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Biomedical subjects

A S Huang

Publications and source records attributed to A S Huang.

At least 19 recordsLinked to original sources
PubMed1991

Replication-defective viruses modulate immune responses.

By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.

Animals↗
PubMed1991

Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system.

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.

Animals↗
PubMed1990

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.

Animals↗
PubMed1990

Characterization of the polymerase activity associated with cultured peripheral blood mononuclear cells from patients with Kawasaki disease.

The particulate fractions of culture supernatants from peripheral blood mononuclear cells from 39 patients with Kawasaki disease (KD) were examined for the presence of particle-associated reverse transcriptase activity. The peak polymerase activity was significantly higher in cultures from KD patients compared to controls (mean = 6.4 versus 3.6 pmol of dTMP incorporated, p = 0.001). PBMC cultured between the 3rd and 9th wk after onset of fever were most likely to be associated with reverse transcriptase activity. Peak polymerase activity was positively associated with older age (r = 0.41, p = 0.01) and greater magnitude of the serum IgA response at 7-14 d after onset of fever (r = 0.45, p = 0.01) and IgM response at 6-9 wk after onset of fever (r = 0.46, p = 0.01). The appearance of enzyme activity was not associated with a decrease in viability of the cultured cells. A purified enzyme preparation showed radiolabel incorporation only with an RNA template with DNA primer. These data suggest that circulating mononuclear cells from KD patients may harbor a polymerase-associated agent and that these cells can be most readily detected in the early convalescent phase of KD from older patients who mount a marked humoral immune response.

Age Factors↗
PubMed1979

RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA.

The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.

Base Sequence↗
PubMed1979

Synthesis of a small RNA in cells coinfected by standard and defective interfering particles of vesicular stomatitis virus.

A small RNA, containing approximately 50 nucleotides, is synthesized by cells coinfected with standard vesicular stomatitis virus and its defective interfering (DI) particles. Infection of cells by standard virus or DI particles alone does not lead to synthesis of significant amounts of small RNA. The RNA is initiated at its 5' end with (p)ppXp and is not polyadenylylated at the 3' end despite a content of 51% adenosine. It has sequences complementary to the genome of a DI particle. The synthesis of the small RNA correlates with the replication of the genome of DI particles with molar ratio small RNA/genome RNA of DI particles greater than 50. When replication of DI genomes is prevented by the addition of cycloheximide or prior UV irradiation of DI particles, small RNA is not synthesized in coinfected cells. These results indicate that the small RNA is not the result of transcriptional initiation and that it may relate to interference mediated by DI particles.

Animals↗
PubMed1979

Surface analysis by bacterial adherence to virus-infected cells.

A rapid and inexpensive assay has been developed for the examination of new antigens on the surface of whole eucaryotic cells. This assay is based on the specific tagging of antigen-antibody complexes at the cell surface with Staphylococcus aureus. Cells that had been infected with vesicular stomatitis virus were exposed to specific antiserum, were then exposed to the bacteria, and finally were examined in the light microscope. Almost 100% of infected cells reacted positively. Two temperature-sensitive mutants of vesicular stomatitis virus were employed to determine which virus-specific antigen interacted with antibody and resulted in adherence of bacteria to the cells. Examination of cells infected with either ts G41 or ts 045 at the nonpermissive temperature showed that the assay by bacterial adherence measured the presence of only the viral glycoprotein at the cell surface. Internal viral antigens or readsorbed extracellular virions did not result in bacterial adherence. This assay has potential widespread significance for diagnostic medicine as well as for rapid and sensitive analyses of cell surfaces.

Animals↗
PubMed1978

The matrix (M) protein of vesicular stomatitis virus regulates transcription.

Temperature-sensitive mutants of vesicular stomatitis virus (VSV) belonging to complementation group III contain a lesion in the matrix (M) protein. This results in a 2--5 fold increase in transcription at the nonpermissive temperature. Co-infection of cells with one of these mutants and wild-type virus reverses this mutant phenotype. Separation of the transcriptional and translational products from mutant-infected cells reveals an overall increase in each of the viral mRNA species concomitant with degradation of the M protein at the nonpermissive temperature. The increase in mRNA, however, does not lead to increased synthesis of viral proteins. Quantitation of individual mRNA species indicates that M protein acts as a direct inhibitor of transcription as well as an attenuator of sequential transcription.

Genes, Dominant↗
PubMed1978

Shedding of the glycoprotein from vesicular stomatitis virus-infected cells.

In a culture of Chinese hamster ovary cells infected with vesicular stomatitis virus, there is specific shedding of viral antigens into the medium. This shedding appears to be unrelated to progeny formation or to cell lysis. Although all five of the virus-specific proteins are detected in the extracellular soluble fraction, the major antigen is the Gs protein. This protein has a molecular weight of 54,000. Indirect analysis of the content of sialic acid as well as peptide analysis of the Gs and G proteins of vesicular stomatitis virus suggest that the Gs protein is derived from the G protein by proteolysis. Both proteins are hydrophobic when analyzed by charge-shift electrophoresis. The presence of phenylmethylsulfonyl fluoride in the culture medium or the removal of serum from the culture medium partially reduces the shedding of Gs protein. Increased shedding of the Gs protein is seen when there is an unstable M or matrix protein synthesized by a temperature-sensitive mutant, tsG31. These results indicate that the G protein is cleaved at the cell surface, thus releasing Gs protein into the medium. Furthermore, the stability of G protein at the cell surface appears to be dependent on its association with the M protein.

Antigens, Viral↗
PubMed1978

Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores.

The proteins of vesicular stomatitis virus (VSV) were analyzed on the basis of charge as well as size in polyacrylamide gels containing urea and acetic acid. The phosphorprotein NS was resolved into two major species. The less phosphorylated NS1 species contained about 10% fewer phosphate residues than the second species, NS2. These two phosphorylated forms were compartmentalized both in the virus and in the infected cell cytoplasm. Cores from virions and the core-containing fraction of the infected cell cytoplasm contained only the NS1 form. All of the more highly phosphorylated NS2 form and some of the NS1 form were found to be free of cores, whether they were derived from virions or from the infected cell. Therefore, the degree of phosphorylation appeared to determine whether or not the NS protein became bound to VSV cores. Moreover, the amount of bound NS1 protein relative to nucleocapsids increased as the pH of the culture medium was raised from 6.6 to 7.4. Because an increased in pH increases VSV replication (Fiszman et al., J. Virol. 13:801-808, 1974; Palma and Huang, in W.S. Robinson and C.F. Fox, ed., Mechanisms of Virus Disease, ICN-UCLA Symposia, p. 87-100, 1974), the NS1 protein may either regulate overall VSV RNA synthesis or regulate the switch between transcription and replication.

Cell Line↗
PubMed1977

Effects of temperature and antibody on the cyclid growth of vesicular stomatitis virus.

To see the effects of temperature on the interrelated cyclic production of standard and defective interfering (DI) particles of vesicular stomatitis virus, a temperature-sensitive (ts) G114 mutant was passaged successively at different temperatures and the production of the two types of viral particles as well as the ability of Chinese hamster ovary cells to survive each passage was continuously monitored. When the temperature was nonpermissive for standard virus, the synthesis of both standard and defective interfering particles was inhibited. When revertants appeared in the population, their ability to take over the infection depended on the permissiveness of the temperature for the temperature-sensitive mutant. At permissive temperatures periodic inhibition of both types of standard viruses was maintained by the production of defective interfering particles. Reverents did not become a majority of the population due to this periodic inhibition. When the conditions were nonpermissive for the mutant, revertants became the major standard virus in the population within a few passages. These findings can be understood if conditions of high and low multiplicities are dissected out together with a thorough understanding of the individual properties of each of the viral particles and of the result of interactions between them. In the presence of antiserum which neutralized only 90% of the viral particles, cyclic production of standard virus occurred, with a decline in the total amount of virus produced after each cycle. Therefore, in the presence of limiting concentrations of antiserum, the virus appeared to be able to establish a persistent cyclic growth pattern.

Animals↗
PubMed1977

RNA synthesis of vesicular stomatitis virus. VII. Complete separation of the mRNA's of vesicular stomatitis virus by duplex formation.

Full-length virion RNA and complementary mRNA's of vesicular stomatitis virus can be annealed to each other, digested with RNases, and then separated as five unique duplex RNA molecules on polyacrylamide slab gels. Similar RNA duplexes were detected whether mRNA or virion RNA was the radioactive component and whether the mRNA was synthesized in vitro or in vivo. The sharp banding pattern of these RNA molecules was dependent on treatment with RNase T2, suggesting that removal of poly(A) is necessary. Identification of the coding region contained in each RNA duplex was based on their previous identification as single-stranded mRNA on formamide-containing, polyacrylamide gels. Because the two smallest mRNA'S had not been previously separated, their identification was based on their in vitro transcriptional gene order. In the order of increasing mobilities on the slab gels, the RNA duplexes are identified as the hybrid of the region of the genome RNA hybridized to the complementary mRNA coding for the large protein, the glycoprotein, the nucleocapsid protein, the core-associated NS protein, and the matrix protein (L,G,N,NS, and M). Several lines of evidence support the presence of undegraded complete mRNA, excluding poly(A), in these RNA duplexes. Also, the two smallest mRNA's, separated by duplex formation, were denatured, and their individual oligonucleotide fingerprints were determined. From chemical length determinations, the molecular weights of the mRNA, minus poly(A), are 2.78 X 10(5) and 2.5 X 10(5), respectively, for the mRNA's of the NS and M proteins.

Cell Line↗