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PubMed2004

Portal donor-specific blood transfusion and mycophenolate mofetil allow steroid avoidance and tacrolimus dose reduction with sustained levels of chimerism in a pig model of intestinal transplantation.

BACKGROUND: In a pig model of intestinal transplantation, we previously showed that hepatic conditioning through portal donor-specific blood transfusion (pDSBT), high-dose tacrolimus (TAC), and steroids prevented rejection and increased survival Our current study tests a protocol of pDSBT, short-term mycophenolate mofetil (MMF), and low-dose TAC to eliminate the use of steroids, reduce TAC dosage, and increase the level of chimerism in the peripheral blood. MATERIALS AND METHODS: Four groups of outbred, mixed lymphocyte culture (MLC)-reactive pigs underwent bowel transplants and pDSBT. Immunosuppression (group 1, high-dose TAC and steroids; group 2, low-dose TAC and MMF; group 3, low-dose TAC, MMF, and aminoguanidine; group 4, low-dose TAC, MMF, and arginine) was discontinued after 28 days. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements. Chimerism levels were determined using a Q-PCR analysis. RESULTS: Pig survival and death rates due to rejection did not significantly differ between the four groups. Chimerism levels determined by Q-PCR analysis were not different until day 28. After discontinuation of immunosuppression, we noted a trend (P = 0.15) toward higher mean chimerism levels on day 60 for groups 2, 3, and 4 (9%) vs. group 1 (0.5%). Tissue cytokine and serum nitrate levels did not significantly differ between the four groups. Attempts to modify nitric oxide synthase activity offered no added benefit. CONCLUSIONS: The combination of pDSBT, MMF, and low-dose TAC (vs. high-dose TAC and steroids) allowed sustained levels of mixed chimerism to develop after discontinuation of immunosuppression.

Animals↗
PubMed2001

Tolerance, mixed chimerism and protection against graft-versus-host disease after total lymphoid irradiation.

Total lymphoid irradiation (TLI), originally developed as a non-myeloablative treatment for Hodgkin's disease, has been adapted for the induction of immune tolerance to organ allografts in rodents, dogs and non-human primates. Moreover, pretransplantation TLI has been used in prospective studies to demonstrate the feasibility of the induction of tolerance to cadaveric kidney allografts in humans. Two types of tolerance, chimeric and non-chimeric, develop after TLI treatment of hosts depending on whether donor bone marrow cells are transplanted along with the organ allograft. An advantageous feature of TLI for combined marrow and organ transplantation is the protection against graft-versus-host disease (GVHD) and facilitation of chimerism afforded by the predominance of CD4+ NK1.1(+) -like T cells in the irradiated host lymphoid tissues. Recently, a completely post-transplantation TLI regimen has been developed resulting in stable mixed chimerism and tolerance that is enhanced by a brief course of cyclosporine. The post-transplantation protocol is suitable for clinical cadaveric kidney transplantation. This review summarizes the evolution of TLI protocols for eventual application to human clinical transplantation and discusses the mechanisms involved in the induction of mixed chimerism and protection from GVHD.

Animals↗
PubMed1989

Characterization of a novel human respiratory syncytial virus chimeric FG glycoprotein expressed using a baculovirus vector.

Human respiratory syncytial virus (RSV) codes for two glycoproteins (F and G) which have been shown to the major targets for the host antibody response. We have expressed a novel chimeric glycoprotein (FG) in insect cells using a baculovirus vector. The chimeric glycoprotein contains the signal and extracellular regions of the RSV F glycoprotein linked to the extracellular region of the RSV G glycoprotein. Beginning at the amino terminus, the chimeric glycoprotein consists of amino acids 1 to 489 from RSV F followed by amino acids 97 to 279 from RSV G. The chimeric FG glycoprotein did not contain an anchor region and was efficiently secreted into the medium of recombinant baculovirus-infected insect cells. The FG glycoprotein ranged in size from 69K to 91K and was heterogeneous with respect to isoelectric point. The cleavage site present on the F glycoprotein was recognized on the chimeric FG, and the glycoprotein appeared to be antigenically similar to the native RSV F and G glycoproteins.

Antigens, Viral↗
PubMed2001

Characterization of chimeric enzymes between caprine arthritis--encephalitis virus, maedi--visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli.

In order to investigate the functions of the three putative lentiviral integrase (IN) protein domains on viral DNA specificity and target site selection, enzymatically active chimeric enzymes were constructed using the three wild-type IN proteins of caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1). The chimeric enzymes were expressed in Escherichia coli, purified by affinity chromatography and analysed in vitro for IN-specific endonuclease and integration activities on various DNA substrates. Of the 21 purified chimeric IN proteins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integration reaction. Analysis of the chimeric IN proteins revealed that the central domain together with the C terminus determines the activity and substrate specificity of the enzyme. The N terminus appears to have no considerable influence. Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the reaction conditions that support optimal enzyme activities of CAEV IN. Also, under the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibited endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments. Thus, the following report presents a detailed characterization of the activities of CAEV IN in vitro as well as the analysis of functional chimeric lentiviral IN proteins.

Arthritis-Encephalitis Virus, Caprine↗
PubMed2002

Reversible heat-induced inactivation of chimeric beta-glucuronidase in transgenic plants.

We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to beta-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5'-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions.

Adaptation, Physiological↗
PubMed2002

Clinicopathologic comparison between the API2-MALT1 chimeric transcript-positive and -negative gastric low-grade B-cell lymphoma of mucosa-associated lymphoid tissue type.

Little is known about the clinicopathological differences between API2-MALT1 chimeric transcript-positive and -negative gastric low-grade B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) type. The aim of this study was to clarify those differences in gastric MALT lymphoma. Twenty-three patients with gastric MALT lymphoma were enrolled in a unicenter study. Helicobacter pylori (H. pylori) infection status and clinical stages were investigated. Antibacterial treatment was performed for every patient. Responsiveness of MALT lymphoma to this treatment was assessed by means of regular follow-up endoscopy combined with biopsy. All cases were examined for API2-MALT1 chimeric transcript by means of RT-PCR and sequencing analyses. H. pylori infection status was assessed as positive in 20 patients and negative in three. With regard to responsiveness to antibacterial treatment, complete remission was observed in two patients, partial remission in 12 and no change in nine. API2-MALT1 chimeric transcript was detected in seven patients, all of whom showed no change in response to antibacterial treatment. API2-MALT1 positivity was found to be significantly correlated with responsiveness to antibacterial treatment (P = 0.0001), absence of H. pylori infection (P = 0.0198), and gross cobblestone mucosa observed endoscopically (P = 0.0198). For the other factors (age, sex, dominant site of lesion, high-grade component, infiltrated layer of gastric wall, nodal involvement or clinical stages), there were no differences between API2-MALT1 chimeric transcript-positive and -negative cases. Gastric API2-MALT1 chimeric transcript-positive MALT lymphoma generally features unresponsiveness to antibacterial treatment, and is thought to be unrelated to H. pylori infection in its pathogenesis. Our findings indicate the presence of different clinical subtypes in gastric MALT lymphomas.

Adult↗
PubMed1993

In vivo treatment with a monoclonal chimeric anti-CD4 antibody results in prolonged depletion of circulating CD4+ cells in chimpanzees.

Chimeric M-T412 (cM-T412), an anti-CD4 antibody, was tolerated in chimpanzees at a dosage of 5 mg/kg per day for up to 7 consecutive days, or 5 mg/kg per dose, twice weekly for 4 weeks. All cM-T412-treated chimpanzees showed a prolonged CD4-cell depression. Weak chimpanzee antibody responses to chimeric M-T412 were observed. One of the chimpanzees on the biweekly dosage regimen exhibited a hypersensitivity reaction immediately after receiving its seventh dose. Following supportive treatment, the animal recovered and remained asymptomatic during the non-treatment observation period. The hypersensitivity reaction was not an unexpected response considering the animal received repeated intermittent i.v. administration of a foreign protein. This animal also showed a chimpanzee antibody response to chimeric M-T412 after the seventh dose. Chimeric M-T412 also induced an anti-cM-T412 response in some of the other animals. The level of this response was lower than the anti-mouse responses observed in animals treated with murine anti-CD4. Moreover, the anti-cM-T412 response was mainly directed to idiotypic determinants. The decrease in CD4+ cells observed for all chimeric M-T412-treated chimpanzees is an expected effect of the anti-CD4 antibody. The duration of this CD4+ cell decrease is, however, much longer than observed for other CD4-specific MoAbs described. No selective loss of either memory or naive CD4+ cells was observed after either the single, 7-day or twice-weekly treatments. The CD4+ cell depression was reversible, although individual variation in time to recovery was observed. Therefore, cM-T412 could be a good candidate for clinical use in autoimmune conditions.

Animals↗
PubMed1994

Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions.

The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TM2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed 'smooth', biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.

Adaptation, Physiological↗
PubMed2004

Mixed chimerism is frequent after allogeneic peripheral blood stem cell transplantation with positive CD34 selection, and is not reverted by low doses of donor T-cells add-back.

UNLABELLED: The incidence of full donor chimerism (full DC) after CD34+ -selected peripheral blood stem cell transplantation (CD34+ -PBSCT) is controversial. Whereas the initial reports suggested a high incidence of full DC (hypothetically because of the high number of CD34+ cells infused) more recent works describe a high incidence of mixed lymphoid chimerism. There are no data concerning the ability of low-dose donor T-lymphocyte add-back on conversion to full DC. METHODS: We prospectively monitored the chimerism status of 25 patients undergoing CD34+ -PBSCT and the effect on chimerism of delayed low doses of donor T-cell add-back (TCAB). One, two or three doses of TCAB were administered on days +28 (2 x 10(5) CD3+/kg), +60 (2 x 10(5) CD3+/kg) and +90 (2 x 10(6) CD3+/kg), respectively, when on cyclosporine A prophylaxis. RESULTS: Incidence of full DC on day +20 was 56%. However, all but two patients progressed to MC. Fifteen patients were scheduled to TCAB. Six patients with initial MC did not convert to full DC after TCAB. Moreover, seven patients with full DC status progressed to mixed chimerism. CONCLUSIONS: Our results indicate that low doses of TCAB administered under cyclosporine A prophylaxis have no effect on the eradication of the recipient cells. We believe that a high dose of CD34+ cells in the grafts of CD34+ -PBSCT is not enough to achieve stable full DC unless a minimum number of CD3+ cells are infused, or more intensified transplant conditioning regimens are employed.

Adolescent↗
PubMed2000

Interactions of alcuronium, TMB-8, and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors.

A series of ligands that allosterically modulate the binding of classical ligands to muscarinic receptors was evaluated at wild-type and chimeric receptors. All of the ligands studied had highest affinity toward the M(2) subtype and lowest affinity toward the M(5) subtype. The chimeric receptors were mostly M(5) sequence; the amount of M(2) sequence ranged from about 6 to just under 30%. Alcuronium and TMB-8 had much higher affinity for the chimeric receptor that included the M(2) second outer loop of the receptor plus flanking regions of TM4 and TM5 than for any of the other chimeric receptors (the affinities of which remained similar to that of the M(5) subtype). However, this chimera retained the negative cooperativity between alcuronium and the classical antagonist N-methylscopolamine that is characteristic of M(5) (these ligands are positively cooperative at M(2)). Verapamil, tetrahydroaminoacridine, and d-tubocurarine were also sensitive to that chimeric substitution, although verapamil and tetrahydroaminoacridine had even higher affinity for a chimera with M(2) sequence in TM7. None of these ligands shared gallamine's sensitivity to a region of the third outer loop, but studies in which obidoxime reversed the allosteric effects of gallamine and other ligands suggested that they nevertheless compete for a common site. In summary, although the present data are consistent with previous studies that have suggested that allosteric ligands bind to the outermost regions of muscarinic receptors, it appears that different allosteric ligands may derive subtype selectivity from different regions of the receptor.

Acetylcholine↗
PubMed2003

Ligand specific up-regulation of a Renilla reniformis luciferase-tagged, structurally unstable muscarinic M3 chimeric G protein-coupled receptor.

The rat muscarinic acetylcholine receptor subtype 3 was modified by swapping the third intracellular loop with the corresponding region of a constitutively active mutant human beta2-adrenergic receptor and attaching Renilla reniformis luciferase to its C terminus. The chimeric fusion receptor displayed constitutive Gq- and Gs-coupled activity as demonstrated in nuclear factor of activated T cell and cAMP response element reporter gene assays. The chimeric receptor displayed a pharmacological binding profile comparable with that of the wild-type receptor for agonists, antagonists, and inverse agonists but showed a large decrease in expression in both human embryonic kidney 293 and COS-7 cells. Long-term treatment of cells expressing the chimeric receptor with agonists, antagonists, and inverse agonists resulted in a concentration-dependent up-regulation in the steady-state levels that was not observed for the wild-type receptor. The EC50 of neutral antagonists and inverse agonists was significantly correlated to their binding affinities at the wild-type receptor, whereas agonists demonstrated greater EC50 values for the chimeric receptor. To validate the approach as a means of discovering novel receptor modulators, a cell-based, high-throughput screening assay was developed and used to screen a small molecule compound collection against the chimeric fusion receptor. Several novel hits were identified and confirmed by ligand binding assay and functional assays using the wild-type rat muscarinic acetylcholine receptor subtype 3.

Amino Acid Sequence↗
PubMed1997

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention.

Actinomycetales↗
PubMed1998

Recombinant mouse-human chimeric antibodies as calibrators in immunoassays that measure antibodies to Toxoplasma gondii.

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was > or =0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

Amino Acid Sequence↗
PubMed1992

A chimeric avian retrovirus containing the influenza virus hemagglutinin gene has an expanded host range.

We have investigated what protein sequences are necessary for glycoprotein incorporation into Rous sarcoma virus (RSV) virions by utilizing the hemagglutinin (HA) protein of influenza virus. Two chimeric HA genes were constructed. In the first the coding sequence for the signal peptide of the RSV env gene product was fused in frame to the entire HA structural gene, and in the second the hydrophobic anchor and cytoplasmic domain sequences of the HA gene were also replaced with those from the RSV env gene. Both chimeric genes, expressed from a simian virus 40 expression vector in CV-1 cells, yielded functional HA proteins that were transported to the cell surface and were able to bind to erythrocytes. When the genes were expressed in combination with the RSV gag-pol gene region in QT6 cells by using a vaccinia virus-T7 expression/complementation system, virions that efficiently incorporated either chimeric protein were assembled. This result indicated that the presence of the RSV env membrane anchor and cytoplasmic sequences did not facilitate HA glycoprotein incorporation into virions. The presence of the RSV env signal sequence allowed the chimeric HA genes to be substituted into the RSV-derived BH-RCAN.HiSV viral genome in place of the RSV env gene. Both chimeric genomes yielded infectious virus that could infect human and avian cells with equal efficiency. These experiments demonstrate that a foreign glycoprotein, efficiently incorporated into virions lacking a native glycoprotein, can confer a broadened host range on the virus. Moreover, because the HA of influenza virus requires the acidic pH of the endosome in order to be activated, these results imply that foreign proteins can modify the normal route of entry of this avian retrovirus.

Amino Acid Sequence↗
PubMed1993

5' sequences of rubella virus RNA stimulate translation of chimeric RNAs and specifically interact with two host-encoded proteins.

Sequences at the 5' and 3' ends of the rubella virus (RV) genomic RNA can potentially form stable stem-loop (SL) structures that are postulated to be involved in virus replication. We have analyzed the function of these putative SL structures in RNA translation by constructing chimeric chloramphenicol acetyltransferase (CAT) RNAs, flanked either by both 5'- and 3'-terminal sequence domains from the RV genome or several deletion derivatives of the same sequences. After in vitro transcription of chimeric RNAs, the translational efficiencies of these RNAs were compared by the rabbit reticulocyte lysate translation system. For in vivo translation studies, the level of CAT activity was measured for chimeric RV/CAT RNAs expressed in transfected cells by the adenovirus major late promoter. Both in vivo and in vitro translation activities of the chimeric RNAs revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct (+) orientation and context [5'(+)SL and 3'(+)SL, respectively] was necessary for efficient translation of chimeric RV/CAT RNAs. The presence of the RV 5'(+)SL sequence had the primary enhancing effect on translation. To identify host proteins which interact with the 5'(+)SL which may be involved in RV RNA translation, RNA gel-shift and UV cross-linking assays were employed. Two host proteins 59 and 52 kDa in size, present in cytosolic extracts from both uninfected and RV-infected cells, specifically interacted with the RV 5'(+)SL RNA. Direct binding comparisons between wild-type and mutant 5'(+)SL RNAs demonstrated that sequences in and around the bulge region of the terminal stem domain of this structure constituted a protein binding determinant. Human serum, qualified for anti-Ro/SS-A antigen specificity, immunoprecipitated 59- and 52-kDa protein-RNA complexes containing the RV 5'(+)SL RNA. However, poly- and monoclonal antisera raised against the recombinant 60- and 52-kDa Ro proteins failed to precipitate complexes containing the 5'(+)SL RNA. The identity of the proteins binding this RV cis-acting element remains to be determined; however, their role in RV translation is discussed.

Animals↗
PubMed1996

Analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the R peptide.

It was previously reported that truncation or proteolytic removal of the C-terminal 16 amino acids (the R peptide) from the cytoplasmic tail of the murine leukemia virus (MuLV) envelope protein greatly increases its fusion activity. In this study, to investigate the specificity of the effect of the R peptide on the fusion activity of viral envelope proteins, we expressed simian immunodeficiency virus (SIV)-MuLV chimeric proteins in which the entire cytoplasmic tail of the SIV envelope protein was replaced by either the full-length MuLV cytoplasmic tail or a truncated MuLV cytoplasmic tail with the R peptide deleted. Extensive fusion of CD4-positive cells with the chimeric protein containing a truncated MuLV cytoplasmic tail was observed. In contrast, no cell fusion activity was found for the chimeric protein with a full-length MuLV cytoplasmic tail. We constructed another SIV-MuLV chimeric protein in which the MuLV R peptide was added to an SIV envelope protein cytoplasmic tail 17 amino acids from its membrane-spanning domain. No fusion activity was observed within this construct, while the corresponding truncated SIV envelope protein lacking the R peptide showed extensive fusion activity. No significant difference in the transport or surface expression was observed among the various SIV-MuLV chimeric proteins and the truncated SIV envelope protein. Our results thus demonstrate that the MuLV R peptide has profound inhibitory effects on virus-induced cell fusion, not only with MuLV but also in a distantly related retroviral envelope protein which utilizes a different receptor and fuses different cell types.

Amino Acid Sequence↗
PubMed1996

Interactions between PE2, E1, and 6K required for assembly of alphaviruses studied with chimeric viruses.

During the assembly of alphaviruses, a preassembled nucleocapsid buds through the cell plasma membrane to acquire an envelope containing two virally encoded glycoproteins, E2 and E1. Using two chimeric viruses, we have studied interactions between E1, E2, and a viral peptide called 6K, which are required for budding. A chimeric Sindbis virus (SIN) in which the 6K gene had been replaced with that from Ross River virus (RR) produced wild-type levels of nucleocapsids and abundant PE2/E1 heterodimers that were processed and transported to the cell surface. However, only about 10% as much chimeric virus as wild-type virus was assembled, demonstrating that there is a sequence-specific interaction between 6K and the glycoproteins required for efficient virus assembly. In addition, the conformation of E1 in the E2/E1 heterodimer on the cell surface was different for the chimeric virus from that for the wild type, suggesting that one function of 6K is to promote proper folding of E1 in the heterodimer. A second chimeric SIN, in which both the 6K and E1 genes, as well as the 3' nontranslated region, were replaced with the corresponding regions of RR also resulted in the production of large numbers of intracellular nucleocapsids and of PE2/E1 heterodimers that were cleaved and transported to the cell surface. Budding of this chimera was severely impaired, however, and the yield of the chimera was only approximately 10(-7) of the SIN yield in a parallel infection. The conformation of the SIN E2/RR E1 heterodimer on the cell surface was different from that of the SIN E2/SIN E1 heterodimer, and no interaction between viral glycoproteins and nucleocapsids at the cell plasma membrane could be detected in the electron microscope. We suggest that proper folding of the E2/E1 heterodimer must occur before the E2 tail is positioned properly in the cytoplasm for budding and before heterodimer trimerization can occur to drive virus budding.

Animals↗
PubMed1996

Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions.

An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to cultured eukaryotic cells. Mutant particles carrying the tripeptide sequence RGE in place of RGD and the use of a competitive peptide, GRGDS, confirmed the critical involvement of the RGD sequence in this binding. The chimeric particles also bound to purified integrins, and inhibition by chain-specific anti-integrin monoclonal antibodies implicated alpha 5 beta 1 as a candidate cell receptor for both the chimeric particle and FMDV. Some serotypes of FMDV bound to beta 1 integrins in solid- phase assays, and the chimeric particles competed with FMDV for binding to susceptible eukaryotic cells. Thus, HBc particles may provide a simple, general system for exploring the interactions of specific peptide sequences with cellular receptors.

Animals↗