Fc receptor-like activity of mouse hepatitis virus E2 glycoprotein.
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Animals↗
Biomedical subjects
J L Leibowitz
Publications and source records attributed to J L Leibowitz.
Mouse hepatitis virus ORF 2a is expressed in the cytosol of infected mouse fibroblasts.
A 266-bp fragment of cDNA from within gene B, ORF 2a, of MHV-A59 was used to construct a vector encoding a bacterial/viral fusion protein. Antiserum raised against this fusion protein specifically immunoprecipitates a 30K protein from infected 17Cl-1 mouse fibroblasts. The protein is localized primarily in the cytosol and not in the membranes. This is consistent with its predicted sequence and potential role as an RNA binding protein.
Animals↗
Immunoglobulin Fc binding activity is associated with the mouse hepatitis virus E2 peplomer protein.
Antigenic variation among murine coronaviruses is associated primarily with the surface peplomer protein E2 (180,000 Da). E2 is responsible for attachment of the virus to the host cell, MHV-induced cell fusion, and eliciting neutralizing antibody. We report here the molecular mimicry between E2 and Fc gamma receptor (Fc gamma R). Molecular mimicry between E2 and Fc gamma R may allow the escape of virus-infected cells from destruction by immunological mechanisms. Rabbit IgG, monoclonal rat IgG1 and IgG2b, monoclonal mouse IgG2a and IgG2b, and the rat anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-infected cells a polypeptide with a molecular mass identical to that immunoprecipitated by anti-E2 antibodies. F(ab')2 fragments of rabbit IgG did not immunoprecipitate any proteins from MHV-infected cells. All of these antibodies did not immunoprecipitate any proteins from uninfected cells. The anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-, MHV-3-, or MHV-A59-infected L-2 cells and 17CL-1 cells, or MHV-JHM-infected cultures of neonatal BALB/c brain cells, a protein with a molecular weight identical to that of MHV-JHM E2. The anti-Fc gamma R monoclonal antibody did not immunoprecipitate any proteins from uninfected cells. Furthermore, the 2.4G2 monoclonal antibody (mab), unrelated rat and mouse monoclonal antibodies, and a goat antiserum against E2, but not normal goat serum, immunoprecipitated a 75,000- to 77,000-Da molecule from uninfected WEHI-3 cells, a Fc gamma R bearing cell line. Several lines of evidence demonstrated that the protein immunoprecipitated by the anti-Fc gamma R mab from MHV-JHM-infected cells is the E2 glycoprotein: (1) Partial proteolytic maps obtained by Staphylococcus aureus V-8 protease treatment of the 180,000-Da proteins immunoprecipitated by the anti Fc gamma R mab and the anti-E2 mab were identical. (2) Sequential immunoprecipitation experiments from MHV-JHM-infected cells revealed that the same polypeptide chain was recognized by the anti-E2 mab and by the anti-Fc gamma R mab 2.4G2, (3) Actinomycin D did not influence the induction and expression of the 180,000-Da polypeptide chain that was immunoprecipitated by the anti-Fc gamma R mab, demonstrating that this protein is of viral origin.
Amino Acid Sequence↗
Persistent infection of a glioma cell line generates a Theiler's virus variant which fails to induce demyelinating disease in SJL/J mice.
Theiler's murine encephalomyelitis virus (TMEV) induces demyelinating disease which is associated with persistent virus infection of the central nervous system. To study the interaction between TMEV and host cells, we infected the G26-20 glioma cell line in vitro, and this resulted in a lytic infection in which most, but not all, cells were killed. Surviving cells divided and formed a viable monolayer in which a small proportion of cells displayed viral cytopathic effects. Levels of virus produced by these cultures over a 6 month period fluctuated between 6 and 8 log10 p.f.u./ml as measured by viral plaque assay. Similarly, the percentage of cells producing both viral antigen and viral RNA, as measured by a simultaneous immunoperoxidase/in situ hybridization technique, varied between 5 and 30%. Although persistently infected cultures were susceptible to challenge by both vesicular stomatitis virus and herpes simplex virus, they were resistant to infection by homologous viruses. Interferon activity was not identified. TMEV isolated from passage 12 produced smaller plaques than wild-type Daniels strain virus (wt-DAV) on L-2 cell monolayers. In contrast to demyelination induced in SJL/J mice after intracerebral inoculation with wt-DAV, mice infected with the small plaque variant virus failed to develop viral persistence or chronic demyelination. However, following immunosuppression by total body irradiation, SJL/J mice infected with the small plaque variant developed viral persistence but no demyelination. Characterization of the biochemical and molecular determinants of the variant will lead to a better understanding of determinants important in viral persistence.
Animals↗
A general method for the induction and screening of antisera for cDNA-encoded polypeptides: antibodies specific for a coronavirus putative polymerase-encoding gene.
A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59). The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus. Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein. Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum. The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene. This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA. This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA. Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs.
Animals↗
Lymphocyte-virus interactions. Identification of a restriction fragment permitting virus replication in lymphocytes.
We are interested in understanding the effects of virus infection on lymphocyte function. To approach this question, we used a unique system of two genetically related leporipoxviruses to generate recombinants. One of these viruses, malignant fibroma virus (MV), replicates in many different cell types, including lymphocytes. The other, Shope fibroma virus (SFV), replicates principally in fibroblasts, but cannot replicate in lymphocytes. Fibroblasts infected with SFV received restriction fragments from MV by transfection. Recombinant viruses were selected in vitro for their ability to replicate in lymphocytes. By these means we have identified one restriction fragment, the 10.8-kb BamHI "C" fragment, capable of transferring from MV to SFV the ability to replicate in lymphocytes. A family of recombinants bearing different sized inserts of this restriction fragment has been isolated and is being characterized. Lymphocytotropic recombinants bearing portions of this restriction fragment produce colony morphology in vitro intermediate between MV's plaques and SFV's foci. On the basis of their ability to grow in and suppress mitogen responsiveness of lymphocytes, these recombinants may be classified into four different groups. Group 1 viruses are the most immunosuppressive, whereas those of group 4 are least. These traits correlate with ability to replicate in lymphocytes. Genetic analysis of recombinants indicates that the most immunosuppressive recombinants do not necessarily contain the most fragment C DNA. Therefore, we have identified a restriction fragment, one or more of the genes of which are sufficient to allow an otherwise nonlymphocytotropic virus to replicate in lymphocytes. Additional genetic and immunologic analysis should permit us to determine the structure and function of the protein responsible for this effect.
Animals↗
Synthesis of virus-specific RNA in permeabilized murine coronavirus-infected cells.
We have developed a permeabilized cell system for assaying mouse hepatitis virus-specific RNA polymerase activity. This activity was characterized as to its requirements for mono- and divalent cations, requirements for an exogenous energy source, and pH optimum. This system faithfully reflects MHV-specific RNA synthesis in the intact cell, with regard to both its time of appearance during the course of infection and the products synthesized. The system is efficient and the RNA products were identical to those observed in intact MHV-infected cells as judged by agarose gel electrophoresis and hybridization. Permeabilized cells appear to be an ideal system for studying coronavirus RNA synthesis since they closely mimic in vivo conditions while allowing much of the experimental flexibility of truly cell-free systems.
Cell Line↗
Detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame.
Mouse hepatitis virus (MHV) gene 5 contains two open reading frames. We have expressed the second open reading frame of this gene (gene 5 ORF 2) in an Escherichia coli expression system. This system utilized a plasmid which contained the promoter and the first 36 codons of the recA gene fused in frame with the MHV gene 5 ORF 2, which is fused in turn to the beta-galactosidase gene. The protein product of this gene fusion was used to raise antibody to gene 5 ORF 2. The specificity of the antibody was verified by immunoprecipitation of the in vitro transcribed and translated protein product of gene 5 ORF 2. The second reading frame of MHV gene 5 was shown to be expressed during the course of infection by immunocytochemistry and radioimmunoprecipitation using the antibody raised against the E. coli fusion protein and by two-dimensional gel electrophoresis.
Genes↗
Isolation and characterization of two plaque size variants of Theiler's murine encephalomyelitis virus (DA strain).
We have isolated two plaque size variants of Theiler's murine encephalomyelitis virus (TMEV) strain DA. One variant, TMEV-CL (CL), produced large plaques, while the other, TMEV-DS (DS), produced small plaques in L-2 cells. The DS variant yielded a lower titre in BHK cells and had a significantly slower growth rate when compared to CL and DA. In contrast, DS replicated to a higher titre in the central nervous system (CNS) of infected mice than the large plaque counterpart and DA. Furthermore, the DS (but not CL) variant was temperature-sensitive, replicating 130- to 500-fold more at 37 degrees C than at 39 degrees C. Although DS, CL and DA were able to establish persistent CNS infections in mice, only the DS variant and DA induced demyelinating disease in SJL/J mice. Therefore persistence of TMEV in the CNS is not sufficient to produce demyelinating disease. These two variants of the DA strain of TMEV will be useful for study of the viral genetic elements important in the mechanism of virus-induced demyelination.
Animals↗
Increased hepatotropism of mutants of MHV, strain JHM, selected with monoclonal antibodies.
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Animals↗
Growth of malignant rabbit fibroma virus in lymphoid cells.
To understand better the immunosuppressive capacity of malignant rabbit fibroma virus (MV), we characterized MV growth in lymphoid cells. Replication of MV occurs in unstimulated normal spleen cells in vitro and is enhanced by adding T- or B-lymphocyte mitogens. In splenic T-lymphocyte preparations, comparable results are found: virus growth in the absence of mitogen, augmented by adding Con A. Unlike mature T cells, thymic lymphocytes support MV replication only when mitogen is added. When spleen cells from rabbits infected with MV in vivo are removed and cultured without mitogen, MV growth is again observed, with virus titer increasing about 10-fold per day of culture. In spleen cell populations from MV tumor-bearing rabbits, MV grows best in T lymphocytes, moderately in B lymphocytes, and least efficiently in adherent cells. When spleen cells are examined immediately following sacrifice, MV antigens are expressed solely on T lymphocytes from rabbits infected in vivo with MV 7 days previously. However, following overnight incubation in vitro a population of non-T lymphocytes displays cell membrane virus antigens. MV adapts itself somewhat to growth in lymphocytes, showing significantly greater growth in lymphocytes following passage in lymphocytes than is observed for non-lymphocyte-propagated virus. MV-infected lymphocytes also elaborate a factor that enhances MV growth in lymphocytes. Thus, MV replicates preferentially in mature T lymphocytes but will grow well in B cells as well. In vivo infection produces relatively small amounts of recoverable virus. However, when these lymphocytes are cultured in vitro virus replicates very well without added mitogens. These growth patterns may help to understand MV-induced immunologic dysfunction.
Animals↗
Virus-lymphocyte interactions during the course of immunosuppressive virus infection.
Malignant rabbit fibroma virus (MV) is a lymphocytotropic leporipoxvirus which produces profound immunological dysfunction and lethal fibromyxosarcoma. We examined virus recovery from splenic lymphocytes as a function of time after inoculation in vivo, and correlated this with both immunological function and expression of virus-induced host suppressor activity. MV was most abundant in lymphocytes obtained 4 days following inoculation. At that time, immune function was relatively normal and host suppressor activity was not observed. By 7 days after infection, when active host immunosuppressor functions were observed, virus recovery was decreased. Eleven days post-inoculation host immune function began to recover despite increasing virus-induced tumours and developing opportunistic infection. Simultaneously, MV was no longer recoverable from spleen cells. Spleen cells from day 11 tumour-bearing rabbits did not support MV replication as efficiently as did normal or day 4 or 7 splenic lymphocytes, but they did not alter the ability of MV to grow in the latter cells. By fluorescence examination and cytofluorography, splenic lymphocytes bearing MV antigens were abundant 7 days after infection but disappeared by 11 days. This was temporally related to production of neutralizing antibody to MV, and development of virus-specific lymphocyte proliferative activity. The composition of splenic lymphocytes changed as well: the normal ratio of about 1:1 for B and T cells changed to 1:2 by day 7, and then inverted to almost 2:1 by day 11. Rabbits infected with MV thus appear to recover their immune function, concurrently eliminate virus-infected lymphocytes, and elaborate high titres of neutralizing serum antibodies despite progressive infections and tumour development.
Antibodies, Viral↗
Inhibition of epidermal growth factor-induced cellular proliferation.
The authors tested whether two oncogenic poxviruses, Shope fibroma virus (SFV) and malignant rabbit fibroma virus (MV), coded for an epidermal growth factor (EGF)-like protein. Virus-free lysates of SFV or MV-infected rabbit kidney cells do not appreciably affect proliferation of EGF-unresponsive RK-13 cells. Comparable lysates inhibit the background proliferation of two EGF-responsive cell lines, human foreskin fibroblasts and normal rat kidney cells. Inhibition of EGF-stimulated proliferation is also observed in a dose-dependent fashion. This inhibition is greatest when the virus lysate preparations are added either simultaneously with or before EGF. When EGF is added to cultures 24 hours before the SFV or MV preparations, the latter then have little or no effect on EGF-induced target cell proliferation. The inhibitory factor competed with radiolabeled EGF for its receptor site. Electroblotting shows a protein of 35 kd molecular weight in the lysates of virus-infected RK-13 cells which reacts with anti-EGF antibody. These findings interpreted to indicate that SFV and MV code for an inhibitor of EGF activity, and that this inhibition occurs at least in part by competitive inhibition of EGF-EGF receptor interactions.
Cell Division↗
Immunosuppression in viral oncogenesis. III. Effects of virus infection on interleukin 1 and interleukin 2 generation and responsiveness.
Malignant rabbit fibroma virus (MV) is an oncogenic immunosuppressive leporipoxvirus. We studied the effects of MV infection and MV-associated tumor-induced suppressor factor (TISF) on the production of and responsiveness to interleukins 1 and 2. Adherent cells from MV tumor-bearing rabbits elaborate adequate amounts of IL 1 in response to E. coli endotoxin. Neither live virus nor TISF alters the production or the responsiveness to IL 1. However, when we examined spleen cells from rabbits 7 days after MV inoculation, we noted that their ability to produce and respond to IL 2 is deficient. Despite their relatively poor capacity to produce IL 2, these spleen cells express receptor for IL 2 in normal amounts, as measured by the monoclonal antibody 7D4. TISF derived from T lymphocytes from MV tumor-bearing rabbits is by itself capable of inhibiting partially normal secretion of IL 2 and also the response of the cloned murine T cell line HT-2 to added IL 2. Full expression of the immunosuppressive capacity of spleen cells from MV tumor-bearing rabbits requires cell-cell contact, however, and cannot be replaced by either live virus or spleen cell supernatants. Such spleen cells inhibit normal mitogen responsiveness, a defect not remedied by adding exogenous IL 2. Immunologic dysfunction induced by MV infection is transient, and by 11 days after virus inoculation, actively mediated recovery from immunosuppression is observed. We found that spleen cells from rabbits studied 11 days postinoculation secreted IL 2 normally. Thus, immunologic dysfunction secondary to infection with malignant rabbit fibroma virus reflects deficiencies in both elaboration of and response to IL 2, and return of immune function later in the course of the infection is associated with return of the ability of lymphocytes to secrete IL 2.
Animals↗
Reversal of virus-induced immune suppression.
We studied the immunosuppressive capacity of splenic lymphocytes from rabbits at different stages of progressive myxosarcoma induced by malignant rabbit fibroma virus (MV). Spleen cells taken from rabbits 7 days after virus inoculation proliferate poorly in response to Con A, and suppress normal responses to the mitogen. Those from animals 11 days after virus injection have recovered partially from MV-induced suppression. Further, their Con A responses are no longer suppressed by day 7 spleen cells. Supernatants from cultures of spleen cells from rabbits given MV 7 days previously suppress both antibody-producing and proliferative responses to unrelated antigens. Comparable supernatants from rabbits receiving MV 11 days before sacrifice neither suppress nor augment such responses. Mixing cells from 7 or 11 day MV rabbits with normal spleen cells gives similar results. When supernatants from spleen cell of rabbits with tumors induced 7 and 11 days previously are mixed, the supernatants from rabbits with 11-day-old tumors inhibit the suppressive capacity of those from animals with 7-day-old tumors. Similarly, mixing spleen cells from rabbits given MV 7 and 11 days previously results in culture supernatants that do not suppress normal antibody and proliferative responses. The ability of cells from rabbits given MV 11 days before to inhibit the effects of cells from rabbits given MV 7 days previously does not involve the production of interferon. Thus, despite progressive tumor burden, immunologic recovery is observed in rabbits 11 days after tumor virus inoculation. One factor in this recovery may be the generation of active inhibitors of virus-induced immunosuppression. Similar mechanisms may apply to recovery of immunologic function in other virus infections as well.
Animals↗
Transcriptional mapping of early RNA from regions of the Shope fibroma and malignant rabbit fibroma virus genomes.
Malignant rabbit fibroma virus (MV) is a recombinant poxvirus derived from Shope fibroma virus (SFV) and rabbit myxoma virus (D. S. Strayer, E. Skaletsky, G. F. Cabirac, P. A. Sharp, L. B. Corbeil, S. Sell, and J. L. Leibowitz, 1983a, J. Immunol. 130, 399-404; W. Block, C. Upton, and G. McFadden, 1984, Virology 140, 113-124). We report here the transcriptional mapping of early RNAs transcribed from the SFV sequences within MV and from the corresponding regions in SFV. Hybridization analysis and S1 nuclease mapping of RNA using viral DNA probes were used to define 5' and 3' ends of the various transcripts. The RNAs described here are transcribed in one direction in a densely arranged head to tail fashion similar to that described for some vaccinia virus early transcriptional units. At late times of infection the early SFV RNAs are not detected whereas the early MV RNAs are present in minor amounts. The early SFV and MV transcripts range in size from 3170 to 425 nucleotides (nt) long. All of the longer transcripts are produced as a result of read through transcription. Three MV transcripts contain fused SFV and rabbit myxoma virus sequences due to transcription through the recombination junction region in the MV genome. Two other MV transcripts are transcribed from a unique initiation site near another recombination junction region resulting in RNAs that are composed of SFV sequences having unique 5' ends.
Animals↗
Inhibition of virus replication does not alter malignant rabbit fibroma virus-induced immunosuppression.
Malignant rabbit fibroma virus (MV) directly suppresses generation of antibody responses and mitogen induced T and B lymphocyte proliferation. We investigated whether this phenomenon required expression of the complete viral genome. Phosphonoacetic acid (PAA) inhibits poxvirus specific DNA polymerases. Adding PAA to cultures reduces both MV replication and mitogen-driven rabbit lymphocyte proliferation in a dose-dependent fashion. A dose of PAA adequate to inhibit MV replication by about 97%, but insufficient to reduce lymphocyte proliferation appreciably, does not affect the ability of MV to suppress lymphocyte proliferation or initiation of antibody production. Spleen cells from MV tumour-bearing rabbits contain very little virus, but inhibit the proliferative and antibody forming responses of normal spleen cells. This activity is shown here to reflect the production by T lymphocytes of a soluble mediator of greater than 25 kD molecular weight. Adding PAA to these mixed spleen cell cultures does not alter the ability of MV to induce T suppressor activity in host lymphocytes. Thus, these immunosuppressive capabilities of MV appear to reflect early MV gene functions.
Animals↗
In vitro growth of two related leporipoxviruses in lymphoid cells.
We examined the in vitro growth patterns of two leporipoxviruses, malignant rabbit fibroma virus (MV) and Shope fibroma virus (SFV), in lymphoid cells. MV replicates well in normal spleen cells in vitro. At low m.o.i. (0.001), dramatic virus growth occurs in unstimulated cell cultures. This growth is enhanced by addition of the T lymphocyte mitogen, concanavilin A, or the B lymphocyte mitogen, Escherichia coli lipopolysaccharide. Shope fibroma virus does not grow in lymphocytes in culture, with or without mitogen stimulation. MV itself profoundly inhibits lymphocyte mitogenesis, while SFV does not. MV and SFV added to normal lymphocytes do not appear to alter their viability in culture. Thus, MV appears to be novel in its ability to replicate to high titer in resting lymphocytes. This growth pattern may be useful in understanding MV-induced immunologic dysfunction.
Animals↗