sec61 complex and its effectors and were termed Sec61-channelopathies.     

Nucleotide sequence of the gene encoding infectious laryngotracheitis virus glycoprotein B.

The nucleotide sequence of the infectious laryngotracheitis virus (ILTV) gene encoding the 205K complex glycoprotein (gp205) was determined. The gene is contained within a 3-kb EcoRI restriction fragment mapping at approximately map coordinates 0.23 to 0.25 in the UL region of the ILTV genome and is transcribed from right to left. Nucleotide sequence analysis of the DNA fragment identified a single, long open reading frame capable of encoding 873 amino acids. The predicted precursor polypeptide derived from this open reading frame would have a calculated Mr of 98,895 Da and contains nine potential glycosylation sites. Hydropathic analysis indicates the presence of an amino terminal hydrophobic sequence and hydrophobic carboxyl terminal domain which may function as a signal peptide and a membrane anchor sequence, respectively. Comparison of the predicted ILTV gp205 protein sequence with those of other herpesviruses revealed a significant sequence similarity with gB-like glycoproteins. Extensive homology was observed throughout the molecule except for the amino and carboxyl termini. The high homology in predicted primary and secondary structures is consistent with the essential role of the gB family of proteins for viral infectivity and pathogenesis.     

Characterization of three proteins expressed from the virulence region of plasmid pSDL2 in Salmonella dublin.

Infection of both cattle and humans with Salmonella dublin can result in septicemia and death. Like many nontyphoid Salmonella species that cause disease, S. dublin contains a cryptic plasmid (pSDL2) that is required for the full expression of virulence. Transposon mutagenesis of pSDL2 defined a 4.1-kb EcoRI region that is necessary for the development of a systemic infection in BALB/c mice. This EcoRI fragment was cloned into an expression vector (pEL11), and three proteins produced from this region with apparent molecular weights of 30,500, 76,000, and 27,000 were identified. Because bacterial proteins that play a role in virulence are often associated with the outer membrane, we were interested in establishing whether the proteins expressed from the EcoRI fragment are located in the membrane. Transposon mutagenesis of pEL11 with TnphoA defined the order of the genes along the fragment and suggested that the proteins may be exported out of the cytoplasm. Sucrose gradient cell fractionation was done to identify the cellular location of each of the three proteins. The 30-kDa protein was identified in the outer membrane fraction, and the 76-kDa protein was located in the cytosolic fraction. The 27-kDa protein was identified in both the cytosolic and the outer membrane fractions. The outer membrane contained less than 10% of the activity of enzymes known to be located in the cytoplasm, periplasm, and inner membrane. Sequence data of the 4.1-kb EcoRI region revealed that both the 30- and the 27-kDa proteins lack a typical signal sequence for export out of the cytoplasm (M. Krause, C. Roudier, J. Fierer, J. Harwood, and D. G. Guiney, Mol. Microbiol. 5:307, 1991). The outer membrane location of these proteins suggests that they may be exported out of the cytoplasm by an unusual mechanism.