Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope.

Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 x LD50) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.

Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.

Virucidal activity of an oral fluid human immunodeficiency virus-1 antibody preservative.

Diagnosis of human immunodeficiency virus-1 (HIV-1) infection requires the collection of either serum or oral fluid that is subsequently tested for the presence of antibodies to HIV-1. The effective use of oral fluid for the detection of HIV antibodies is contingent on stabilization of immunoglobulins in the sample through the use of preservatives. Oral fluid preservatives also contain agents that can disrupt and inactivate viruses. This study demonstrates the virucidal activity of a commercially available oral fluid preservative against HIV-1 using a sensitive 28-day cell culture assay designed to detect infectious virus. The results demonstrate that a 5-log reduction in viral titer is obtained when equal volumes of HIV-1 viral stocks and the preservative are mixed. The data provide strong evidence that preserved oral fluid samples from infected individuals are noninfectious for HIV-1.

Future applications of oral fluid specimen technology.

Research has demonstrated that oral mucosal transudate (OMT), a serum-derived fluid that enters saliva from the gingival crevice and across oral mucosal surfaces, can be preferentially concentrated by a novel collecting system to yield detectable levels of immunoglobulins (i.e., IgG and IgM antibodies) against various bacterial and viral diseases. Assays based on OMT can aid in the diagnosis of disease and in the management of therapeutic drugs. A reliable and accurate OMT-based test to detect human immunodeficiency virus (HIV) antibodies is commercially available. Additional tests based on similar technologies may aid in the diagnosis of viral hepatitis, measles, mumps, and rubella as well as in monitoring levels of therapeutic drugs such as theophylline. The future use of OMT-based testing will likely increase because of the inherent advantages of this technology: convenience; avoidance of inadvertent transmission of blood-borne pathogens; ease of use in pediatric and geriatric populations; as well as the potential for blood-free home and workplace collection of patient samples.

Evaluation of a system using oral mucosal transudate for HIV-1 antibody screening and confirmatory testing. OraSure HIV Clinical Trials Group.

OBJECTIVE: To determine accuracy of a human immunodeficiency virus type 1 (HIV-1) antibody testing system using a device to collect and stabilize oral mucosal transudate (OMT), a fluid with increased levels of IgG; an enzyme immunoassay (EIA) screening test optimized for OMT; and a Western blot confirmatory test designed for use with OMT. DESIGN: The OMT specimens were tested by EIA and, if indicated, confirmatory Western blot according to a standard testing algorithm. The OMT results were compared with true HIV status as determined by serum testing and/or clinical diagnosis. PATIENTS: Specimens from 3570 subjects (2382 at low risk, 698 at high risk, 242 with acquired immunodeficiency syndrome [AIDS], and 248 "nonspecificity" [persons with diseases associated with an increased frequency of false-positive results in HIV testing]) were collected at 11 geographically diverse sites (including blood banks, public health clinics, general medical clinics, HIV clinics, sexually transmitted disease clinics, and a hemophilia center) in the United States. MAIN OUTCOME MEASURES: Overall accuracy of testing OMT for HIV-1 antibodies compared with true HIV-1 antibody status; sensitivity and specificity of OMT EIA and Western blot. RESULTS: Sensitivity of OMT EIA testing in 673 true-positive subjects was 99.9% (672/673). The OMT Western blot results in the 673 true-positive subjects were positive in 665 and indeterminate in 8. The EIA followed by Western blot (if EIA was repeatedly reactive) yielded a negative result in 99.9% (2893/2897) of OMT samples from true negatives and an indeterminate result in 4. The OMT testing system provided the correct result or would trigger appropriate follow-up testing in 3569 (>99.9%) of 3570 cases. CONCLUSION: HIV-1 antibody testing of OMT samples is a highly accurate alternative to serum testing.

Studies of coat protein-mediated resistance to TMV. I. The PM2 assembly defective mutant confers resistance to TMV.

Tobacco mosaic virus mutant PM2 contains two amino acid changes in coat protein sequence relative to the sequence of the coat protein of TMV U1. This results in unstable infectivity, inability to cause normal systemic infection, and accumulation of elongated open helixes of coat protein. Using site-directed mutagenesis we demonstrated that the characteristics of PM2 are due to the change of Thr28-->Ile, while the second change, Glu95-->Asp, had no apparent effect on virion structure or infectivity. Transgenic Nicotiana tabacum cv Xanthi NN and Xanthi nn plants that accumulate coat protein that contains one or both of the amino acid changes are as resistant to TMV infection as transgenic plants that contain wild-type TMV coat protein. The implication of these results on a model for coat protein-mediated resistance that involves the interaction of transgenic coat protein with the challenge virus is discussed.

Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica.

S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine. We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica. The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT. Rapid amplification of the 5' end of the cDNA indicated that the full-length cDNA of SMT consisted of 1,458 nucleotides that encoded 381 amino acids. When the full-length cDNA was expressed in E. coli, the molecular mass of the expressed SMT was greater than that of native SMT in Coptis cells. This result suggests that SMT might be produced in a pre-mature form and processed post-translationally. SMT was also found to exhibit sequence homology to other O-methyltransferases from plants and N-terminal region of the SMT polypeptide appeared to be necessary for enzymatic activity.

Theophylline in oral mucosal transudate. A practical method for monitoring outpatient therapy.

We tested the utility of a novel collection system that allows measurement of theophylline in oral mucosal transudate (OMT) to calculate serum theophylline concentrations. In 25 adult patients, theophylline levels in OMT correlated better than expectorated saliva with serum theophylline (r = 0.927 for OMT versus r = 0.831 for expectorated saliva, each p < 0.0001). In a subsequent study of 128 patients (118 adults and 10 children aged 4 to 12 yr), OMT and serum theophylline were measured and polynomial regression analysis performed to allow calculation of serum level for any given OMT level. Theophylline levels calculated from OMT values closely followed measured serum theophylline in two normal subjects after administration of either intravenous or oral theophylline. OMT samples collected by 24 patients at home were mailed to the laboratory for testing. Theophylline values from the home collection samples correlated closely (r = 0.930, p < 0.0001) with serum theophylline levels obtained at the same dose of theophylline. These findings suggest that once the relationship of serum to OMT theophylline is established in a given laboratory, the latter can be used to monitor outpatient theophylline therapy in adults (and possibly children) at times of the day otherwise inaccessible to serum sampling.

Genetically engineered protection against viruses in transgenic plants.

Transgenic plants carrying nucleotide sequences derived from plant viruses can exhibit increased resistance to viral disease. Many viral sequences confer some level of either resistance to infection or suppression of disease symptoms (tolerance). These include segments of viral genomes encoding capsid or coat proteins, sequences encoding proteins that are or may be subunits of the viral replicase, sequences incapable of encoding proteins, entire genomes of defective interfering viruses and satellite viruses, and complete genomes of mild strains of virus. The transgene may act on initiation of infection, replication of virus, spread of the infection throughout the plant, and symptom development. More than one of these processes can be impaired by a single transgene derived from a single viral gene. The level of protection ranges from very low to high, while the breadth of protection ranges from very narrow, where protection is only observed against closely related strains of the virus from which the transgene was derived, to moderately broad, extending to other viruses. Data are insufficient to establish a molecular mechanism of resistance for most of the described examples. In addition, although the use of a particular segment of the viral genome confers resistance in one virus-host system, analogous sequences from a different virus in another host may be ineffective.

Synergistic activity of 5-trifluoromethylthioribose and inhibitors of methionine synthesis against Klebsiella pneumoniae.

5-Methylthioribose (MTR) is an intermediate in the methionine recycling pathway of organisms containing the enzyme MTR kinase. Analogs of MTR have been proposed as a new class of antimicrobial agents because of their ability to perturb the growth of MTR kinase-containing pathogens through inhibition of methionine salvage or by conversion to toxic products. One such analog, 5-trifluoromethylthioribose (TFMTR), has demonstrated potent inhibitory effects on the growth of Klebsiella pneumoniae (A. G. Gianotti, P. A. Tower, J. H. Sheley, P. A. Conte, C. Spiro, J. H. Fitchen, and M. K. Riscoe, J. Biol. Chem. 265:831-837, 1990). Although the mode of action of TFMTR has yet to be determined, it is believed that the drug is converted to the toxic products trifluoromethionine or carbonothioic difluoride via MTR kinase and the methionine recycling pathway. On the basis of this assumption, we theorized that blocking de novo methionine synthesis would increase dependence on the methionine salvage pathway and lead to an increased rate of synthesis of toxic metabolites from TFMTR. In this report, we show that three separate inhibitors of de novo methionine synthesis (1,2,4-triazole, azaserine, and propargylglycine) act synergistically with TFMTR in inhibiting the growth of K. pneumoniae.

Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure.

To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.

Selective killing of Klebsiella pneumoniae by 5-trifluoromethylthioribose. Chemotherapeutic exploitation of the enzyme 5-methylthioribose kinase.

5'-Deoxy-5'-methylthioadenosine (MTA), an important intermediate in methionine recycling, can be metabolized by one of two mechanisms that appear to be mutually exclusive. In human cells, MTA is degraded in one step to adenine and 5-methylthioribose 1-phosphate (MTR-1-P) via MTA phosphorylase. In contrast, certain microbes metabolize MTA in two steps: first to 5-methylthioribose (MTR) followed by conversion to MTR-1-P. The enzymes involved in this two-step conversion are MTA nucleosidase and MTR kinase. In both cases, MTR-1-P is subsequently recycled to methionine. Because MTR kinase is "unique" to microbes (it is also found in plant tissue) and since it is essential to microbial methionine salvage, we hypothesized that MTR kinase is a promising target for chemotherapeutic exploitation. We demonstrate that 5-trifluoromethylthioribose (TFMTR), a structural analog of MTR, is a potent inhibitor of the MTR kinase-containing organism Klebsiella pneumoniae. TFMTR not only inhibits the growth of K. pneumoniae in a dose-dependent manner (50% inhibition at approximately 40 nM) but also competitively inhibits MTR kinase activity (Ki approximately 7 microM). Furthermore, TFMTR is shown to be a substrate for MTR kinase (Km = 1.7 microM), suggesting that the drug could be converted to toxic products (e.g. trifluoromethionine or carbonothionic difluoride) in enzyme-containing organisms. Structural analogs of MTR represent a new class of compounds with the potential for treating diseases caused by MTR kinase-containing microorganisms.

Methionine recycling as a target for antiprotozoal drug development.

The development of new and effective ontiprotozool drugs has been difficult because of the close metabolic relationship between protozoa and mammalian cells. In this article, Michael Riscoe, Al Ferro and john Fitchen present their hypothesis for chemotherapeutic exploitation of methylthioribose (MTR) kinase, an enzyme critical to methionine salvage in certain protozoa. They propose that analogues of MTR if properly designed, would be converted to toxic products in organisms that contain MTR kinase but not in mammalian cells, which lack this enzyme.

Purine metabolism as a target for leukemia chemotherapy.

This article focuses on the chemotherapeutic agents which alter purine metabolism as a means to achieve selective killing of leukemic cells. We present an overview of purine metabolism in order to highlight enzymatic steps which are targeted by antileukemic drugs. Purine antimetabolites used in the treatment of leukemia can be grouped into three classes: (1) structural analogs of normal purines (6-mercaptopurine and 6-thioguanine); (2) inhibitors of de novo purine biosynthesis (methotrexate and hydroxyurea); and (3) inhibitors of purine salvage (2'-deoxycoformycin). In addition, a number of investigational drugs (trimetrexate, fludarabine and 2'-chlorodeoxyadenosine) have been recently introduced and show promise in early clinical trials. Purine antimetabolites are active in a variety of lymphoid and myeloid leukemias and represent an important component of the therapy of these disorders. Several of the drugs have been developed with the specific intent of perturbing enzymes involved in purine metabolism. Refinements in our understanding of purine biochemistry in normal and leukemic cells may aid future efforts to design more effective drugs.

Analogs of 5-methylthioribose, a novel class of antiprotozoal agents.

Since drug resistance and toxicity limit the use of available antiprotozoal agents, it is important that new drugs be developed as soon as possible. In this study, the method by which several protozoa degrade 5'-methylthioadenosine (MTA) was shown to differ from MTA catabolism in human cells. To exploit this metabolic difference, two analogs of methylthioribose (MTR), an MTA catabolite, were synthesized and found to be cytocidal to Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis in vitro. In contrast, these analogs had no effect on cultured mammalian cells. Analogs of MTR represent a potential new class of antiprotozoal drugs.

Methylthioadenosine phosphorylase deficiency in acute leukemia: pathologic, cytogenetic, and clinical features.

Blast cells from 100 cases of acute leukemia were evaluated for the presence of methylthioadenosine phosphorylase (MTAase), an enzyme important in polyamine metabolism. Ten cases (10%) had undetectable levels of MTAase activity. Of the 10, 5 had acute lymphoblastic leukemia (ALL), 3 had acute myeloblastic leukemia (AML) and 2 expressed mixed lineage markers as determined by immunophenotyping. A relatively high frequency (38%) of MTAase deficiency was seen in ALL of T-cell origin. Nonmalignant hematopoietic cells from three patients with MTAase-deficient leukemias had readily detectable enzyme activity. Chromosomal abnormalities were detected in four of the seven MTAase-deficient cases in which karyotypic analysis was performed. No consistent karyotypic defect was apparent, and only one case displayed changes in chromosome 9, the putative location of the MTAase structural gene. The clinical findings among the enzyme-deficient cases were unremarkable except that all patients were male (P less than .01). Only one patient had "lymphomatous" features. We conclude that MTAase deficiency occurs in a wide variety of acute leukemias, that the lack of enzyme activity is specific to the malignant cells, and that an increased incidence occurs in ALL of T-cell origin. Furthermore, no specific gross chromosomal abnormality is associated with the enzyme deficiency. The marked male predominance in patients with MTAase-deficient acute leukemias suggests involvement of the X chromosome in the loss of enzyme activity. The absence of MTAase in some leukemias may be therapeutically exploitable.