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Biomedical subjects

S Gillam

Publications and source records attributed to S Gillam.

At least 73 records · Page 4Linked to original sources
PubMed1992

Identification of T-cell epitopes on E2 protein of rubella virus, as recognized by human T-cell lines and clones.

T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell cloned isolated from one T-cell line responded strongly in proliferation assays with peptide E2-4 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HLA DR7.

Amino Acid Sequence↗
PubMed1992

Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy.

Glycosylated, membrane-associated E1 (58-kDa) and E2 (47- to 49-kDa) rubella virus proteins and unglycosylated nucleoprotein C (33 kDa), from separately expressed vaccinia virus recombinants, were injected into golden Syrian hamsters. Rubella virus E1 and E2 glycoproteins consistently induced an organ-specific autoimmune disease, autoimmune lymphocytic hypophysitis, which was evidenced by the induction of autoantibodies against pituitary cells and by lymphocytic infiltration of the pituitary. Neonatal thymectomy prevented the disease. In contrast, rubella virus nucleoprotein C did not induce either autoantibodies against pituitary cells or lymphocytic infiltration of the pituitary. This finding raises the possibility that virus-specific protein itself can induce an organ-specific autoimmune disease in certain circumstances.

Animals↗
PubMed1992

Analysis of T- and B-cell epitopes of capsid protein of rubella virus by using synthetic peptides.

A nested set of 11 overlapping synthetic peptides covering the entire sequence of rubella virus capsid protein was synthesized, purified, and tested against human rubella virus-specific T-cell lines and rubella virus-seropositive sera. T-cell lines derived from four donors responded strongly to four synthetic peptides containing residues 96 to 123, 119 to 152, 205 to 233, and 255 to 280. Only one peptide (residues 255 to 280) was recognized by all four T-cell lines. Two human immunodominant linear B-cell epitopes were mapped to residues 1 to 30 and 96 to 123 by using peptide-specific enzyme-linked immunosorbent assay. All 11 synthetic peptides were highly immunogenic and induced strong antibody responses in rabbits against the respective immunized peptides. Seven of the 11 rabbit antipeptide antisera (anti-1-30, -74-100, -96-123, -119-152, -205-233, -231-257, and -255-280) specifically recognized the capsid protein on immunoblots. Identification of these T- and B-cell epitopes represents the first step toward rational design of synthetic vaccines against rubella.

Amino Acid Sequence↗
PubMed1992

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site.

Animals↗
PubMed1991

The influence of capsid protein cleavage on the processing of E2 and E1 glycoproteins of rubella virus.

The structural polyprotein of rubella virus is cotranslationally processed by host cell signal peptidase. Oligonucleotide-directed mutagenesis was used to alter the cleavage site between capsid and E2 proteins and to examine the importance of this cleavage for the transport and processing of E2 and E1 glycoproteins. The in vitro and in vivo expression of the cleavage site mutant revealed that the E2 polypeptide can cross the endoplasmic reticulum membrane without the cleavage of its signal peptide, while the transport of E2 beyond the endoplasmic reticulum requires the cleavage of E2 from capsid. We have shown that capsid protein does not appear to undergo further proteolytic processing after it is cleaved from E2 by signal peptidase. Some of the requirements for the cleavage by signal peptidase between capsid and E2 were examined by the in vitro analysis of wild-type and mutant cDNAs.

Animals↗
PubMed1991

Analysis of rubella virus E1 glycosylation mutants expressed in COS cells.

cDNA clones encoding the envelope glycoprotein E1 of rubella virus (RV) were altered by site-directed mutagenesis at consensus sites for addition of N-linked glycans. The resulting plasmids were introduced into COS cells and the mutant E1 proteins were analyzed by indirect immunofluorescence, radioimmunoprecipitation, and immunoblotting. We found that RV E1 contains three N-linked oligosaccharides, each approximately 2 kDa in size. Although lack of glycosylation did not appear to affect targeting of E1 to the Golgi region, mutants lacking N-linked glycans at Asn 177 and Asn 209 failed to bind anti-E1 antibodies under nonreducing conditions. Our results suggest that glycosylation may be important for expression of important immunologic epitopes on RV E1.

Animals↗
PubMed1991

pH-dependent solubility shift of rubella virus capsid protein.

The mechanism of capsid uncoating in rubella virus and other togaviridae is not well understood. This study presents data which suggest that rubella virus capsid undergoes a structural change from having hydrophilic to hydrophobic properties, between pH 5 and 5.5. Such a conformational change would allow capsid uncoating to occur within the lysosome, allowing RNA penetration to occur upon fusion of the viral envelope with the limiting membrane of the lysosome.

Animals↗
PubMed1990

Processing and intracellular transport of rubella virus structural proteins in COS cells.

Plasmids encoding rubella virus (RV) structural proteins C-E2-E1, E2-E1, E2, and E1 have been constructed in the eukaryotic expression vector pCMV5. The processing and intracellular transport of these proteins have been examined by transient expression of the cDNAs in COS cells. Compared to alphaviruses, processing of RV glycoprotein moieties occurred relatively slowly and the transport of glycoproteins E2 and E1 to the plasma membrane was inefficient. Indirect immunofluorescence revealed that the majority of RV antigen in transfected and infected COS cells was localized to the Golgi region, including the capsid protein. Accumulation of capsid protein in the juxtanuclear region was determined to be RV glycoprotein dependent. Unlike alphaviruses, RV E1 did not require E2 for targeting to the Golgi where it was retained. E2 was however necessary for cell surface expression of E1. This study revealed that the processing and transport of RV structural proteins is quite different from alphaviruses and that the accumulation of antigens in the Golgi region may be significant in light of previous reports which suggest that RV buds from the internal membranes in some cell types.

Acylation↗
PubMed1989

In vitro and in vivo expression of rubella virus glycoprotein E2: the signal peptide is contained in the C-terminal region of capsid protein.

The 24 subgenomic mRNA of rubella virus (RV) specifies a polyprotein which is post-translationally processed to three structural protein species E1, E2, and capsid. E1 and E2 are membrane glycoproteins forming the virion spikes. In the polyprotein, E2 and E1 are both preceded by stretches of uncharged, mainly nonpolar amino acids which probably function as signal peptides mediating translocation into the endoplasmic reticulum. We have previously shown that translocation of E1 is reinitiated by a signal peptide located in the carboxy-terminus of E2 (Hobman et al., 1988, J. Virol. 62, 4259-4264). A cDNA from RV encoding the entire E2 gene fused to the capsid N-terminus has been constructed, allowing expression of RV E2 in vitro and in vivo. The resulting protein is efficiently translocated into canine pancreatic microsomes and is glycosylated when expressed in vitro. In vivo some of the N-linked sugars are processed to complex types. Cell surface immunofluorescence indicates that RV E2 is transported to the plasma membrane in COS cells. Oligonucleotide-directed mutagenesis was used to create a cDNA lacking 163 nucleotides immediately 5' to the E2 coding region. This deletion mutant failed undergo translocation into microsomes in vitro and was unstable when expressed in COS cells. The results imply that a signal peptide domain for RV E2 is contained in the carboxyl terminus of the capsid.

Animals↗
PubMed1988

Expression of rubella virus cDNA coding for the structural proteins.

A cDNA clone encoding the precursor polypeptide (Mr 115,000) to the nucleocapsid C (Mr 30,000) and two envelope glycoproteins E1 (Mr 58,000) and E2 (Mr 42,000-47,000) of rubella virus was inserted into a simian virus 40-derived eukaryotic expression vector. When the plasmid was introduced into COS cells, three proteins were synthesized. The expressed proteins were antigenically similar and identical in size to the authentic structural proteins of rubella virus. Expression in the presence of tunicamycin confirmed that E1 and E2 are glycoproteins. Unglycosylated E1 and E2 had Mrs of about 53,000 and 30,000, respectively. The mobility of the nucleocapsid protein was unaffected by tunicamycin. The locations of the translation start and stop codons for synthesis of the precursor to the structural proteins of rubella virus were determined by in vitro and in vivo expression studies. It was found that the first AUG codon at the 5' end of the rubella virus 24S cDNA acts as a start codon for translation. The stop codon was found to be 3183 bp from the start codon.

Animals↗
PubMed1988

In situ detection of rubella RNA and antigens in cultured cells.

We developed in situ hybridization and immunofluorescent procedures to detect rubella RNA and antigens in tissue-cultured cells infected with rubella virus. cDNA fragments of the rubella virus E1 structural gene were used as probes for in situ hybridization to detect rubella RNA sequences in Vero cells infected with rubella virus. Using antibodies against rubella proteins, indirect immunofluorescence detected rubella virus structural proteins in Vero cells infected with rubella virus. The immunofluorescence method has also been applied to study the expression of rubella polypeptide E1 in transfected COS cells and may be applied to the detection and study of persistent rubella virus infection in human tissues.

Animals↗
PubMed1988

Translocation of rubella virus glycoprotein E1 into the endoplasmic reticulum.

Rubella virus (RV) contains four structural proteins, C (capsid), E2a, E2b, and E1, which are derived from posttranslational processing of a single polyprotein precursor, p110. C protein is nonglycosylated and is thought to interact with RV RNA to form a nucleocapsid. E1 and E2 are membrane glycoproteins that form the spike complexes located on the virion exterior. Two different E1 cDNAs were used to analyze the requirements for translocation of E1 into the endoplasmic reticulum. Analysis of expression of these cDNAs both in vivo and in vitro showed that RV E1 was stably expressed and glycosylated in COS cells and correctly targeted into microsomes in the absence of E2 glycoprotein. The results provide experimental evidence that translocation of RV E1 glycoprotein into the endoplasmic reticulum is mediated by a signal peptide contained within the 69 carboxyl-terminal residues of E2.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic messenger RNA encoding the structural proteins E1, E2 and C.

The complete nucleotide sequence of the 24S subgenomic mRNA of wild-type M33 strain rubella virus has been determined. This RNA is 3,383 nucleotides in length excluding the 3'-terminal poly(A) tract. After the three multiple in-phase termination codons clustered in the 5' terminus of this RNA, there are 81 nucleotides of nontranslated nucleic acid followed by a reading frame of 2,978 nucleotides that encodes the 110 kD precursor of the structural proteins. The 3'-untranslated region is 263 nucleotides. The 110 kD polyprotein is processed to produce nucleocapsid C, the glycoproteins E2 and E1 in that order. Sites of post-translational cleavage to produce E2 and E1 were located using available N-terminal amino acid sequences. RNAs synthesized by transcription in vitro are effective messengers in the rabbit reticulocyte cell-free translation system. Post-translational processing of the structural proteins was observed in the cell-free system supplemented with microsomes from dog pancreas.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli.

A 3240-base-pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced. The sequence contains two open-reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase alpha and beta subunits, respectively. The coding sequences are preceded by a promoter-like structure and are most likely co-transcribed. Each coding sequence is preceded by a Shine-Dalgarno sequence. The amino-terminal amino acid sequences were determined from the purified alpha and beta subunits of the transhydrogenase. These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes. The predicted relative molecular masses of 53906 (alpha) and 48667 (beta) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit. These results indicate that transhydrogenase is an intrinsic membrane protein.

Amino Acid Sequence↗
PubMed1985

Structural analysis of a new GC-specific insertion element IS186.

A new insertion sequence, IS186, has been identified from Escherichia coli and sequenced. It contains 1336 base pairs with a terminal inverted repeat of 22 nucleotides. A long open reading frame, from an ATG codon at position 55 to a TAG termination codon at 1327, could code for a polypeptide of 424 amino acids. This element recognizes GC-rich regions as target sites for insertion.

Base Composition↗