DBT Cells Research Articles

Knockout immune regulator FGL2 in tumor cells impairs tumor progression in the CNS by facilitating CD103+ dendritic cell differentiation

Abstract Although many studies link brain tumor progression to oncogene activation and tumor-suppressor gene inactivation in tumor cells, few studies implicate immune regulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout (FGL2KO) in GL261, DBT, and LLC tumor cells did not affect tumor cell proliferation in vitro or tumor progression in immunodeficient NSG mice, but completely impaired GBM progression in immune-competent C57bl/6 mice. This impairment was reversed in mice with a defect in Batf3 (a key transcription factor for CD103+ DCs differentiation). Mechanistic studies revealed that FGL2KO in tumor cells induces CD103+ DCs differentiation in both the central nervous system (CNS) and in tumor draining lymph nodes (TDLN). The increased CD103+ DCs population in the CNS and TDLNs induce CD8+ T cells priming and activation and thereby gliomas regress. More specifically, the presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

TCR-pMHC encounter differentially regulates transcriptomes of tissue-resident CD8 Tcells.

To investigate the role of TCR-pMHC interaction in regulating lung CD8 tissue-resident Tcell (TR ) differentiation, polyclonal responses were compared against NP366-374 /Db and PA224-233 /Db , two immunodominant epitopes that arise during influenza A infection in mice. Memory niches distinct from iBALTs develop within the lamina propria, supporting CD103+ and CD103- CD8 TR generation and intraepithelial translocation. Gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) identify dominant TCR, adherens junction, RIG-I-like and NOD-like pattern recognition receptor as well as TGF-β signaling pathways and memory signatures among PA224-233 /Db Tcells consistent with T resident memory (TRM ) status. In contrast, NP366-374 /Db Tcells exhibit enrichment of effector signatures, upregulating pro-inflammatory mediators even among TRM . While NP366-374 /Db Tcells manifest transcripts linked to canonical exhaustion pathways, PA224-233 /Db Tcells exploit P2rx7 purinoreceptor attenuation. The NP366-374 /Db CD103+ subset expresses the antimicrobial lactotransferrin whereas PA224-233 /Db CD103+ utilizes pore-forming mpeg-1, with <22% of genes correspondingly upregulated in CD103+ (or CD103- ) subsets of both specificities. Thus, TCR-pMHC interactions among TR and antigen presenting cells in a tissue milieu strongly impact CD8 Tcell biology.

Abstract 1598: Isothiocyanates promote cell death and sensitize glioblastoma cells to radiation.

Abstract Sulforaphane (SFN) and beta-phenylethyl isothiocyanate (PEITC), are organic isothiocyanate compounds found in dietary cruciferous vegetables with previously characterized anti-proliferative properties; and more recently, pro-apoptosis capabilities in several tumor types. The present study was designed to determine the role of isothiocyanates, particularly SFN and PEITC in vitro with human glioblastomas cells; and in vivo with a murine orthotropic glioblastoma tumor model. Cultured glioblastoma cells were treated with SFN in increasing doses to 80 μM and PEITC to 20 μM. Cells were examined for changes in morphology characteristic of apoptosis and with MTT for cell survival. Cells were exposed to both isothiocyanates either alone or in combination. Our results indicate that SFN inhibits cell proliferation as well as the activation of apoptosis in three GBM cell lines, LD50 for SFN was at 25 μM and essentially kill all the cells at 50 μM 24 hours post-treatment. In three cell lines the LD50 of PEITC is 5 μM and loss of all the cells at 10μM. Treatment with SFN 10 μM and PEITC 5 μM promoted cell death to 80% in which cause 10% and 30% die in U87 cells respectively. Furthermore, exposure of DBT cells to SFN at 10 μM as well as radiation at 2.5Gy, 5Gy and 7.5 Gy resulted in remarkably decreased of clones formation comparing to radiation only. Yield rate of control only, SFN only, 2.5Gy only and SFN+2.5GY are 70.5, 12.7%, 4.8% and 0.62% respectively. While visible clones were existed at 7.5 Gy only, it was barely existing at 7.5Gy with SFN. In vivo studies with subcutaneous murine DBT glioblastoma tumors which treated with SFN daily IP injection for 5 days at dose 12.5mg/kg, decreased tumor size based on weight by 28% compared with those treated with vehicle only. PEITC at 25mg/kg only had failed to decrease the tumor on weight. However, SFN combine with PEITC decreased the GBM on weight by 68% (P =0.016, less than 0.05). This decrease in tumor weight was significantly different as assessed with a two-tailed Students’ t-test for independent variables. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was assessed for apoptosis. The results are: vehicle-only (0.4+0.06%)(n=8); vehicle-only+SFN (1.9+0.3%)(n=8); PEITC (0.9+1.2%)(n=8); and SFN+PEITC (3.6+0.1%)(n=9), demonstrated a significantly greater incidence of apoptosis compared with the vehicle-only group as determined with a two-tailed Students’ t-test for independent variables. These studies supported SFN as a potential anti-tumor drug to promote cell death and enhance of radiosensitivity in glioblastomas in vivo. Our findings suggest that, in addition to the known effects on cancer prevention, isothiocyanates should be viewed as a therapeutic advantage in established malignant glioma. Citation Format: Liya Yuan, Xuan Ren, Jerry Jaboin, Jonathan McConathy, Keith M. Rich. Isothiocyanates promote cell death and sensitize glioblastoma cells to radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2013-1598

Radiosynthesis and biological evaluation of alpha-[F-18]fluoromethyl phenylalanine for brain tumor imaging

Radiolabeled amino acids have proven utility for imaging brain tumors in humans, particularly those that target system L amino acid transport. We have prepared the novel phenylalanine analogue, (FMePhe, 9), as part of an effort to develop new system L tracers that can be prepared in high radiochemical yield through nucleophilic [(18)F]fluorination. The tumor imaging properties of both enantiomers of this new tracer were evaluated through cell uptake, biodistribution and microPET studies in the mouse DBT model of high grade glioma. The non-radioactive form of 9 and the cyclic sulfamidate labeling precursor were prepared from commercially available racemic α-benzylserine. Racemic [(18)F]9 was prepared from the labeling precursor in two steps using standard[(18)F]fluoride nucleophilic reaction conditions followed by acidic deprotection. The individual enantiomers [(18)F]9a and [(18)F]9b were isolated using preparative chiral HPLC. In vitro uptake inhibition assays were performed with each enantiomer using DBT cells. Biodistribution and microPET/CT studies were performed with each enantiomer in male BALB/c mice at approximately 2 weeks after implantation of DBT tumor cells. Radiolabeling of the cyclic sulfamidate precursor 5 provides racemic [(18)F]9 in high radiochemical yield (60%-70%, n=4) and high radiochemical purity (>96%, n=4). In vitro uptake assays demonstrate that both [(18)F]9a and [(18)F]9b undergo tumor cell uptake through system L transport. The biodistribution studies using the single enantiomers [(18)F]9a and [(18)F]9b demonstrated good tumor uptake with lower uptake in most normal tissues, and [(18)F]9a had higher tumor uptake than [(18)F]9b. MicroPET imaging demonstrated good tumor visualization within 10 min of injection, rapid uptake of radioactivity, and tumor to brain ratios of approximately 6:1 at 60 min postinjection. The novel PET tracer, [(18)F]FMePhe, is readily synthesized in good yield from a cyclic sulfamidate precursor. Biodistribution and microPET studies in the DBT model demonstrate good tumor to tissue ratios and tumor visualization, with enantiomer [(18)F]9a having higher tumor uptake. However, the brain availability of both enantiomers was lower than expected for system L substrates, suggesting the [(18)F]fluorine group in the β-position affects uptake of these compounds by system L transporters.

Coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus.

The focus of this protocol is mouse hepatitis virus (MHV), with occasional references to other coronaviruses. Many of these protocols can be easily adapted to other coronaviruses. Protocols for propagating MHV in DBT and 17CL‐1 cells; the storage and titration of viral stocks; purification of MHV on sucrose gradients; and the generation of recombinant viruses by a cDNA assembly method and by targeted recombination will be presented. Protocols are also included for the propagation of DBT, 17CL‐1, and L2 cells used for growing and titrating MHV, and for the growth of BHK‐R cells and FCWF cells. The latter two cell lines are used for regenerating infectious MHV by an in vitro cDNA assembly protocol and by a targeted recombination protocol, respectively, allowing reverse genetic manipulation of these viruses. An additional protocol for the maintenance of the large plasmids used for generating recombinant MHVs will also be presented. Curr. Protoc. Microbiol. 21:15E.1.1‐15E.1.46. © 2011 by John Wiley & Sons, Inc.

Characterization of a Variant Virus from Ascitic Fluid of Subacute Granulomatous Serositis in Interferon-γ-Deficient C57BL/6 Mice Persistently Infected with Murine Coronavirus Strain JHM

Previously, we showed that intraperitoneal infection with murine coronavirus strain JHM (JHMV) established a persistent infection with subacute granulomatous serositis in interferon-gamma-deficient C57BL/6 (B6-GKO) mice. Herein, we characterize a variant virus from B6-GKO mice persistently infected with JHMV. Viruses were isolated from ascites at 25 d post-infection and cloned by limiting dilution on DBT cells; one variant was named 25V16G. To compare pathogenicity in vivo, we inoculated 25V16G and JHMV intraperitoneally into 8- to 12-week-old B6-GKO mice. Whereas nearly all of the B6-GKO mice infected with JHMV survived over 14 d, all of those infected with 25V16G died by 9 d post-infection. Histopathological examination revealed that 25V16G induced acute fulminant hepatitis in B6-GKO mice, whereas JHMV caused severe but focal hepatitis. The virus titer of 25V16G in the liver was 50- and 250-fold higher than that of JHMV at 5 and 7 d post-infection, respectively. However, there was no significant difference in viral growth between 25V16G and JHMV in cell lines cultured in vitro. Nucleotide sequencing of the S gene of 25V16G and JHMV revealed a deletion of 29 amino acids encompassing S(511-539), which covers a major cytotoxic T lymphocyte (CTL) epitope in C57BL/6 mice, and two point mutations resulting in amino acid changes in the S protein of 25V16G. One explanation for the greater pathogenicity of 25V16G is that 25V16G escapes CTL-mediated protection in B6-GKO mice. This experimental model may be used to assess the role of IFN-gamma in viral persistence in vivo.

Medicinal herbal extracts of Sophorae radix Acanthopanacis cortex Sanguisorbae radix and Torilis fructus inhibit coronavirus replication in vitro

Cimicifuga rhizome, Meliae cortex, Coptidis rhizome and Phellodendron cortex have been previously shown to exhibit anti-coronavirus activity. Here, an additional 19 traditional medicinal herbal extracts were evaluated for antiviral activities in vitro. A plaque assay was used to evaluate the effects of 19 extracts, and the concentration of extract required to inhibit 50% of the replication (EC(50)) of mouse hepatitis virus (MHV) A59 strain (MHV-A59) was determined. The 50% cytotoxic concentration (CC(50)) of each extract was also determined. Northern and western blot analyses were conducted to evaluate antiviral activity on viral entry, viral RNA and protein expression, and release in MHV-infected DBT cells. Sophorae radix, Acanthopanacis cortex and Torilis fructus reduced intracellular viral RNA levels with comparable reductions in viral proteins and MHV-A59 production. The extracts also reduced the replication of the John Howard Mueller strain of MHV, porcine epidemic diarrhoea virus and vesicular stomatitis virus in vitro. Sanguisorbae radix reduced coronavirus production, partly as a result of decreased protein synthesis, but without a significant reduction in intracellular viral RNA levels. The EC(50) values of the four extracts ranged from 0.8 to 3.7 microg/ml, whereas the CC(50) values ranged from 156.5 to 556.8 microg/ml. Acanthopanacis cortex and Torilis fructus might exert their antiviral activities in MHV-A59-infected cells by inducing cyclooxygenase-2 expression via the activation of extracellular signal-related kinase (ERK) and p38 or ERK alone, respectively. Sophorae radix, Acanthopanacis cortex, Sanguisorbae radix and Torilis fructus might be considered as promising novel anti-coronavirus drug candidates.

High Expression of the PAX3-FKHR Oncoprotein Is Required to Promote Tumorigenesis of Human Myoblasts

PAX3-FKHR is a fusion oncoprotein generated by the 2;13 chromosomal translocation in alveolar rhabdomyosarcoma (ARMS), a cancer associated with the skeletal muscle lineage. Previous studies determined that high-level PAX3-FKHR expression is a consistent feature in ARMS tumors. To investigate the relationship between expression and phenotype in human myogenic cells, PAX3-FKHR was introduced into immortalized human myoblasts to produce a low overall PAX3-FKHR expression level. Although PAX3-FKHR alone failed to exert transforming activity, a combination of PAX3-FKHR and MYCN induced transforming activity in cell culture assays. Furthermore, myoblasts expressing PAX3-FKHR with or without MYCN formed tumors in SCID mice. These tumors demonstrated invasive features and expressed myogenic markers, consistent with rhabdomyosarcoma. Comparisons of tumor and parental cells revealed that only a subset of parental cells developed into tumors and that tumor cells expressed high PAX3-FKHR levels compared with transduced parental cells. Subcloning of parental PAX3-FKHR/MYCN-transduced myoblasts identified rare high PAX3-FKHR-expressing subclones with high transforming and tumorigenic activity; however, most subclones expressed low PAX3-FKHR and showed neither transforming nor tumorigenic activity. Finally, RNA interference experiments in myoblast-derived tumor and ARMS cells revealed that high PAX3-FKHR expression plays a crucial role in regulating proliferation, transformation, and differentiation. These findings support the premise that high PAX3-FKHR-expressing cells are selected during tumorigenesis.

The Spike Protein of Murine Coronavirus Regulates Viral Genome Transport from the Cell Surface to the Endoplasmic Reticulum during Infection

We observed that the nonfusogenic mouse hepatitis virus (MHV) strain MHV-2 reached a titer of approximately 2 log10 higher than that of the fusogenic strain A59 in astrocytoma DBT cells. To determine whether the spike protein is responsible for the difference, a recombinant virus, Penn-98-1, that contains the A59 genome with a spike from MHV-2 was used to infect DBT cells. Results showed that Penn-98-1 behaved like MHV-2, thus establishing a role for the spike protein in viral growth. The inverse correlation between viral fusogenicity and growth was further established in four different cell types and with a fusogenic mutant, the S757R mutant, derived from isogenic Penn-98-1. While both A59 and Penn-98-1 entered cells at similar levels, viral RNA and protein syntheses were significantly delayed for A59. Interestingly, when the genomic RNAs were delivered directly into the cells via transfection, the levels of gene expression for these viruses were similar. Furthermore, cell fractionation experiments revealed that significantly more genomic RNAs for the nonfusogenic MHVs were detected in the endoplasmic reticulum (ER) within the first 2 h after infection than for the fusogenic MHVs. Pretreatment of Penn-98-1 with trypsin reversed its properties in syncytium formation, virus production, and genome transport to the ER. These findings identified a novel role for the spike protein in regulating the uncoating and delivery of the viral genome to the ER after internalization.

Development of a Chimeric DNA-RNA Hammerhead Ribozyme Targeting SARS Virus

Objective: Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). Method: Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To confirm its activity, in vitro cleavage reactions were performed with the synthesized ribozyme. Effects of the chimeric ribozyme were evaluated on multiplication of MHV. Effects of the chimeric ribozyme on expression of SARS-CoV were evaluated in cultured 3T3 cells. Result: The synthetic ribozyme cleaved the synthetic target MHV and SARS-CoV RNA into fragments of predicted length. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited the expression of SARS-CoV RNA in 3T3 cells transfected with the recombinant plasmid. The chimeric DNA-RNA ribozyme targeting SARS-CoV significantly inhibited MHV viral activity and expression of recombinant SARS RNA in vitro. Conclusion: These findings indicate that the synthetic chimeric DNA-RNA ribozyme could provide a feasible treatment for SARS.

In vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, Cimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, and Phellodendron cortex

BackgroundA search for new anti-coronaviral drugs to treat coronaviral infections was motivated by an outbreak of severe acute respiratory syndrome (SARS). ObjectivesIn order to find drugs that treat coronavirus infections, including SARS, we screened traditional medicinal herbal extracts and evaluated their antiviral activities on coronavirus replication. Study designWe employed a plaque assay to evaluate the effect of 22 medicinal herbal extracts on virus replication. We determined the 50% effective concentration (EC50) of each extract that was necessary to inhibit the replication of mouse hepatitis virus A59 (MHV-A59); we also determined 50% cytotoxic concentrations (CC50) for each extract. Northern and Western blot analyzes were performed to investigate antiviral activity in MHV-infected DBT cells, including virus entry, viral RNA and protein expression, and virus release. Coronavirus specific inhibition was also demonstrated using porcine epidemic diarrhea virus (PEDV). ResultsCimicifuga rhizoma, Meliae cortex, Coptidis rhizoma, Phellodendron cortex and Sophora subprostrata radix decreased the MHV production and the intracellular viral RNA and protein expression with EC50 values ranging from 2.0 to 27.5μg/ml. These extracts also significantly decreased PEDV production and less dramatically decreased vesicular stomatitis virus (VSV) production in vitro. ConclusionsThe extracts selected strongly inhibited MHV replication and could be potential candidates for new anti-coronavirus drugs.

Amino Acid Substitutions in the S2 Subunit of Mouse Hepatitis Virus Variant V51 Encode Determinants of Host Range Expansion

We previously described mouse hepatitis virus (MHV) variant V51 derived from a persistent infection of murine DBT cells with an expanded host range (R. S. Baric, E. Sullivan, L. Hensley, B. Yount, and W. Chen, J. Virol. 73:638-649, 1999). Sequencing of the V51 spike gene, the mediator of virus entry, revealed 13 amino acid substitutions relative to the originating MHV A59 strain. Seven substitutions were located in the amino-terminal S1 cleavage subunit, and six were located in the carboxy-terminal S2 cleavage subunit. Using targeted RNA recombination, we constructed a panel of recombinant viruses to map the mediators of host range to the six substitutions in S2, with a subgroup of four changes of particular interest. This subgroup maps to two previously identified domains within S2, a putative fusion peptide and a heptad repeat, both conserved features of class I fusion proteins. In addition to an altered host range, V51 displayed altered utilization of CEACAM1a, the high-affinity receptor for A59. Interestingly, a recombinant with S1 from A59 and S2 from V51 was severely debilitated in its ability to productively infect cells via CEACAM1a, while the inverse recombinant was not. This result suggests that the S2 substitutions exert powerful effects on the fusion trigger that normally passes from S1 to S2. These novel findings play against the existing data that suggest that MHV host range determinants are located in the S1 subunit, which harbors the receptor binding domain, or involve coordinating changes in both S1 and S2. Mounting evidence also suggests that the class I fusion mechanism may possess some innate plasticity that regulates viral host range.

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase.

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G(0)/G(1) phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G(0) phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G(1) and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21(Cip1), p27(Kip1), and p16(INK4a) did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G(1) cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G(1) cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G(0)/G(1) phase.

Down-regulation of transcription of the proapoptotic gene BNip3 in cultured astrocytes by murine coronavirus infection

Murine coronavirus mouse hepatitis virus (MHV) causes encephalitis and demyelination in the central nervous system of susceptible rodents. Astrocytes are the major target for MHV persistence. However, the mechanisms by which astrocytes survive MHV infection and permit viral persistence are not known. Here we performed DNA microarray analysis on differential gene expression in astrocyte DBT cells by MHV infection and found that the mRNA of the proapoptotic gene BNip3 was significantly decreased following MHV infection. This finding was further confirmed by quantitative reverse transcription–polymerase chain reaction, Western blot analysis, and BNip3-promoter-luciferase reporter system. Interestingly, infection with live and ultraviolet light-inactivated viruses equally repressed BNip3 expression, indicating that the down-regulation of BNip3 expression does not require virus replication and is mediated during cell entry. Furthermore, treatment of cells with chloroquine, which blocks the acidification of endosomes, significantly inhibited the repression of the BNip3 promoter activity induced by the acidic pH-dependent MHV mutant OBLV60, which enters cells via endocytosis, indicating that the down-regulation of BNip3 expression is mediated by fusion between viral envelope and cell membranes during entry. Deletion analysis showed that the sequence between nucleotides 262 and 550 of the 588-base-pair BNip3 promoter is necessary and sufficient for driving the BNip3 expression and that it contains signals that are responsible for MHV-induced down-regulation of BNip3 expression in DBT cells. These results may provide insights into the mechanisms by which MHV evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes.

Antiviral Activity of Limitin against Encephalomyocarditis Virus, Herpes Simplex Virus, and Mouse Hepatitis Virus: Diverse Requirements by Limitin and Alpha Interferon for Interferon Regulatory Factor 1

Limitin has sequence homology with alpha interferon (IFN-alpha) and IFN-beta and utilizes the IFN-alpha/beta receptor. However, it has no influence on the proliferation of normal myeloid and erythroid progenitors. In this study, we show that limitin has antiviral activity in vitro as well as in vivo. Limitin inhibited not only cytopathic effects in encephalomyocarditis virus- or herpes simplex virus (HSV) type 1-infected L929 cells, but also plaque formation in mouse hepatitis virus (MHV) type 2-infected DBT cells. In addition, administration of limitin to mice suppressed MHV-induced hepatitis and HSV-induced death. The antiviral activity may be mediated in part by 2',5'-oligoadenylate synthetase, RNA-dependent protein kinase, and Mx protein, which inhibit viral replication or degrade viral components, because limitin induced their mRNA expression and enzyme activity. While limitin has antiviral activity as strong as that of IFN-alpha in vitro (the concentration that provided 50% inhibition of cytopathic effect is approximately 30 pg/ml), IFN regulatory factor 1 (IRF-1) dependencies for induction of an antiviral state were different for limitin and IFN-alpha. In IRF-1-deficient fibroblasts, a higher concentration of limitin than of IFN-alpha was required for the induction of antiviral activity and the transcription of proteins from IFN-stimulated response element. The unique signals and the fewer properties of myelosuppression suggest that a human homolog of limitin may be used as a new antiviral drug.

Analysis of the antitumoral mechanisms of lipopolysaccharide against glioblastoma multiforme.

Our objective was to analyze the lipopolysaccharide (LPS) antitumoral effect upon glioblastoma, including whether the lipid A subunit alone can elicit glioblastoma regression, whether dexamethasone suppresses this response to LPS, whether B and T lymphocytes factor in this response, and whether this antitumoral effect of LPS provides resistance against subsequent challenge with glioblastoma. Mice (BALB/c, nude or SCID) implanted with s.c. DBT glioblastomas were treated with LPS (with or without dexamethasone) or with lipid A. A subset of BALB/c mice in which s.c. DBT glioblastomas had previously been eradicated using LPS were re-implanted with s.c. or intracranial (i.c.) DBT cells. For mice with s.c. tumors, mean tumor masses (MTM) were compared between groups. Survival was compared for mice with i.c. tumors. Lipid A caused near complete tumor regression of DBT glioblastomas in BALB/c mice (p<0.0001). Dexamethasone did not alter the antitumoral effect of LPS (p=0.48). LPS reduced the MTM of s.c. glioblastomas in T lymphocyte-deficient nude mice, but not as effectively as in immunocompetent mice. The antitumoral response to LPS for T and B lymphocyte-deficient SCID mice bearing DBT glioblastomas was similar to that for nude mice. Eradication of s.c. DBT glioblastoma in BALB/c provided partial resistance to subsequent challenge with s.c. or i.c. glioblastoma. We conclude that the LPS-mediated antitumoral response against glioblastoma is dependent upon the lipid A subunit of LPS, partially dependent upon T lymphocytes, independent of B lymphocytes, unaffected by dexamethasone and provides partial protection against subsequent challenges with glioblastoma.

Soluble receptor potentiates receptor-independent infection by murine coronavirus.

Mouse hepatitis virus (MHV) infection spreads from MHV-infected DBT cells, which express the MHV receptor CEACAM1 (MHVR), to BHK cells, which are devoid of the receptor, by intercellular membrane fusion (MHVR-independent fusion). This mode of infection is a property of wild-type (wt) JHMV cl-2 virus but is not seen in cultures infected with the mutant virus JHMV srr7. In this study, we show that soluble MHVR (soMHVR) potentiates MHVR-independent fusion in JHMV srr7-infected cultures. Thus, in the presence of soMHVR, JHMV srr7-infected DBT cells overlaid onto BHK cells induce BHK cell syncytia and the spread of JHMV srr7 infection. This does not occur in the absence of soMHVR. soMHVR also enhanced wt virus MHVR-independent fusion. These effects were dependent on the concentration of soMHVR in the culture and were specifically blocked by the anti-MHVR monoclonal antibody CC1. Together with these observations, direct binding of soMHVR to the virus spike (S) glycoprotein as revealed by coimmunoprecipitation demonstrated that the effect is mediated by the binding of soMHVR to the S protein. Furthermore, fusion of BHK cells expressing the JHMV srr7 S protein was also induced by soMHVR. These results indicated that the binding of soMHVR to the S protein expressed on the DBT cell surface potentiates the fusion of MHV-infected DBT cells with nonpermissive BHK cells. We conclude that the binding of soMHVR to the S protein converts the S protein to a fusion-active form competent to mediate cell-cell fusion, in a fashion similar to the fusion of virus and cell membranes.

Quantifying viral propagation in vitro: toward a method for characterization of complex phenotypes.

For a eukaryotic virus to successfully infect and propagate in cultured cells several events must occur: the virion must identify and bind to its cellular receptor, become internalized, uncoat, synthesize viral proteins, replicate its genome, assemble progeny virions, and exit the host cell. While these events are taking place, intrinsic host defenses activate in order to defeat the virus, e.g., activation of the interferon system, induction of apoptosis, and attempted elicitation of immune responses via chemokine and cytokine production. As a first step in developing an imaging methodology to facilitate direct observation of such complex host/virus dynamics, we have designed an immunofluorescence-based system that extends the traditional plaque assay, permitting simultaneous quantification of the rate of viral spread, as indicated by the presence of a labeled viral protein, and cell death in vitro, as indicated by cell loss. We propose that our propagation and cell death profiles serve as phenotypic read-outs, complementing genetic analysis of viral strains. As our virus/host system we used vesicular stomatitis virus (VSV) propagating in hamster kidney epithelial (BHK-21) and murine astrocytoma (DBT) cell lines. Viral propagation and death profiles were strikingly different in these two cell lines, displaying both very different initial titer and cell age effects. The rate of viral spread and cell death tracked reliably in both cell lines. In BHK-21 cells, the rate of viral propagation, as well as maximal spread, was relatively insensitive to initial titer and was roughly linear over several days. In contrast, viral plaque expansion in DBT cells was contained early in the infections with high titers, while low titer infections spread in a manner similar to the BHK-21 cells. The effect of cell age on infection spread was negligible in BHK-21 cells but not in DBTs. Neither of these effects was clearly observed by plaque assay.

RNase L-independent specific 28S rRNA cleavage in murine coronavirus-infected cells.

We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.

Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes.

The coronavirus replicase gene (gene 1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene 1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopic studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not in the fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of these populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.