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S A Stohlman

Publications and source records attributed to S A Stohlman.

At least 109 records · Page 6Linked to original sources
PubMed1987

Analysis of intracellular small RNAs of mouse hepatitis virus: evidence for discontinuous transcription.

We have previously shown the presence of multiple small leader-containing RNA species in mouse hepatitis virus (MHV)-infected cells. In this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small RNAs. Using cDNA probes specific for leader RNA and genes A, D, and F, we demonstrate that subsets of these small RNAs were derived from the various viral genes. These subsets have discrete and reproducible sizes, varying with the gene from which they are derived. The size of each subset correlates with regions of secondary structure, whose free energy ranges from -1.6 to -77.1 kcal/mol, in each of the mRNAs examined. In addition, identical subsets were detected on the replicative intermediate (RI) RNA, suggesting that they represent functional transcriptional intermediates. The biological significance of these small RNAs is further supported by the detection of leader-containing RNAs of 47, 50, and 57 nucleotides in length, which correspond to the crossover sites in two MHV recombinant viruses. These data, coupled with the high frequency of RNA recombination during MHV infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing RNA intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant RNAs. These data suggest that MHV transcription uses a discontinuous and nonprocessive mechanism in which RNA polymerase allows the partial RNA products to be dissociated from the template temporarily during the process of transcription.

DNA↗
PubMed1987

Experimental demyelination induced by coronavirus JHM (MHV-4): molecular identification of a viral determinant of paralytic disease.

The molecular basis for demyelination induced by the neurotropic murine coronavirus JHM (JHMV or MHV4) is unknown. We have attempted to explore this issue by using neutralizing monoclonal antibodies specific for the major JHMV glycoprotein (E2) to select sets of neutralization resistant (NR) antigenic variant viruses. Monoclonal antibodies J.7.2 and J.2.2 bind to topographically distinct sites on E2. NR variants selected with J.7.2, like parental JHMV, predominantly cause a fatal encephalitis when given intracerebrally to mice, while J.2.2-selected NR variants cause a subacute disease characterized by paralysis and severe demyelination. We report here that consecutive selection with both J.2.2 and J.7.2 monoclonal antibodies results in NR variants which are markedly attenuated in both encephalitic potential and ability to induce demyelination. Analysis of the different variants suggests that the subregion of E2 bound by monoclonal antibody J.7.2 may be a critical viral determinant of paralysis and demyelination in this model system.

Animals↗
PubMed1987

RNA recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers.

Murine coronaviruses undergo RNA recombination at a very high frequency. We have obtained a series of recombinant viruses using neutralizing monoclonal antibodies in conjunction with temperature-sensitive markers. All of the recombinants obtained have a crossover within gene C, which encodes the peplomer protein of the virus. The genetic structure of these recombinants suggests that the antigenic regions recognized by these neutralizing monoclonal antibodies are localized on the carboxyl-terminal one-third of the peplomer protein. Since the two monoclonal antibodies used are also associated with the critical determinants of virus neuropathogenicity, we conclude that both the neutralizing antibody binding sites and determinants of pathogenicity are localized at the carboxyl-terminal one-third of the peplomer. The variation of crossover sites in different recombinant viruses also allowed precise mapping of additional antigenic sites. RNA recombination thus presents a powerful genetic tool, and the carboxyl-terminal localization of the biological functions of peplomers suggests a distinct conformation of these viral membrane proteins.

Animals↗
PubMed1987

Characterization of the structural proteins of the murine coronavirus strain A59 using monoclonal antibodies.

Monoclonal antibodies reacting with the A59 strain of mouse hepatitis virus (MHV-A59) were characterized and those specific to the E2 major envelope glycoprotein were studied in detail. Antibodies were tested for their ability to neutralize viral infectivity (N+ characteristic) and prevent viral-induced cell-to-cell fusion (F+ characteristic). All four possible combinations of activities reflecting E2 functions were found, i.e., N+F+, N-F-, N+F-, and N-F+. In addition, competitive binding studies with these monoclonal antibodies revealed two nonoverlapping antigenic regions. The first region, designated A, was recognized by antibodies which included each of the four functional types. Region B was identified by a single monoclonal antibody with N-F- activities. The existence of antibodies which only neutralize virus or only block viral-induced fusion implies that the structures on E2 which serve as targets for neutralization and which induce fusion are not identical. The critical determinants for neutralization and fusion must be closely related topographically on E2 since both N+F- and N-F+ antibodies recognize the same antigenic region.

Antibodies, Monoclonal↗
PubMed1986

Phosphorylation of the mouse hepatitis virus nucleocapsid protein.

Analysis of the radiolabeled tryptic peptides derived from the nucleocapsid proteins of two serotypes of mouse hepatitis virus showed each to have a small number of unique peptides; however, two biologically distinct variants of the JHM strain appeared identical. Analysis of [32P]-labeled nucleocapsid-derived peptides showed that phosphorylation occurs at only a few sites and that all three viruses differed in the sites of phosphorylation. No differences in the sites of phosphorylation were found between the nucleocapsid proteins derived from purified virions and the membranes or the cytosol of infected cells, suggesting that post-translational phosphorylation plays no role in the regulation of viral assembly. These data show unequivocal evidence that the nucleocapsid proteins of mouse hepatitis virus strains differ in the sites of phosphorylation.

Capsid↗
PubMed1986

In vivo effects of coronavirus-specific T cell clones: DTH inducer cells prevent a lethal infection but do not inhibit virus replication.

A lethal infection by neurotropic JHM strain of mouse hepatitis virus was prevented by the local adoptive transfer of three virus-specific Lyt-1+2-, L3T4+ T cell clones. The transfer of Lyt-1+ T cells specific for an unrelated antigen (hen egg lysozyme) did not protect. Protection required I region compatibility between the T cells and the recipients, and was reversed either by irradiation of the cells before transfer or by pretreatment of the recipients with cyclophosphamide. Adoptive transfer prevented death due to JHM virus infection but did not result in altered antiviral immunoglobulin synthesis or the suppression of viral replication in the central nervous system (CNS). The data presented implicate a local DTH response in the protection of the host from lethal infection of the CNS by a neurotropic virus, but clearly imply that other antiviral effector mechanisms are necessary for the suppression of viral replication.

Animals↗
PubMed1986

Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription.

Mouse hepatitis virus (MHV), which replicates in cytoplasm of infected cells, contains an identical leader RNA sequence at the 5' end of each of the virus-specific mRNAs. Previous studies suggested that the synthesis of these mRNAs does not involve conventional RNA splicing and may instead require priming by a free leader RNA. In this communication, we demonstrate that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the mRNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments. This finding demonstrates that MHV mRNA transcription utilizes independently transcribed leader RNA species that possess the trans-acting property. This study thus provides further evidence in support of the unique model of "leader-primed transcription" for coronavirus mRNA synthesis.

Murine hepatitis virus↗
PubMed1986

High-frequency RNA recombination of murine coronaviruses.

The RNA genome of coronaviruses consists of a single species of nonsegmented RNA. In this communication, we demonstrate that the RNA genomes of different strains of murine coronaviruses recombine during mixed infection at a very high frequency. Susceptible cells were coinfected with a temperature-sensitive mutant of one strain of mouse hepatitis virus (MHV) and a wild-type virus of a different strain. Of 21 randomly isolated viruses released from the coinfected cells at the nonpermissive temperature, 2 were recombinants which differed in the site of recombination. After three serial passages of the original virus pool derived from the mixed infection, the majority of the progeny viruses were recombinants. These recombinant viruses represented at least five different recombination sites between the two parental MHV strains. Such a high-frequency recombination between nonsegmented RNA genomes of MHV suggests that segmented RNA intermediates might be generated during MHV replication. We propose that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy-choice mechanism.

Base Sequence↗
PubMed1986

Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies.

To analyze the pathogenesis of the neurotropic murine coronavirus JHMV, we used monoclonal antibodies to the E2 viral glycoprotein to select antigenic variant viruses. Monoclonal antibodies J.7.2 and J.2.2 were shown to bind to topographically distinct regions of the E2 molecule, and the variants selected with the two antibodies demonstrated very different disease pictures in mice. Variants selected with J.7.2 were, like the parental virus, highly virulent and caused an acute encephalitic illness. By contrast, J.2.2-selected variants predominantly caused a subacute paralytic disease clinically and extensive demyelination histologically. Antigenic differences among the variants and parental virus were readily demonstrable with anti-E2 monoclonal antibodies. However, no differences between the viruses could be shown in binding studies with monoclonal antibodies directed against either E1 or N, the other two JHMV structural proteins. Since only J.2.2 selected demyelinating variants with reduced neurovirulence, it is likely that this monoclonal antibody recognizes a subregion of the E2 molecule that is particularly important in JHMV pathogenesis.

Animals↗
PubMed1985

The defect in delayed-type hypersensitivity of young adult SJL mice is due to a lack of functional antigen-presenting cells.

SJL mice exhibit a strain-specific age-dependent delay in the maturation of delayed-type hypersensitivity (DTH) responsiveness. They do not attain "adult" levels of DTH responsiveness until the 10th week of age, which is 4 to 6 weeks later than the other strains of mice tested. In this report we demonstrate that spleen cells, resident peritoneal cells and thioglycollate-elicited peritoneal exudate cells are all able to transfer DTH responsiveness from naive 12-week-old DTH responders to 6-week-old nonresponders. Transfer prior to immunization was more efficient at eliciting a response than transfer after immunization. As few as 5 X 10(4) cells from 12-week-old SJL mice can adoptively transfer responsiveness to unresponsive 6-week-old animals. The active cell was found to be adherent, radiation (2000 rds) resistant, I-A+, Thy-1- and Mac-1+. I-A compatibility between the adoptively transferred population and the nonresponder mice is required. These data suggest that young adult SJL mice lack a functional population of antigen-presenting cells specific for DTH and that the appearance of these cells is under maturational control.

Aging↗
PubMed1985

Characterization of leader-related small RNAs in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription.

Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNAs containing an identical stretch of leader RNA sequences at the 5'-end of each RNA. The leader-coding sequences at the 5'-end of genomic RNA are at least 72 nucleotides in length and are joined to the viral mRNAs by a unique mechanism. Utilizing a leader-specific cDNA probe, we have detected several free leader RNA species ranging from 70 to 82 nucleotides in length. The predominant leader RNA was approximately 75 nucleotides. In addition, larger distinct leader-containing RNAs were also detected ranging from 130 to 250 nucleotides in length. The 70-82-nucleotide leader-related RNAs were present in both the cytosol and membrane fractions of infected cells. They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA. These data suggest that the leader RNAs were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template. We have also identified a temperature-sensitive mutant, which synthesizes only leader RNA but not mRNAs at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNAs. These data support the leader-primed mechanism for coronavirus transcription and suggest that one or more free leader RNAs are used as primers of mRNA synthesis.

Animals↗
PubMed1985

Recombination between nonsegmented RNA genomes of murine coronaviruses.

We have isolated a recombinant virus between the A59 and JHM strains of mouse hepatitis virus, which contain a single species of nonsegmented RNA genome. This recombinant was derived by mixed infection of DBT cells with temperature-sensitive mutants of A59 and JHM at nonpermissive temperature. Viruses recovered at this temperature were screened by oligonucleotide fingerprinting of their genomic RNAs. One recombinant virus, B1, was found to contain mostly A59-derived sequences, but the 3 kilobases at the 5' end of the genomic RNA was derived from JHM. Thus, the crossover point in the B1 genome is located within gene A, which codes for the viral RNA polymerases. The study of the intracellular RNA species of B1 virus revealed that probably all of the virus-specific subgenomic mRNA species contained the body sequences of strain A59 but the leader sequences of JHM. This result indicates that the JHM leader RNA, which differs from the A59 leader RNA, could be fused to the mRNAs of a different virus strain during RNA transcription. Furthermore, B1 virus-infected cells contain an additional subgenomic mRNA species which is transcribed from a new initiation site within gene C, suggesting that the leader RNA could determine the site of initiation for coronavirus mRNAs. These data represent a first report of RNA recombination between viruses, other than picornaviruses, which contain nonsegmented RNA genomes.

Base Sequence↗
PubMed1984

Studies on the mechanism of RNA synthesis of a murine coronavirus.

The mechanism of viral RNA replication in mouse hepatitis virus (MHV)-infected cells was studied by oligonucleotide mapping of every mRNA. We discovered that an oligonucleotide, No. 10, was localized at the 5'-end of every mRNA, and was not colinear with the sequences of the virion genomic RNA. This result indicates that all of the mRNAs contain a leader sequence which is joined to the body sequences of the mRNAs. We have also studied the structure of the replicative intermediate (RI) RNA in the MHV-infected cells. This RI RNA consists of a single species corresponding to the MHV genomic RNA. No subgenomic RI structures were detected. Furthermore, the nascent RNA chains in the RI structure contained the leader sequences, suggesting that the leader RNA was not added to the mRNA post- transcriptionally , but rather, it was probably synthesized independently and then used as a primer for the synthesis of mRNAs. We have also shown that the poly (A) sequences in the MHV genome were transcribed from the poly (U) sequences present in the negative-strand template. The RNA polymerases involved in the MHV RNA synthesis were also characterized. The early polymerase synthesizes a single negative-stranded, full-length RNA. The late polymerases could be separated into two activities, one synthesizing positive-stranded genomic RNA, and the other synthesizing genomic as well as subgenomic RNAs. Thus, the replication and transcription functions of MHV could probably be separated. A plausible model of MHV replication is presented.

Animals↗
PubMed1984

Further characterization of mouse hepatitis virus RNA-dependent RNA polymerases.

Two temporally and enzymatically distinct RNA-dependent RNA polymerase activities associated with membranes of the mouse hepatitis virus (MHV)-infected cells have been identified previously [Brayton et al., J. Virol. 42, 847-853 (1982)]. In this paper, the subcellular distribution and functions of these two polymerases were examined. Fractionation of the postnuclear membranes by sucrose gradient sedimentation showed that the early polymerase activity (detected at 1 hr p.i.) was homogeneous, while the late polymerase (6 hr p.i.) was associated with two distinct membrane fractions. The early polymerase synthesized a single RNA species of viral genomic size and negative sense. In contrast, the light peak of the late polymerase synthesized genomic-sized RNA of positive sense, while the heavy peak of the activity synthesized positive-sensed genomic and subgenomic mRNAs. These findings suggest that the light peak of the late polymerase represents a replication complex while the heavy peak represents a transcription complex. They also establish the essential features of the mode of replication of MHV.

Animals↗
PubMed1984

Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus.

Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs. We have sequenced this leader RNA by synthesizing cDNA from a synthetic oligodeoxyribonucleotide primer (15-mer) that is complementary to the sequences at the junction site between the leader and body sequences of the mRNAs. The leader sequences on each mRNA have exactly the same size, which span approximately equal to 70 nucleotides. Leader cDNA fragments obtained from several mRNA species were sequenced and found to be identical. Computer analysis of the leader RNA sequences shows that they share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions. This is a novel case of spliced leader sequences in the mRNAs of a cytoplasmic virus. An identical leader sequence is also present at the 5' end of the virion genomic RNA. The leader RNA is thus probably encoded by the virion genomic RNA template and is fused to the different body sequences of the various mRNAs. Since conventional RNA splicing is not involved, a novel mechanism for fusing two noncontiguous RNA segments in the cytoplasm must be utilized during viral transcription. Several minor cDNA bands longer than the leader were also synthesized, suggesting the possible presence of partially homologous sequences in other parts of the genome RNA.

Base Sequence↗
PubMed1984

Production and characterization of T cell clones specific for mouse hepatitis virus, strain JHM: in vivo and in vitro analysis.

Mouse hepatitis virus, strain JHM, is a coronavirus capable of inducing both acute and chronic central nervous system disease. To characterize the cell-mediated immune response directed toward JHMV antigens, we have generated and analyzed the properties of four T cell clones reactive to JHMV antigens. All four clones are of the Ly-1+2- phenotype and show an antigen-specific, I-Ab-restricted proliferative response that is dependent on antigen-presenting cells (APC). The fine specificity of I-A restriction was revealed by their differential recognition of JHMV antigen in the context of mutant I-A molecules present on B6.C-H-2bm12 APC. Two clones were unable to recognize JHMV antigen presented on bm12 APC, whereas a third clone showed an equivalent response with both bm12 and wild type B6 APC. In the analysis of the viral specificity, three of the clones showed distinct cross-reactions with MHV-2 and very little if any cross-reaction with MHV-A59. In addition, three of three clones tested responded well to sucrose gradient-purified, UV-inactivated JHMV, indicating that their specificity is directed toward virus structural rather than nonstructural proteins. Finally, two of the clones were analyzed for their ability to induce a DTH reaction when injected together with antigen into the footpads of various mouse strains. Consistent with the in vitro data, both clones produced a marked DTH response only in those strains carrying the I-Ab genotype. These data demonstrate, both in vivo and in vitro, that at least one component of the cell-mediated immune response to JHMV is characterized by an Ly-1+2-, I-A-restricted T cell specific for JHMV structural proteins.

Animals↗
PubMed1983

Synthesis and subcellular localization of the murine coronavirus nucleocapsid protein.

The synthesis and processing of the nucleocapsid protein (pp60) of the JHM strain of murine coronaviruses were examined. Pulse-chase experiments showed that pp60 was synthesized initially as a protein of approximately 57,000 in molecular weight (p57). Immunoprecipitation using mouse anti-JHMV antiserum indicated that p57 was virus specific. Immunoprecipitation with monoclonal antibodies specific for pp60 showed that p57 was antigenically related to pp60 and was not phosphorylated, while the intracellular protein that comigrated with the virion nucleocapsid protein, pp60, was phosphorylated. The p57 was found exclusively in the cytosol while the majority of pp60 was associated with the membrane fraction but pp60 was not an integral membrane protein.

Animals↗