Biomedical subjects

S R Weiss

Publications and source records attributed to S R Weiss.

At least 91 records · Page 5Linked to original sources

PubMed1995

Characterization of the leader papain-like proteinase of MHV-A59: identification of a new in vitro cleavage site.

Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteinases encoded within the first open reading frame (ORF 1a) of the replicase gene. The more 5' of these domains, the leader papain-like proteinase, is responsible for the cleavage of the amino terminal protein, p28. The core of this proteinase domain was defined to between amino acids 1084 and 1316 from the beginning of ORF 1a. Through the use of deletion analysis coupled with in vitro expression, we studied the role of the coding region between p28 and the leader papain-like proteinase on the cleavage of p28 itself. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels. Reduced p28 production resulting from a 0.4-kb deletion positioned between p28 and the proteinase domain suggests an involvement of this region in catalytic processing. Some mutants displayed cleavage patterns indicative of a second cleavage site. Interestingly, this new cleavage site identified in vitro maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59-infected cells. Mutagenesis of the catalytic His1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like proteinase encoded in ORF 1a.

Amino Acid Sequence↗

PubMed1995

Identification and characterization of a 65-kDa protein processed from the gene 1 polyprotein of the murine coronavirus MHV-A59.

A 65-kDa protein has been detected in mouse hepatitis virus A59 (MHV-A59)-infected DBT cells using polyclonal antibodies directed against polypeptides encoded by the 5' 1.8 kb of gene 1. The presence of this 65-kDa protein (p65) was previously predicted from immunoprecipitation studies of gene 1 expression in MHV-A59-infected DBT cells with other antisera (1). p65 was rapidly labeled in virus-infected cells at late times of infection; however, its cleavage from the polyprotein was significantly delayed compared to the amino-terminal gene 1 polyprotein cleavage product, p28. Similar to p28, p65 was cleaved from the growing polyprotein without detectable intermediate precursors. Kinetic analysis of p65 with specific antibodies indicates that p65 is immediately adjacent to p28 in the gene 1 polyprotein. The proteolytic activity responsible for the carboxy-terminal cleavage of p65, as well as the function of the p65 protein, remains to be determined.

Animals↗

PubMed1995

Intracellular localization of polypeptides encoded in mouse hepatitis virus open reading frame 1A.

We have investigated the intracellular localization of several of the proteolytic cleavage products derived from the 5' portion of mouse hepatitis virus (MHV) gene 1. Antisera UP1 recognizes the N-terminal ORF1a cleavage product p28. Immunofluorescent staining of cells with this antisera resulted in a diffuse punctate pattern of cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm. Immunofluorescent staining of infected cells with antisera which recognize polypeptides p240 and p290 stained discrete vesicular perinuclear structures suggesting that these proteins localized to the Golgi. This was confirmed by double immunofluorescent staining of BHK cells expressing the MHV receptor (BHK-R) with a Golgi specific antibody in addition to our anti-MHV ORF1a antibodies. Antisera UP102 recognizes p28 and the immediately downstream p65 gene product. Double immunofluorescent staining of MHV infected BHK-R cells with UP102 labeled discrete vesicular structures overlapping the Golgi complex. In addition there was punctate staining more widely distributed in the cytoplasm. The simplest explanation for this pattern is that p65 is also localized to the Golgi region of the cell, whereas p28 is more widespread. Plasmids containing the first 4.7 and 6.75 kb of ORF 1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Images obtained by immunofluorescent staining of transfectants with our anti-ORF1a antisera are similar to those obtained during infection with A59. These studies indicate that the signals which direct p290 to the Golgi are likely contained between the C-terminus of p28 and ORF1a residue 1494.

Animals↗

PubMed1995

Characterization of the leader papain-like protease of MHV-A59.

Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteases encoded within the first open reading frame of the replicase gene. The more 5' of these domains, the leader papain-like protease, is responsible for the cleavage of the amino terminal protein, p28. We have defined the core of this protease to between amino acids 1075 and 1344 from the beginning of ORF 1a. Deletion analysis coupled with in vitro expression, was used to study p28 cleavage by this leader protease. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels in some of them. Reduced p28 production resulting from a 0.4 kb deletion positioned between p28 and the protease domain suggests an involvement of this region in catalytic processing. Some mutants display cleavage patterns indicative of a second cleavage site. Interestingly, this newly identified cleavage site maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59 infected cells. Mutagenesis of the catalytic H1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like protease encoded in ORF 1a.

Animals↗

PubMed1995

Genomic regions associated with neurotropism identified in MHV by RNA-RNA recombination.

Infection of mice with a neurotropic strain of MHV (MHV-A59), a non-neurotropic strain of MHV (MHV-2), and a set of recombinant viruses (kindly provided by Dr. Michael Lai) were used to map genetic determinants of viral neurotropism and demyelination. Following intracerebral (IC) inoculation of 4-week old C57B1/6 mice, 1LD50 of MHV-A59 produced acute meningoencephalitis and hepatitis, and subsequently chronic CNS demyelinating disease. IC inoculation of 1LD50 of MHV-2 produced acute hepatitis without CNS disease. Recombinants ML-3, ML-11, ML-7, ML-8, ML-9 and ML-10 produced acute encephalitis similar to MHV-A59. According to previous oligonucleotide fingerprinting analysis the only common denominator of the neurotropic recombinant viruses was an M gene derived from MHV-A59. Sequencing of PCR-amplified viral S and M genes confirmed that the M genes of neurotropic viruses are derived from A59 while the S genes of neurotropic viruses are either derived from MHV-2 or from A59. In tissue culture, ML-11, ML-3 and MHV-2 are fusion negative, while A59, ML-7, ML-8 and ML-10 are fusion positive. Thus, neurotropism in MHVs is not linked to fusion or the S gene. Moreover, the M gene may be a significant determinant of neurotropism and acute encephalitis.

Animals↗

PubMed1995

Identification and analysis of MHV-A59 P28 cleavage site.

During translation of Murine hepatitis virus (MHV-A59) ORF1a, p28, the N-terminal polypeptide is cleaved from the growing polypeptide chain. Amino terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly247 and Val248. Site directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg246 (P2) and Gly247 (P1) nearly eliminated cleavage of p28. Single amino acid substitutions of other residues between P7 and P2' were generally permissive for cleavage although a few changes did greatly reduce proteolysis. The amino acids around the p28 cleavage site represent a new sequence recognized by a virus encoded papain-like proteinase.

Amino Acid Sequence↗

PubMed1995

Hepatitis mutants of mouse hepatitis virus strain A59.

MHV-A59 causes acute meningoencephalitis and hepatitis in susceptible mice, and a persistent productive, but nonlytic, infection of cultured glial cells. We have shown previously that viruses isolated from persistently infected glial cell cultures have a fusion-defective phenotype and were impaired in their abilities to cause hepatitis compared to wild-type MHV-A59. Two mutants chosen for detailed study, B11 and C12, display two distinct hepatitis phenotypes. The ability of B11 to replicate in the liver was dependent on infectious dose and route of inoculation, while C12 consistently displayed decreased liver titers regardless of dose and route of inoculation. Sequence analysis of wild-type, mutant and revertant S proteins indicates that 1) a mutation in the N terminal subunit of S, resulting in a glutamine to leucine amino acid substitution (Q159L), may affect ability to cause hepatitis and 2) a cleavage site mutation (H716D) which determines fusogenicity is not responsible for the altered hepatitis phenotype. Sequence analysis indicated that hepatitis-producing revertants did not revert at mutation Q159L, although it is possible that a mutation in the heptad repeat domain of S2 may compensate for the mutation in S1. Since B11, C12 and a nonattenuated fusion mutant (B12) have identical S protein sequences, there must be additional mutations outside of S which influence both virulence and ability to replicate in the liver.

Animals↗

PubMed1995

Contingent tolerance to the anticonvulsant effects of carbamazepine: relationship to loss of endogenous adaptive mechanisms.

Contingent tolerance to the anticonvulsant effects of carbamazepine on amygdala kindled seizures develops when the drug is repeatedly given prior to but not after the electrical stimulation. Such tolerance can be reversed by kindling the rats for several days without drug or even by continuing to give the drug but after each seizure has occurred. Contingent tolerance can be slowed by reducing the electrical stimulus intensity and by chronic continuous (as opposed to repeated paired) drug administration. Contingent cross-tolerance has been demonstrated from carbamazepine to PK11195 (a drug active at peripheral-type benzodiazepine receptors) and valproate, but not to clonazepam and diazepam (two drugs active at central-type benzodiazepine receptors) or phenytoin. Endogenous physiological changes occur in conjunction with contingent tolerance, exemplified by the decrease in seizure threshold that returns to normal upon reversal of tolerance. We suggest that contingent tolerance is associated with a loss of seizure-induced adaptations, since many biochemical changes that occur following seizures (or in non-tolerant animals given drug after seizures) are not observed in tolerant animals. These include a loss of seizure-induced up-regulation of GABAA receptors and a loss of increases in mRNA expression for corticotropin-releasing-factor (CRF), thyrotropin-releasing-hormone (TRH), neuropeptide Y (NPY), glucocorticoid receptors and brain-derived neurotrophic factor (BDNF). Thus, several putative seizure-induced anticonvulsant adaptations, such as increases in GABAA receptors and TRH and NPY mRNA fail to occur in tolerant animals. These findings are consistent with the novel observations that, paradoxically, seizures themselves appear to facilitate the anticonvulsant effects of carbamazepine or diazepam on amygdala kindled seizures. That is, animals given a 'vacation' from seizures show a decreased response to these agents, a phenomenon we have called the 'time-off seizure' effect. Thus, seizures are postulated to induce adaptive changes that influence seizure thresholds and potentiate the anticonvulsant effects of exogenously administered drugs such as carbamazepine and diazepam. Taken together, these data suggest that seizures are associated with endogenous adaptations lasting days to weeks and that a selective failure of some of these to occur during contingent drug administration may underlie the development of contingent tolerance. These observations suggest tht endogenous illness-related mechanisms may participate both in the therapeutic responses of some agents and that their failure to occur could relate to loss of drug efficacy via tolerance; these processes may reveal new potential targets for therapeutic intervention.

Adaptation, Physiological↗

PubMed1995

Mouse hepatitis virus A59-induced demyelination can occur in the absence of CD8+ T cells.

Mouse hepatitis virus causes a chronic demyelinating disease in C57BL/6 mice. While early studies suggested demyelination is due to direct cytolytic effects of virus on oligodendrocytes, there is increasing evidence for the involvement of the immune system in the mechanism of demyelination. In this study we have asked whether demyelination can occur in the absence of functional MHC class I expression and CD8+ T cells. We infected transgenic mice lacking expression of beta 2 microglobulin (beta 2 M -/- mice) with MHV-A59. In beta 2M-/- mice, virus was much more lethal than in either of the parental strains used to produce the mice; furthermore, while clearance from the CNS did occur in beta 2M-/- mice, it was slower than in C57BL/6 mice. This is consistent with the importance of CD8+ cells in viral clearance. Because of the increased sensitivity of the beta 2M-/- mice to infection, only low levels of virus could be used to evaluate chronic disease. Even at these low levels, demyelination did occur in some animals. To compare infection in beta 2M-/- and C57BL/6 mice we used a higher dose of an attenuated variant of MHV-A59, C12. The attenuated variant induced less demyelination in C57BL/6 mice compared to wild type A59, but the levels observed were not significantly different from those seen in beta 2M-/- mice. Thus, MHV-induced demyelination can occur in some animals in the absence of MHC class I and CD8+ cells.

Animals↗

PubMed1995

Identification of the murine coronavirus p28 cleavage site.

Mouse hepatitis virus strain A59 encodes a papain-like cysteine proteinase (PLP-1) that, during translation of ORF1a, cleaves p28 from the amino terminus of the growing polypeptide chain. In order to determine the amino acid sequences surrounding the p28 cleavage site, the first 4.6 kb of murine hepatitis virus strain A59 ORF1a was expressed in a cell-free transcription-translation system. Amino-terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly-247 and Val-248. Site-directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg-246 (P2) and Gly-247 (P1) nearly eliminated cleavage of p28. Single-amino-acid substitutions of other residues between P7 and P2' were generally permissive for cleavage, although a few changes did greatly reduce proteolysis. The relationship between the p28 cleavage site and other viral and cellular papain proteinase cleavage sites is discussed.

Amino Acid Sequence↗

PubMed1995

Reduction of LDL cholesterol by 25% to 60% in patients with primary hypercholesterolemia by atorvastatin, a new HMG-CoA reductase inhibitor.

This 6-week, double-blind clinical trial evaluated lipid parameter responses to different dosages of atorvastatin in patients with primary hypercholesterolemia. Atorvastatin is a new 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor under development. After completing an 8-week placebo-baseline dietary phase, 81 patients were randomly assigned to receive either placebo or 2.5, 5, 10, 20, 40, or 80 mg atorvastatin once daily for 6 weeks. Plasma LDL cholesterol reductions from baseline were dose related, with 25% to 61% reduction from the minimum dose to the maximum dose of 80 mg atorvastatin once a day. Plasma total cholesterol and apo B reductions were also dose related. Previously, reductions in LDL cholesterol of the magnitude observed in this study have been seen only with combination drug therapy. In this study, atorvastatin was well tolerated by hyperlipidemic patients, had an acceptable safety profile, and provided greater reduction in cholesterol than other previously reported HMG-CoA reductase inhibitors.

Adolescent↗

PubMed1994

Mouse hepatitis virus gene 5b protein is a new virion envelope protein.

Highly purified radiolabeled mouse hepatitis virus (MHV) A59 contained a previously overlooked protein which coelectrophoreses with the gene 5b product immunoprecipitated from infected cells. The gene 5b protein is post-translationally acylated. Rabbit antibody raised against a recombinant gene 5b protein expressed in Escherichia coli neutralized viral infectivity in the presence of complement, although not in the absence of complement. Immunofluorescent staining of MHV-infected cells with two anti-peptide antibodies revealed that the gene 5b product is membrane-associated and is transported to the cell surface, findings consistent with the prediction of a membrane-spanning segment in the gene 5b polypeptide. These results suggest strongly that the gene 5b polypeptide represents a new MHV virion envelope protein which is homologous to the TGEV ORF 4 and IBV 3c proteins.

Antigens, Viral↗

PubMed1994

Contingent tolerance to carbamazepine: alterations in TRH mRNA and TRH receptor binding in limbic structures.

Tolerance to carbamazepine's anticonvulsant effects on amygdala kindled seizures occurs contingently, that is, only when carbamazepine is given prior to, but not after the seizure occurs. Biological correlates of contingent tolerance were examined using in situ hybridization and receptor binding techniques for thyrotropin-releasing hormone (TRH) mRNA and TRH receptor binding. Rats were fully kindled and given daily injections of carbamazepine (15 mg/kg, i.p.) either 15 min before (CBZ-before) or after (CBZ-after) amygdala stimulation until the CBZ-before rats became tolerant. Kindled rats were matched so that the two groups had an equal number of seizures and doses of CBZ. Three other groups were also used for comparison: kindled rats that received vehicle injections, and sham-kindled animals treated with either vehicle or CBZ. Rats were sacrificed 4 h after the last seizure or sham stimulation. Both sham-kindled rat groups had barely detectable levels of TRH mRNA. In the CBZ-after (non-tolerant) and vehicle-kindled rats, TRH mRNA levels were increased in the dentate gyrus, pyriform, entorhinal, and perirhinal cortices. In contrast to the other kindled animals, the CBZ-before rats (tolerant) had dramatically diminished TRH mRNA levels bilaterally in the dentate gyrus and pyriform cortex, and ipsilateral to the stimulation in the entorhinal cortex. Decreases in TRH receptor binding were demonstrated autoradiographically in the dentate gyrus and perirhinal cortex in all of the kindled groups with no differences between tolerant and non-tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Glucocorticoid treatment increases the ability of CRH to induce seizures.

We examined whether glucocorticoids could enhance the ability of corticotropin-releasing hormone (CRH) to induce seizures. Rats were treated with systemic glucocorticoids (dexamethasone, 100 micrograms) or vehicle for either 3 days (chronic) or 2 h (acute) before intracerebroventricular CRH (3 or 10 micrograms) or saline injections and then monitored for 8 h following each injection. Our results suggest that chronic, but not acute, glucocorticoid pretreatment increases the likelihood of CRH-induced seizures.

Aggression↗

PubMed1994

Electrical kindling is associated with increases in amygdala acetylcholine levels: an in vivo microdialysis study.

Using brain microdialysis, acetylcholine (Ach) levels were assessed in the nucleus amygdaloideus lateralis of electrically kindled rats using a cholinesterase inhibitor in the perfusion fluid to prevent Ach breakdown. During kindling development, when the animals displayed afterdischarges lasting more than 30 s but no seizures, a significant increase in Ach levels (+66%) was observed after electrical stimulation. Ach levels were also elevated after a stage 5 kindled seizure (+48%), with no additional increment compared to rats experiencing only afterdischarges. Chronic diazepam administration prevented kindling development and the increment in Ach overflow; however, diazepam itself (following acute administration) tended to decrease Ach levels in the amygdala. These findings are convergent with pharmacological data suggesting that changes in the cholinergic function may be important especially during amygdaloid kindling development.

Acetylcholine↗

PubMed1994

Mouse hepatitis virus strain A59 RNA polymerase gene ORF 1a: heterogeneity among MHV strains.

Gene 1, the putative RNA replicase gene of coronaviruses, is expressed via two large overlapping open reading frames (ORF 1a and ORF 1b). We have determined the nucleotide sequence of ORF 1a, encoded within the first 13.7 kb of gene 1, for the coronavirus mouse hepatitis virus strain A59 (MHV-A59). Putative papain-like protease domains, a picornavirus 3C-like protease domain, two hydrophobic domains, and a domain "X" of unknown function, previously identified in other coronaviruses (1-3), are also present in ORF 1a of MHV-A59. Comparison between the ORF 1a sequence of MHV-A59 and the published sequence of the JHM strain of MHV (2) showed a high degree of similarity with the exception of several short regions. We sequenced one region of MHV-JHM that contained an 18 amino acid insertion relative to A59 and four other regions in which the sequences of the two strains differed. The MHV-2 and MHV-3 strains were also sequenced in some of these regions. Our analysis confirmed the presence of only one heterogeneous region in ORF 1a of MHV-A59 and MHV-JHM which is also present in MHV-2. Our findings indicate the need to modify the published sequence of MHV-JHM.

Amino Acid Sequence↗

PubMed1994

MHV-A59 fusion mutants are attenuated and display altered hepatotropism.

Mouse hepatitis virus strain A59 causes a persistent productive, but nonlytic, infection of cultured glial cells. We have mutants isolated from persistently infected glial cell cultures which have been shown to be fusion-defective due to a histidine to aspartic acid mutation (H716D) near the cleavage site of the peplomer protein, S. Here, we examine the pathogenicity of these mutants and show differences in hepatotropism and virulence compared to wild-type virus (WT). Two mutants chosen for detailed study, B11 and C12, were impaired in their abilities to cause hepatitis and/or replicate in the liver of susceptible mice. Furthermore, B11 and C12 display two separate hepatotropic phenotypes. The ability of B11 to replicate in the liver was dependent on infectious dose and route of inoculation, while C12 consistently displayed decreased hepatotropism regardless of dose and route of inoculation. However, B11 and C12 were shown to replicate in the CNS of infected animals similarly to WT. Like WT, the mutants produced meningoencephalitis during acute infection, with viral antigen exhibiting a similar distribution in the brain, and demyelination during chronic infection. Sequence analysis of wild-type, mutant, and revertant S proteins indicates that (1) a mutation in the N terminal subunit of S (S1), resulting in a glutamine to leucine amino acid substitution (Q159L), may affect hepatotropism and (2) a cleavage site mutation which determines fusogenicity is not responsible for altered hepatotropism. Furthermore, since B11, C12, and a nonattenuated fusion mutant (B12) have identical S protein sequences, there must be additional mutations outside of S which influence both virulence and hepatotropism.

Amino Acid Sequence↗

PubMed1994

Coronavirus polyprotein processing.

MHV gene 1 contains two ORFs in different reading frames. Translation proceeds through ORF 1a into ORF 1b via a translational frame-shift. ORF 1a potentially encodes three protease activities, two papain-like activities and one poliovirus 3C-like activity. Of the three predicted activities, only the more amino terminal papain-like domain has been demonstrated to have protease activity. ORF 1a polypeptides have been detected in infected cells by the use of antibodies. The order of polypeptides encoded from the 5' end of the ORF is p28, p65, p290. p290 is processed into p240 and p50. Processing of ORF1a polypeptides differs during cell free translation of genome RNA and in infected cells, suggesting that different proteases may be active under different conditions. Two RNA negative mutants of MHV-A59 express greatly reduced amounts of p28 and p65 at the non-permissive temperature. These mutants may have defects in one or more viral protease activities. ORF 1b, highly conserved between MHV and IBV, potentially contains polymerase, helicase and zinc finger domains. None of these activities have yet been demonstrated. ORF 1b polypeptides have yet been detected in infected cells.

Animals↗