Measurement of reverse transcriptase of feline immunodeficiency virus by poly A-linked colorimetric assay.

The method of the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) was developed and evaluated for the measurement of Mg(2+)-dependent reverse transcriptase (RT) activity of feline immunodeficiency virus (FIV). PAC-RTA was first evaluated for the detection of RT activity in the culture supernatant of FIV Petaluma strain. The detection limit of RT activity by PAC-RTA was about 10-fold better than that by the conventional non-radioisotopic RT assay kit. Then, PAC-RTA was evaluated for the indication of FIV isolation from cats naturally infected with FIV. FIV was isolated from peripheral blood mononuclear cells of 9 FIV-seropositive cats. The time course appearance of RT activity measured by PAC-RTA corresponded with the analysis of FIV antigen expression by indirect immunofluorescence. Finally, PAC-RTA evaluated the drug susceptibility of FIV. MYA-1 cells (feline T-lymphoblastoid cells) were infected with FIV and were cultured in the presence of various concentrations of anti-human immunodeficiency virus agents such as azidothymidine (AZT) or dextran sulfate. An inverse relationship between the RT activities and the concentrations of these agents in the culture supernatant was confirmed by PAC-RTA. PAC-RTA is easy to perform without using radioactive materials, and one plate can handle 96 samples at one time. By monitoring the RT activity, this assay is a useful method for FIV studies such as viral replication and drug susceptibility.

Human immunodeficiency virus type 1 drug susceptibility during zidovudine (AZT) monotherapy compared with AZT plus 2',3'-dideoxyinosine or AZT plus 2',3'-dideoxycytidine combination therapy. The protocol 34,225-02 Collaborative Group.

Human immunodeficiency virus type 1 (HIV-1) isolates obtained prior to and during a combination therapy trial comparing zidovudine (AZT; 3'-azidothymidine) monotherapy with AZT plus 2',3'-dideoxyinosine (ddI) or AZT plus 2',3'-dideoxycytidine (ddC) were assessed for the development of drug resistance. Drug susceptibility was measured by using two different phenotypic assays, one that requires infection of peripheral blood mononuclear cells with HIV-1 isolated from cocultures and a second based on infection of HeLa CD4+ cells with recombinant virus containing the reverse transcriptase (RT) of the clinical isolate. In addition, genotypic assessment of resistance was obtained by DNA sequencing of the RT coding region. No difference in the development of AZT resistance was noted in isolates from individuals receiving AZT monotherapy or combination therapy. However, a low frequency of ddI or ddC resistance was seen in isolates from the combination arms, which may at least partially explain the enhanced efficacy observed with these drug combinations compared with monotherapy. It was noted from DNA sequencing that a relatively high frequency of the nonnucleoside RT inhibitor resistance mutation, codon 181 changed from encoding Tyr to encoding Cys, was present in some isolates both before and during nucleoside analog combination therapy. Since these patients were unlikely to have access to nonnucleoside RT inhibitors, it is probable that this mutation preexisted at a reasonable level in the wild-type virus population. Comparisons of the AZT susceptibility assays indicated a good correlation between the phenotypic and genotypic determinations. However, direct numerical comparisons between the phenotypic assays were not reliable, suggesting that valid comparisons of different resistance data sets will require the use of the same assay procedure.

The reverse transcriptase assay: problems and practical solutions.

The detection of retroviruses has become critical for addressing the safety concerns associated with biologically derived products, including those derived from blood and cell line substrates, and gene therapy based systems. Most, if not all, retroviruses encode a unique enzyme called reverse transcriptase whose RNA-dependent DNA polymerase activity can be used as a marker for detecting retroviral contamination in test material. In this presentation we document some practical concerns when using the reverse transcriptase assay for detection of retroviruses. We also illustrate important aspects in the assay design by presentation of results that needed supplemental testing to enable accurate assessment of retroviral contamination.

Detection of reverse transcriptase activity by enzyme-linked immunosorbent assay in human immunodeficiency virus type 1.

An enzyme-linked immunosorbent assay (ELISA) using biotin-labelled oligo-dT primer and digoxigenin (Dig)-dUTP was designed to measure the reverse transcriptase (RT) activity of human immunodeficiency virus type 1 (HIV-1). The ELISA system involves the selective detection step of a newly synthesized cDNA by two specific bindings, biotin-streptavidin binding and alkaline phosphatase (AP)-conjugated anti-Dig-Dig binding, and the enzymatic amplification step to increase coloring generated by AP. This method was used to measure the activity of RT in the culture supernatants of peripheral leukocytes obtained from four anti-HIV-1-positive persons cocultivated with those from four anti-HIV-1-negative persons. RT activity was detected in all of four anti-HIV-1-positive culture supernatants but not in those cultivated with anti-HIV-1-negative supernatants alone. Thus, our improved ELISA for detection of HIV-1 appears to be sensitive enough and useful for routine laboratory work. This non-radioactive method will also be useful for detecting other retroviruses and for screening of RT inhibitors.

Production of a highly cytopathic HIV-1 isolate from a human mucosal epithelial cell line cultured on microcarrier beads in serum-free medium.

The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins.

Chemoprevention of mammary tumor virus-induced and chemical carcinogen-induced rodent mammary tumors by natural plant products.

The natural plant products turmeric, beta-carotene, catechin, and betel leaf extract were evaluated for their antitumor effects on mammary tumorigenesis in murine mammary tumor expressing C3H (Jax) mice and in Wistar rats treated with the chemical carcinogen 7-12-dimethylbenz(a)anthracene (DMBA). Administration of turmeric through the diet and of beta-carotene, catechin, and betel leaf extract through the drinking water to virgin female C3H mice resulted in decreased tumor incidence and tumor burden. Administering 5% turmeric in the diet from 2 months of age showed suppression of mammary tumor virus-related reverse transcriptase activity and of preneoplastic changes in the mammary glands. Furthermore, feeding turmeric from 6 months of age resulted in a 100% inhibition of mammary tumors. In the DMBA model of rat mammary tumorigenesis, administration of turmeric, catechin, and betel leaf extract resulted in decreased tumor burden and tumor incidence, and a delay in the onset of mammary tumors.

A semiautomated multiparameter approach for anti-HIV drug screening.

We are implementing a series of complementary assays for initial follow-up confirmation and prioritization of new active anti-HIV compounds identified by the U.S. National Cancer Institute's large-scale in vitro primary anti-HIV screen. Two different kinds of cellular viability assays, in addition to specific assays for total cellular DNA content, supernatant reverse transcriptase activity, p24 core antigen production and the synthesis of infectious HIV virions are all performed from a single well of a 96-well microtiter plate containing human host cells infected with HIV. Antiviral activities of several known prototype HIV inhibitors including 3'-azido,3'-deoxythymidine, 2',3'-dideoxycytidine, dextran sulfate and phorbol myristate acetate were compared in these multiparameter assays as a means of validation. Procedures to automate the method optimally, as well as to maximize the safety of the technicians working with HIV and HIV-infected cells have been emphasized. The resulting semiautomated, highly reproducible battery of assays yields a maximum amount of antiviral and cytotoxicity information from a minimum amount of sample. This is especially crucial when analyzing new synthetic compounds and natural product extracts or fractions where the available amounts of sample may be very limited.

Inhibition of HIV replication by Hyssop officinalis extracts.

Crude extracts of dried leaves of Hyssop officinalis showed strong anti-HIV activity as measured by inhibition of syncytia formation, HIV reverse transcriptase (RT), and p17 and p24 antigen expression, but were non-toxic to the uninfected Molt-3 cells. Ether extracts from direct extraction (Procedure I), after removal of tannins (Procedure II), or from the residue after dialysis of the crude extract (Procedure III), showed good antiviral activity. Methanol extracts, subsequent to ether, chloroform and chloroform ethanol extractions, derived from procedure I or II, but not III, also showed very strong anti-HIV activity. In addition, the residual material after methanol extractions still showed strong activity. Caffeic acid was identified in the ether extract of procedure I by HPLC and UV spectroscopy. Commercial caffeic acid showed good antiviral activity in the RT assay and high to moderate activity in the syncytia assay and the p17 and p24 antigen expression. Tannic acid and gallic acid, common to other teas, could not be identified in our extracts. When commercial products of these two acids were tested in our assay systems, they showed high to moderate activity against HIV-1. Hyssop officinalis extracts contain caffeic acid, unidentified tannins, and possibly a third class of unidentified higher molecular weight compounds that exhibit strong anti-HIV activity, and may be useful in the treatment of patients with AIDS.

Proteins of Rous-associated virus type 61: polypeptide stoichiometry and evidence that glycoprotein gp35 is not a cleavage product of gp85.

The two glycoproteins, gp85 and gp35, of Rous-associated virus type 61 (RAV-61), were isolated from radiolabeled virions by gel electrophoresis and digested with trypsin. The chromatographic profile of the gp35 digest revealed no peaks in common with that of gp85; therefore, the smaller glycoprotein is not a cleavage product of gp85. The stoichiometry of radiolabeled RAV-61 proteins was studied by quantitative gel filtration and gel electrophoresis. Among the 11 polypeptides identified were 4 minor ones, including the beta(p91) and alpha(p64) chains of reverse transcriptase and two unidentified chains, p76 and p35; the latter two were unmasked by removing the virions' surface glycoproteins with a protease, bromelain. Virions contained some 15 to 30 molecules of reverse transcriptase.

Intracisternal A particles from FLOPC-1 BALB/c myeloma: presence of high-molecular-weight RNA and RNA-dependent DNA polymerase.

Intracisternal A particles from the FLOPC-1 line of BALB/c myeloma have been shown to contain high-molecular-weight RNA (60 to 70S) that is sensitive to RNase, alkali degradation, and heat but resistant to Pronase treatment. The intracisternal A-particle RNA contains tract of poly (A) approximately 180 nucleotides long. As shown in a reconstitution experiment, by antigenic analysis of A-particle preparation and the SC cytopathogenicity assay, the 70S RNA was not due to contamination by type C virus particles. The FLOPC-1 intracisternal A particles also possess an endogenous RNA-dependent DNA polymerase. The enzyme required Mn2+ or Mg2+, dithiothreitol, detergent, and four deoxyribonucleoside triphosphates for maximum activity. Enzymatic activity was maximally stimuated by poly (rC)-oligo (dG)12-18 and less with poly (rG)-oligo (dC)10 or poly (rA)-oligo (dT)12-18 as compare with synthetic DNA/DNA duplex templates such as poly (dA)-oligo (dT)12-18. The enzyme can utilize the A-particle endogenous RNA as template as shown by analysis of the early and late DNA products of the endogenous reaction by CsSO4 isopycnic gradient centrifuation and hybridization of purified 70S or 35S A-particle RNA with the purified complementary DNA product. Approximately 50% of the A-particle complementary DNA also hybridized with oncornavirus RNA.