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L Roux

Publications and source records attributed to L Roux.

At least 55 records · Page 3Linked to original sources
PubMed1990

Selective and transient association of Sendai virus HN glycoprotein with BiP.

From 10-min [35S]methionine pulse-labeled Sendai virus-infected BHK cells, an anti-BiP monoclonal antibody precipitated, along with the BiP protein, the hemagglutinin-neuraminidase protein (SV-HN) fivefold better than the fusion protein (SV-Fo). A minimal estimate of 30% of the newly made HN was complexed to BiP. The majority of the HN in the complex was endo-H sensitive and the molar ratio of BiP:HN was estimated to be 1:2. With time, HN dissociated from BiP, and the rate of dissociation was found to be inversely proportional to the rate at which HN acquired its native structure. It is proposed that association with BiP followed by slow release (i) is responsible for the HN slow maturation and (ii) represents a normal step in its maturation pathway.

Animals↗
PubMed1990

Intracellular stability of nonreplicating paramyxovirus nucleocapsids.

Using Northern blot analysis, we have demonstrated the ability of infectious measles and Sendai virus particles to rescue the intracellular replication of their homologous defective interfering (DI) nucleocapsids up to 3 days and 1 day, respectively, after initial DI infection. The half-life of the paramyxovirus DI nucleocapsids was therefore judged to be similar to that of rhabdoviruses, and to significantly differ from that of orthomyxoviruses. Moreover, we conclude that the intracellular half-life of measles virus DI nucleocapsids makes possible DI replication in the human body after vaccination with a DI-contaminated attenuated live virus, even when this vaccination represents a low multiplicity of infection.

Animals↗
PubMed1990

Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines.

Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.

Blotting, Northern↗
PubMed1989

Budding efficiency of Sendai virus nucleocapsids: influence of size and ends of the RNA.

The budding efficiency of Sendai virus antigenomes, as well as of defective interfering (DI) nucleocapsids of the deletion and copy-back types, was compared to that of the viral genome during infections of baby hamster kidney (BHK) cells. The antigenomes were shown to bud into virus particles as efficiently as the genomes, arguing for the irrelevance of the nucleocapsid-RNA ends in regulating the efficiency of budding. The DI nucleocapsids, however, were restricted in their budding by factors inversely proportional to their size, arguing for an effect of nucleocapsid size in this process. This restriction in budding, however, appeared to be only expressed under conditions of very efficient DI-RNA replication.

Animals↗
PubMed1989

Addition of high-mannose sugars must precede disulfide bond formation for proper folding of Sendai virus glycoproteins.

The role of glycosylation and of disulfide bonds in the formation of the native structure of the Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins was studied. In contrast to the HN and F0 synthesized in vivo, the proteins made from pSP6 transcripts in reticulocyte lysates, whether glycosylated or not, were not recognized by monoclonal antibodies or polyclonal rabbit sera raised against the native proteins; they efficiently reacted only with rabbit antisera raised against the reduced sodium dodecyl sulfate-denatured proteins. These in vitro-made proteins, however, did not contain disulfide bonds. The proteins made in vivo in the presence of tunicamycin, which were also not recognized by the anti-native protein antibodies, did contain disulfide bonds, but they were mainly incorrect interchain disulfide bonds. Moreover, while F0 acquired proper disulfide bonds as soon as it was synthesized under normal conditions in vivo, the disulfides were formed in HN only after a lag of 10 to 30 min. This lag coincides with the delay observed in HN native structure formation. We therefore conclude that the maturation of the HN and F0 proteins depends on the formation of proper intramolecular disulfide bonds, which in turn depends on the previous addition of high-mannose sugars.

Cystine↗
PubMed1988

Sulfated N-linked oligosaccharides in mammalian cells. I. Complex-type chains with sialic acids and O-sulfate esters.

The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines (Freeze, H. H., and Varki, A. (1986) Biochem. Biophys. Res. Commun. 140, 967-973). Here we report improved methods to metabolically label cell lines with 35SO4 and to release sulfated N-linked oligosaccharides with peptide:N-glycosidase F as well as the partial structure of some of these novel oligosaccharides. The released 35SO4-labeled chains from Chinese hamster ovary (CHO) cells and bovine pulmonary artery endothelial cells (CPAE) were characterized by gel filtration, anion exchange and lectin affinity chromatography, and various enzymatic and chemical treatments. Each cell line contains a class of sulfated oligosaccharide chains bearing from two to six negative charges in varying combinations of O-sulfate esters and sialic acids. These molecules represent a significant proportion of both the total 35SO4 label and the total anionic N-linked oligosaccharides. They are also relatively enriched in a CHO mutant that is deficient in glycosaminoglycan chain synthesis. Lectin affinity chromatography of such molecules from CPAE cells indicates that the majority are sialylated multiantennary complex-type chains. The sulfate esters are exclusively of the primary type. Sequential exoglycosidase digestions, including beta-hexosaminidase A treatment at low pH, demonstrate that at least one-third of these sulfate esters are found in the following structure, (formula; see text) where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., Roux, L., Varki, A., and Freeze, H. H. (1988) J. Biol. Chem. 263, 8890-8896).

Animals↗
PubMed1988

Sulfated N-linked oligosaccharides in mammalian cells. II. Identification of glycosaminoglycan-like chains attached to complex-type glycans.

In the preceding paper (Roux, L., Holojda, S., Sundblad, G., Freeze, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889) we described the metabolic labeling and isolation of sulfated N-linked oligosaccharides from mammalian cell lines. All cell lines studied contained a class of sulfated sialylated complex-type chains with 2-6 negative charges. In this paper, we show that bovine pulmonary arterial endothelial (CPAE) and human erythroleukemia (K562) cell lines also contain a class of more highly charged sulfated but less sialylated oligosaccharides. These molecules were further characterized by ion exchange chromatography and various enzymatic and chemical treatments. In both cell lines they contained greater than 6 negative charges, but those from K562 were even more highly charged than those from CPAE. Nitrous acid, heparinase, and heparitinase degradation of K562 oligosaccharides released 88, 64, and 78%, respectively, of 35S label. Combined digestion with the two enzymes resulted in 87% release. The corresponding values for CPAE were 48, 25, and 50% (60% for the two enzymes together). Chondroitinase ABC (or AC) digestion of K562 and CPAE oligosaccharides released 10 and 5%, respectively. About 30% of the 35S-labeled oligosaccharides from CPAE were sensitive to endo-beta-galactosidase, indicating that poly-N-acetyl-lactosamine structures were present on some chains. Highly charged [3H]mannose-labeled sulfated oligosaccharides from CPAE cells became neutral after treatment with heparinase/heparitinase but were resistant to Pronase, further proving that glycosaminoglycan (GAG)-like chains were directly attached to N-linked oligosaccharides. Such neutralized oligosaccharides did not bind to concanavalin A-Sepharose, but some interacted with phytohemagglutinin L4, indicating that they were bi-, tri-, or tetra-antennary complex-type chains. Thus, K562 and CPAE cells contain different types of GAG chains directly attached to asparagine-linked oligosaccharides. Such molecules were not found in many other cell lines that synthesize the more typical O-linked GAG chains. This suggests that the occurrence of these novel N-linked chains is not a random event resulting from accidental initiation of GAG chain synthesis on N-linked intermediates in the Golgi apparatus.

Animals↗
PubMed1988

Direct adverse effects of Sendai virus DI particles on virus budding and on M protein fate and stability.

Upon infections of BHK cells with a mixture of Sendai standard and defective interfering (DI) viruses (mixed virus infection), viral budding was found to be restricted by factors ranging from 5 to more than 20. The reduced viral budding correlated with a high intracellular M protein turnover. M appeared to be degraded shortly after its synthesis, and seemed not to be able to self-associate in a stable way under the plasma membrane as it did in St virus-infected cells. These data, added to the previous findings that infection with DI particles allowed infected cell survival and favored the cell-surface turnover of the hemagglutinin-neuraminidase protein, led to the hypothesis that DI genomes directly act by preventing the stable formation inside the cells of a viral structure composed of M/HN/nucleocapsids. When involved in this structure M would be protected from degradation and HN would be stably anchored in the plasma membrane. Formation of this structure would be necessary for viral budding and would be damaging for the cells. Comparison with results published by other authors shows that such a model is consistent with other data. It can integrate, as well, data obtained in the analysis of mutant viruses involved in persistence.

Defective Viruses↗
PubMed1988

Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine.

By starting from a thrice-purified wild-type measles virus plaque, the generation of detectable subgenomic RNAs was achieved within a series of five serial infections of Vero cells. The evolution of these subgenomic RNAs was followed for seven serial passages and ended with the preparation of a highly interfering viral stock. On the other hand, the detection of discrete subgenomic RNAs was achieved during the first infection of Vero cells with at least one of three measles virus vaccine preparations tested. These subgenomic RNAs, which interfered very efficiently with the replication of the endogenous standard genomes upon vaccine infection but showed a moderate interfering activity with a standard virus stock derived by plaque purification from the vaccine preparation, resulted from the presence of defective interfering particles in the vaccine preparation. The relevance of this finding for the attenuation, stability, and potential capacity for persistent infection of such a vaccine is discussed.

Chromosome Mapping↗
PubMed1986

Glycosylation characteristics of pigmentation-associated antigen (GP75): an intracellular glycoprotein of human melanocytes and malignant melanomas.

Pigmentation-associated antigen (PAA) or gp75 is a glycoprotein localized to the melanosomes of human melanomas and melanocytes to which a mouse monoclonal antibody (AbTA99) has been produced (T. M. Thomson et al. (1985) J. Invest. Dermatol. 85, 169). Treatment of 3H-labeled immunoprecipitated melanoma PAA with alkaline-borohydride, hydrazinolysis, or N-glycanase released three families of carbohydrate chains (I, II, and III). Peak I consists of a major component (Ia) of sialylated triantennary N-linked chains which are partially substituted with fucose on terminal positions as well as on the chitobiose core and a minor component (Ib) which is a sialylated biantennary N-linked species. Peak II was not well characterized but may be a monoantennary complex chain species. Peak III consists of typical N-linked high mannose units with six to seven mannose residues. Melanocyte PAA carbohydrate chains have the same general features as melanoma PAA except that the biantennary complex chain predominates; this difference resembles that observed between the cell surface glycopeptides of transformed animal cells and their nontransformed counterparts. The glycosylation characteristics of this melanosomal glycoprotein are compared with those of glycoproteins from endoplasmic reticulum, Golgi, and lysosomes, and with tyrosinase. It is suggested that the glycosylation pattern is a reflection of the biosynthetic origin and cellular destination of a particular organelle and its constituents.

Carbohydrate Sequence↗
PubMed1986

Reduced temperature can block different glycoproteins at different steps during transport to the plasma membrane.

Reduced temperature has been shown to block the cell surface expression of Sendai virus haemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins at different steps of their intracellular transport. At 20 degrees C, HN was confined to the rough endoplasmic reticulum or cis Golgi compartment, while F0 acquired complete resistance to digestion by endo-beta-N-acetylglucosaminidase-H and therefore was blocked at a more distal location in the pathway of cell surface expression. The significance of these results for different pathways of transport to the cell surface is discussed.

Animals↗
PubMed1986

Drastic immunoreactivity changes between the immature and mature forms of the Sendai virus HN and F0 glycoproteins.

The immunoreactivity of the Sendai virus HN and F0 glycoproteins was shown to mature before reaching the final form exhibited by the native mature proteins. The maturation process differed for the two proteins. The native F0 immunoreactivity was shown to be defined cotranslationally, and the addition of high-mannose sugar residues may represent the final step in defining the maturation of immunoreactivity. On the other hand, native HN immunoreactivity was slowly fashioned during the hour after the completion of protein synthesis. Although addition of high-mannose sugar could constitute a necessary step in this slow maturation process, it was shown not to be sufficient. Processing of high-mannose sugars and HN self-association in homodimers and homotetramers were investigated as possible steps involved in the slow maturation of HN immunoreactivity. They were found not to play a significant role. On the other hand, conformational changes presumably took place during the maturation of HN immunoreactivity. Drastic immunoreactivity differences were also demonstrated between the native and denatured forms of the glycoproteins. Possible implications of these results in defining the pathways of glycoprotein synthesis are discussed.

Animals↗
PubMed1986

Diagnostic and pathogenetic aspects of subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis, usually a rapidly progressive and fatal disease, is a slow virus infection, where measles virus persists in cells of the CNS and in lymphocytes. Four patients, 3 boys and 1 girl, are described, who presented a characteristic disease course, beginning at the age of 12 to 14 years after they had contracted measles infection during infancy or early childhood. The diagnostic criteria including clinical and laboratory CSF findings are summarized, and epidemiologic features related to measles and measles immunization are briefly discussed. In two patients specific measles virus protein antibodies in serum and CSF were analyzed. The results confirm postulated mechanisms for viral persistence in the CNS and suggest in addition a possible role of the viral protein H in the pathogenesis of SSPE.

Adolescent↗
PubMed1985

Sequence determination of the Sendai virus HN gene and its comparison to the influenza virus glycoproteins.

The nucleotide sequence of the Sendai virus (SV) HN (hemagglutinin-neuraminidase) gene was determined. The deduced primary structure of the protein showed only one hydrophobic domain likely to represent the transmembrane region, but at its N terminus. Since the SV F protein is anchored in the membrane at its C terminus, the two SV glycoproteins are thus membrane-anchored in opposite orientations, similar to the two influenza virus (FLU) glycoproteins. Amino acid sequence comparisons of the SV HN and the FLU HA and NA proteins revealed homologies between 100 amino acids of the hemagglutinin region of the FLU HA protein and the C terminus of the SV HN, and between 200 amino acids of the neuraminidase region of the FLU NA and the central region of SV HN. Alignment of the neuraminidase, hemagglutinin, and fusion regions shared by these glycoproteins suggest the structure of a possible ancestral gene.

Amino Acid Sequence↗
PubMed1985

The role of haemagglutinin-neuraminidase glycoprotein cell surface expression in the survival of Sendai virus-infected BHK-21 cells.

A temperature-sensitive mutant of Sendai virus with a lesion in the haemagglutinin-neuraminidase protein (HN) (ts 271) was used to study the effect of HN cell surface expression on the fate of infected BHK-21 cells. The total amount of HN was reduced in ts 271 virus-infected cells at the non-permissive temperature (38 degrees C) presumably due to degradation of the protein. At this temperature, neither HN nor a modified form of HN were found expressed at the surface of the infected cells. BHK-21 cells infected with ts 271 were nevertheless killed by the infection at 38 degrees C as well as at 30 degrees C. These results ruled out the hypothesis that the lack of HN cell surface expression could be the unique requirement allowing BHK cell survival.

Animals↗
PubMed1985

Three variations in the cell surface expression of the haemagglutinin-neuraminidase glycoprotein of Sendai virus.

The fate of the haemagglutinin-neuraminidase glycoprotein (HN) of Sendai virus in three types of infection was studied by measuring its sensitivity to endoglycosidase H and its rate of appearance and turnover at the cell surface. HN behaved differently in the three types of infection. When highly expressed at the surface, as in a lytic standard virus infection, HN accumulated at the surface in a stable form (half-life of disappearance from the surface much greater than 10 h). When moderately expressed, as in a non-lytic standard virus plus defective interfering virus infection, HN reached the membrane normally, but turned over rapidly (half-life about 2 h) and was re-internalized. When poorly expressed, as in long-term persistent infection, HN did not reach the cell surface and appeared to be degraded before reaching it. In contrast to HN, the other viral glycoprotein, F0, exhibited a similar turnover rate at the cell surface in the three situations. However, when compared to surface expression in standard virus-infected cells under standardized conditions, F0 surface expression in persistently infected cells was reduced. This reduction correlates with a decreased maturation rate in these cells.

Animals↗
PubMed1985

Pigmentation-associated glycoprotein of human melanomas and melanocytes: definition with a mouse monoclonal antibody.

Pigmented melanoma cells and cultured melanocytes express a differentiation-related glycoprotein designated as pigmentation-associated antigen (PAA) of Mr 70,000-80,000. As described previously, PAA was initially defined by reactivity with antibodies in the serum of a patient with melanoma. Here we describe the production and characterization of a mouse monoclonal antibody to PAA. This antibody (TA99, an IgG2a) was shown by sequential immunoprecipitation experiments to react with the same component as the human antibody. Ab TA99 immunoprecipitated PAA from lysates of cells radiolabeled with [35S]methionine, [3H]glucosamine, [3H]fucose, and [3H]mannose as well as 125I. Using Ab TA99, the distribution of PAA was examined in frozen sections of 19 normal tissues and quantitatively in 68 tissue culture cell lines. In frozen sections, only melanin-containing cells were positive, including epithelial cells in the basal layer of the epidermis, in which pigment originates from melanocytes by transfer of melanosomes, and pigmented cells of the eye. In tissue culture cell lines, only pigmented melanoma cells were positive. PAA is an intracellular antigen, with a distribution very similar to that of melanosomes. This evidence confirms the close association of PAA with melanin production, and suggests that PAA may be a melanosome component. PAA was shown to be different from tyrosinase, the enzyme involved in melanin synthesis, but it was found to be identical to the previously recognized glycoprotein, gp75, characteristic of pigmented melanomas and melanocytes.

Animals↗