Biomedical subjects

M K Spriggs

Publications and source records attributed to M K Spriggs.

51 records · Page 3Linked to original sources

PubMed1992

Beta 2-microglobulin-, CD8+ T-cell-deficient mice survive inoculation with high doses of vaccinia virus and exhibit altered IgG responses.

Transgenic mice lacking an intact beta 2-microglobulin (beta 2m) gene fail to express major histocompatibility complex (MHC) class I proteins on the cell surface and, as a result, are virtually devoid of CD4- CD8+ lymphocytes. These animals provide a unique model system for directly assessing the role of CD8+ lymphocytes in the modulation of viral infection in vivo. beta 2m- CD8- mice and their normal littermates were inoculated at the base of the tail with the WR strain of vaccinia virus and monitored for serum antibody and lesion formation. Both groups developed similar lesions in response to a broad virus dose range, and all animals had completely recovered by day 28 after inoculation. Isotype-specific immunoglobulin levels were determined for each animal on day 7 and day 14 after primary inoculation, and again 7 days after a virus challenge. The virus-specific IgG1, IgG2a, and IgG2b levels were significantly different in the beta 2m-/- group (20-, 9-, and 30-fold lower, respectively, on day 7 after challenge) compared with the beta 2m+/- group. Virus-specific serum IgM levels for both groups remained similar throughout the experiment. In a separate experiment, beta 2m-/- mice were immunized with a nonviral antigen, 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin, and both total and antigen-specific isotype-specific immunoglobulin titers were determined. Total IgG1, IgG2a, IgG2b, and IgG3 tended to be lower overall in the beta 2m-/- mice compared with beta 2m+/- littermates. In contrast, total and antigen-specific IgE titers were similar in the two groups. These data indicate that CD8+ lymphocytes are not required to clear high doses of vaccinia virus, and they suggest that beta 2m-/- mice are less efficient at antigen-specific IgG production than their beta 2m+/- littermates.

Animals↗

PubMed1992

Molecular characterization of the interleukin-1 receptor (IL-1R) on monocytes and polymorphonuclear cells.

Primary human monocytes and monocytic cells express an interleukin 1 receptor (IL-1R) which is similar in molecular weight and IL-1 binding characteristics to the IL-1R expressed on B lymphocytes (type II). Northern blot analysis of monocytic cells using a cDNA probe from the recently isolated type II IL-1R indicates that this mRNA is detectable by 4 h and accumulates for at least 24 h following treatment with IL-1R inducing drugs. The time course of induction of this mRNA is slower than that of the type I IL-1R mRNA which is also transcribed in monocytic cells but does not appear to be translated. Sequence analysis of a monocyte-derived cDNA corresponding to the type II IL-1R mRNA shows that the monocyte and B-cell mRNAs are identical. Comparison of monocyte IL-1R peptide maps with those of the type II IL-1R suggests that the two surface IL-1R are identical. This was confirmed serologically using a polyclonal antiserum raised against the type II IL-1R. Data are presented which indicate that primary human neutrophils can also be induced to express abundant type II IL-1R.

Blotting, Northern↗

PubMed1990

Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA.

Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.

Cell Line↗

PubMed1990

Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein.

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.

Amino Acid Sequence↗

PubMed1988

Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses.

Vaccines against parainfluenza (PIV) and respiratory syncytial viruses (RSV) that are currently being developed include both live and subunit vaccines. Candidate live PIV vaccines that have been found to be attenuated and efficacious in rodents or primate models are (1) cold-adapted, temperature-sensitive mutants of PIV-type 3 that have been serially passaged at low temperature (20 degrees C) in simian kidney tissue culture; (2) protease-activation mutants (PIV-1-Sendai), which have mutations that decrease the cleavability of their F glycoprotein by host cell protease; (3) an animal virus, bovine PIV-3 virus, which is antigenically related to the human PIV-3 virus, and (4) vaccinia recombinant viruses bearing RSV or PIV-3 glycoproteins. Subunit RSV and PIV-3 viruses are being produced and evaluated as immunogens. A major concern with these vaccines is the possibility of disease potentiation following virus infection as occurred previously with formalin-inactivated measles and RSV vaccines. Studies indicate that PIV-3 and RSV glycoprotein vaccines are immunogenic and efficacious in animals but insufficient data exist to estimate their capacity to potentiate disease. However, since a cotton rat model is available to detect potentiated disease resulting from infection of cotton rats previously immunized with formalin-inactivated RSV vaccine, it is now possible to systematically evaluate new vaccines in experimental animals for disease potentiation before studies are initiated in humans. It is likely within the next several years that one or more of these PIV or RSV vaccines will be tested in humans for safety and immunogenicity.

Animals↗

PubMed1988

Live viral vaccines for respiratory and enteric tract diseases.

In its programme for accelerated development of vaccines for viral respiratory and enteric tract diseases the WHO has assigned a very high priority to respiratory syncytial virus (RSV), parainfluenza viruses and rotaviruses. There is also some interest in alternative approaches to immunization against influenza viruses because of the failure of inactivated vaccines to provide complete and reasonably durable immunity. Current attempts to develop satisfactorily attenuated viruses for use in prevention of disease caused by the above viral pathogens are described.

Gastrointestinal Diseases↗

PubMed1988

Immunization with vaccinia virus recombinants that express the surface glycoproteins of human parainfluenza virus type 3 (PIV3) protects patas monkeys against PIV3 infection.

Patas monkeys (Eryphrocebus patas) were immunized intradermally with two vaccinia virus recombinants that individually express the hemagglutinin-neuraminidase glycoprotein or the fusion glycoprotein of human parainfluenza virus type 3 (PIV3). These immunizations induced a high titer of PIV3 serum-neutralizing antibodies. At 1 month after immunization, monkeys were challenged intratracheally with PIV3. Subsequent virus replication was reduced in these monkeys by 3.2 log10 and 1.9 log10 (mean peak virus titers) in the upper and lower respiratory tracts, respectively, compared with control animals. The average duration of virus shedding was also reduced from 9.0 to 3.4 days in the upper respiratory tract and from 5.3 to 1.2 days in the lower respiratory tract. These findings demonstrate that a single intradermal dose of live recombinant vaccinia viruses can significantly restrict the replication of a virus which primarily infects the epithelial cells of the respiratory tract.

Animals↗

PubMed1987

Gene overlap and site-specific attenuation of transcription of the viral polymerase L gene of human respiratory syncytial virus.

Sequence analysis of the gene encoding polymerase L protein of respiratory syncytial virus showed that L-gene transcription initiates within its upstream neighbor, the gene encoding the 22-kDa protein 22K. This is an exception to the canonical mode of sequential transcription of nonoverlapping genes described for other nonsegmented negative-strand RNA viruses. As a consequence of the gene overlap, the termination/polyadenylylation signal for the 22K gene is located at nucleotides 56-68 within the L gene. L-gene transcription yielded two RNAs: an abundant, truncated, polyadenylylated transcript resulting from termination at the internal signal and a markedly less abundant large polyadenylylated transcript representing the complete L gene. This result showed that the internal termination/polyadenylylation signal is an attenuator of L-gene transcription.

Amino Acid Sequence↗

PubMed1987

The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins.

Two major antigenic subgroups (designated A and B) have been described for human respiratory syncytial virus (RSV). Previously, on the basis of reactivity patterns with monoclonal antibodies, the greatest intersubgroup variation was shown to occur in the G protein, the putative attachment glycoprotein. To delineate the molecular basis for this variation, we have determined the nucleotide and deduced amino acid sequences of the G mRNAs and proteins representing a subgroup A (Long strain) and a subgroup B (18537 strain) virus. These sequences were compared to the available G mRNA sequence for another subgroup A (A2 strain) virus. The Long G protein shared 94% amino acid identity with the A2 G protein. In contrast, the 18537 G protein shared only 53% amino acid identity with the A2 sequence; interestingly, most of the sequence divergence occurred in the proposed extracellular domain of the G protein. This extensive divergence for the G protein was significantly greater than that observed for other RSV proteins. Despite this considerable divarication, the proposed extracellular domains of the G proteins contained a single region of highly conserved sequence and secondary structure that may represent a conserved structural or function domain, perhaps involved in attachment to cellular receptors. Furthermore, this conserved region may comprise part of an epitope that is shared between the two subgroup G proteins and may significantly contribute to the fact that, despite extensive overall amino acid sequence divergence, the RSV G proteins maintain significant antigenic relatedness.

Amino Acid Sequence↗

PubMed1987

Sequence analysis of the matrix protein gene of human parainfluenza virus type 3: extensive sequence homology among paramyxoviruses.

The sequences of the human parainfluenza virus type 3 (PIV3) matrix (M) mRNA [1150 nucleotides exclusive of poly(A)] and predicted M protein (353 amino acids) were determined by sequence analysis of cloned cDNA and viral genomic RNA. The gene-end sequence of the M gene differed from the semi-conserved gene-end sequence of the other PIV3 genes by an apparent insertion of eight nucleotides. The PIV3 M protein shared high sequence homology with Sendai virus and moderate homology with measles virus and canine distemper virus. Statistical analysis of the available sequences showed that the M protein was the most highly conserved parainfluenza viral protein.

Amino Acid Sequence↗

PubMed1987

Expression of the F and HN glycoproteins of human parainfluenza virus type 3 by recombinant vaccinia viruses: contributions of the individual proteins to host immunity.

cDNA clones containing the complete coding sequences for the human parainfluenza virus type 3 (PIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes were inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of the P7.5 early-late vaccinia virus promotor. The recombinant vaccinia viruses, designated vaccinia-F and vaccinia-HN, expressed glycoproteins in cell culture that appeared to be authentic with respect to glycosylation, disulfide linkage, electrophoretic mobility, cell surface expression, and, in the case of the HN protein, biological activity. Cotton rats inoculated intradermally with vaccinia-HN developed serum neutralizing antibody titers equal to that induced by respiratory tract infection with PIV3, whereas animals receiving vaccinia-F had threefold lower neutralizing antibody titers. A single immunization with either recombinant vaccinia virus induced nearly complete resistance in the lower respiratory tract of these animals. With regard to protection in the upper respiratory tract, animals immunized with vaccinia-HN or vaccinia-F exhibited reductions in PIV3 replication of greater than 3,000-fold and 6-fold, respectively. This large difference (greater than 500-fold) in reduction of PIV3 replication in the upper respiratory tract was in contrast to the relatively modest difference (3-fold) in serum neutralizing antibody titers induced by vaccinia-HN versus vaccinia-F. This dissociation between the level of neutralizing antibodies and protection suggested that immunity to PIV3 is complex, and that immune mechanisms other than serum neutralizing antibodies make important contributions to resistance to infection. Overall, under these experimental conditions, vaccinia-HN induced a substantially more protective immune response than did vaccinia-F.

Animals↗

PubMed1986

Fusion glycoprotein of human parainfluenza virus type 3: nucleotide sequence of the gene, direct identification of the cleavage-activation site, and comparison with other paramyxoviruses.

The complete sequences of the fusion (F) mRNA and protein of human parainfluenza virus type 3 (PF3) were determined from overlapping cDNA clones. To confirm the cDNA sequence, the complete sequence of the F gene was determined independently by dideoxynucleotide sequencing of genomic RNA using synthetic oligonucleotide primers. The mRNA contains 1845 nucleotides, exclusive of poly (A), has an unusually long (193-nucleotide) 5' nontranslated region, and encodes an F0 protein of 539 amino acids. The site within F0 of the proteolytic cleavage that activates fusion activity was established by direct amino acid sequencing of the NH2 terminus of the F1 subunit. The PF3 F0 protein shares major structural features with the previously sequenced F0 proteins of Sendai virus (murine parainfluenza type 1) and simian virus 5 (SV5, canine parainfluenza type 2), including: similarity in overall length; similarity in location of the site of the activating proteolytic cleavage; the presence of an NH2-terminal signal peptide and COOH-proximal membrane anchor; strong conservation of the sequence at the NH2 terminus of the F1 subunit; and nearly exact conservation in the number and positions of cysteine residues. Alignment of the F0 protein sequences of PF3 with those of Sendai, SV5, and respiratory syncytial virus (RSV) using a matrix that scores both amino acid matches and mismatches provided highly significant statistical evidence that all four proteins are related. The order of decreasing relatedness to PF3 was found to be: Sendai virus, SV5, and RSV.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequences for the gene junctions of human respiratory syncytial virus reveal distinctive features of intergenic structure and gene order.

Complete sequences for the intergenic regions of the genome of human respiratory syncytial virus were obtained by dideoxynucleotide sequencing using synthetic oligonucleotides. These experiments established that the 10 respiratory syncytial viral genes are arranged, without additional intervening genes, in the order 3' 1C-1B-N-P-M-1A-G-F-22K-L 5'. For the first nine genes, the exact gene boundaries were identified by comparison of the genomic sequences with previously determined mRNA sequences. The intergenic regions varied in length from 1 to 52 nucleotides and lacked any obvious conserved features of primary or secondary structure except that each sequence ended (3' to 5') with an adenosine residue. The exact start site of the 10th gene, the L gene, was not determined. However, RNA blot hybridization using a synthetic oligonucleotide designed from the genomic sequence mapped the L gene to within 54 nucleotides of the end of the penultimate 22K gene. The lack of conservation of chain length and nucleotide sequence for the respiratory syncytial viral intergenic regions, together with the complexity of the genetic map, contrasts with previous observations for other nonsegmented negative-strand viruses.

Base Sequence↗

PubMed1986

Sequence analysis of the P and C protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins.

The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.

Amino Acid Sequence↗

PubMed1986

Human parainfluenza virus type 3: messenger RNAs, polypeptide coding assignments, intergenic sequences, and genetic map.

cDNA clones of mRNAs for the major nucleocapsid protein (NP), the nucleocapsid P protein plus the nonstructural C protein (P+C), and the matrix protein (M) of human parainfluenza virus type 3 (PF3) were identified by hybrid arrest and hybrid selection of in vitro translation. Previously, cDNA clones were identified and sequenced for the hemagglutinin-neuraminidase glycoprotein (HN) and the fusion glycoprotein (F) mRNAs (N. Elango, J. E. Coligan, R. C. Jambou, and S. Venkatesan, J. Virol. 57:481-489, 1986; M. K. Spriggs, R. A. Olmsted, S. Venkatesan, J. E. Coligan, and P. L. Collins, Virology 152:241-251, 1986). Synthetic oligonucleotides, designed from nucleotide sequences of the cDNAs, were used to direct dideoxynucleotide sequencing of gene junctions in PF3 genomic RNA (vRNA). From sequencing of vRNA, a sixth viral gene was detected and identified as the large nucleocapsid protein (L) gene by hybridization of a synthetic oligonucleotide to intracellular PF3 mRNAs separated by gel electrophoresis. The order of the six PF3 genes on vRNA was 3'-NP-P+C-M-F-HN-L-5'. The five intergenic regions consisted of the trinucleotide 3'-GAA. The PF3 genes initiated with semiconserved 10-nucleotide gene-start sequences and terminated with semiconserved 12-nucleotide gene-end sequences. The M gene terminated with an aberrant gene-end sequence; analysis of intracellular mRNA showed that this aberrant sequence correlated with a disproportionately high accumulation of readthrough mRNA. These studies showed that PF3 encodes six unique mRNAs (NP, P+C, M, F, HN, and L) that encode seven proteins (NP, P, C, M, F, HN, and L) and provided evidence of a close relationship between PF3 and Sendai (murine parainfluenza type 1) viruses.

Base Sequence↗