Reverse Transcriptase Assay Research Articles

Fluorometric Measurement of Reverse Transcriptase Activity with 4′,6-Diamidino-2-phenylindole

We describe a rapid fluorometric assay for reverse transcriptase (RT) activity. After RT is incubated in the presence of poly(A) · oligo(dT) and dTTP for up to 1 h, the reaction is stopped with EDTA and aliquots are added to cuvettes containing 4′,6-diamidino-2-phenylindole (DAPI). DAPI fluorescence, which is increased upon binding the RNA · DNA heteroduplex, is measured after 30 min and is linearly dependent on the enzymatic reaction time and the amount of active RT added to the enzyme assay. The increased fluorescence correlates well with the incorporation of [α- 32P]dTTP into DNA ( r 2 = 0.986). However, similar assays with the Klenow fragment using poly(dA) · oligo(dT) did not result in increased fluorescence under conditions wherein incorporation of [α- 32P]dTTP into DNA was documented. Thus, the poly(A) · poly(dT) [RNA · DNA] heteroduplex must differ from the poly(dA) · poly(dT) [DNA · DNA] duplex in a manner that allows for a perturbation of DAPI fluorescence. The relative specific activities of RT in crude preparations measured with the fluorometric assay were comparable to conventional isotopic enzyme assays as were determinations for the type of inhibition and the kinetic constants of purified RT with inhibitors such as zidovudine 5′-triphosphate, nevirapine, and oltipraz. This assay is far more rapid and less labor-intensive than standard isotopic assays for RT activity, and is much less expensive to perform than nonisotopic assays that measure the incorporation of nucleotide into DNA by immunological detection methods. This assay concept should allow for higher throughput screening of agents that inhibit RT, and may be useful for detecting RT activity in biological specimens.

Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.

Micro-carrier-test: evaluating disinfectants for HIV.

To determine the effect of disinfectants against viruses in vitro, I devised the Micro-Carrier-Test of dry-fixed virus-infected cells. Human immunodeficiency virus type 1-infected Molt-4 cells (1 x 10(5) cells in 5 microliters of 10% fetal bovine serum) were dry-fixed at the bottom of each well of a 96-well flat-bottomed microtiter plate for 120 minutes at room temperature. Disinfectants were added and allowed to remain for designated times and the wells were washed three times with PBS. Uninfected Molt-4 cells (1 x 10(4) cells/well) were inoculated and cultured for 4 weeks. The culture supernatant was harvested to measure reverse transcriptase activity by non-radioisotopic reverse transcriptase assay every week. Residual cytotoxicity of the disinfectant was determined by cytotoxicity assay. To evaluate the new method, the virucidal efficacy of several well-known disinfectants was reevaluated. Dose- and time-dependent effects of the disinfectants were determined. The minimal effective concentrations after 5 minutes of contact were 20% ethanol, 0.01% glutaraldehyde and 0.1% sodium hypochlorite. These results are almost the same as those reported previously, but there are some discrepancies. The differences between the present and previous protocols are discussed. This Micro-Carrier-Test promises to be useful in the screening of disinfectants.

Analysis of the VIF Gene of Feline Immunodeficiency Virus

We have examined FIV vif function in the context of molecular clone pF34 (GenBank Accession No. M25729). Rabbit antisera directed against the translation product of the vif gene identified a 29-kDa protein in tissue culture cells infected with FIV-pF34; this protein was not present in cultures of uninfected cells. Thus, the vif gene of this virus strain is expressed in infected cells. Three mutations were made in distinct regions of the vif gene of molecular clone FIV-pF34: (i) deletion of 223 bp from the central portion of the gene, (ii) site-directed mutation of a conserved N-terminal basic region, and (iii) site-directed mutation of a conserved C-terminal motif. FIV proviruses containing each of these mutations were assessed for replication following transfection into two feline adherent cell types, CrFK and G355-5. Reverse transcriptase and p24gag antigen assays of supernatants from transfected cultures revealed that all three vif mutants produced very little cell-free virus or viral protein in both cell types. Results of immunocytochemical staining of these cultures indicated that all three mutants expressed low levels of cell-associated FIV p24gag. These findings suggest that each of the three regions mutated in vif is critical for function. Our observations are consistent with studies showing marked attenuation of HIV-1 vif mutants in certain cell types. We conclude that the vif gene is a critical determinant of FIV-pF34 replication and infectivity in CrFK and G355-5 cells.

Inhibition of HIV Type 1 Reverse Transcriptase Assay by Nucleases Produced by Contaminating Mycoplasmas

Mycoplasmal contamination of HIV-1-infected cells has been found to induce reduction of reverse transcriptase (RT) activity; however, the exact mechanism of this phenomenon was not clearly elucidated. Our results indicate that the apparent reduction in RT activity is due to a calcium-dependent nuclease(s) that is (are) produced by contaminating mycoplasmas. The interference with the RT assay was found to be due to the degradation of products of the RT activity. Addition of EGTA at a 1 mM concentration was sufficient to remove the inhibitory effect. The particular HIV-1-producing cell line that was under study was found to be contaminated with Mycoplasma fermentans and Mycoplasma pirum and the latter was isolated in pure culture. Nuclease activity was also observed with pure cultures of mycoplasmas from different species. The activity was found to be of the endonuclease type because it was active with both supercoiled and linear DNAs.

Characterization of a Mutant HIV-1 Reverse Transcriptase Resistant to (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione (TIBO R82150)

A virus strain resistant to R82150, a non-nucleoside reverse transcriptase (NNRT) inhibitor (tetrahydro-imidazo [4,5, 1- jk] [1,4] benzodiazepine-2(1 H)-thione), was isolated following serial passage of HIV-1 RF in CEM-SS cells. The virus is cross-resistant to another non-nucleoside reverse transcriptase inhibitor, TGG-II-23A [1,4-dimethyl-1-[5,5-dimethyl-2-oxazoionyl]-naphthalen-2-one), but remains susceptible to AZT, DDI, D4T and phosphonoformate (PFA). DNA sequencing of reverse transcriptase genes from resistant virus indicated that R82150 selects for amino acid alterations Y181C and V108I. In vitro mutagenized reverse transcriptase and recombinant HIV-1 (pNL4-3) carrying either of the mutations have been generated. Genotypic and phenotypic analyses identified V108I as an unreported R82150-associated mutation. Both reverse transcriptase and viral resistance assays indicated that the resistance conferred by the V108I mutation is 7-fold less than that conferred by Y181C.

Quantitative micro P30 and reverse transcriptase assays for Moloney murine leukemia virus

An anti-P30 immunohistochemical and a reverse transcriptase assay for Moloney murine leukemia virus (MoMLV) are adapted to 96-well plates. The assay results are shown to be directly proportional to the number of infectious particles, and can therefore be used to estimate the infective titers of a virus preparation. The micro P30 assay yields a direct estimate of infectious centers, and the reverse transcriptase assay quantitates progeny from a single cycle of replication. The semi-automated nature of these assays is well suited to the analysis of a large number of samples and therefore permits the examination of the efficiency of the process of retroviral/MoMLV infection under varied times or conditions.

A putative new retrovirus associated with multiple sclerosis and the possible involvement of Epstein-Barr virus in this disease.

Since tropical spastic paraparesis in 1985 was found to be associated with HTLV-I infection, it has been suggested that a retrovirus might be involved in multiple sclerosis (MS). Our group has studied long-term cultures of cerebrospinal fluid cells and peripheral blood mononuclear cells from MS patients and controls with the purpose of elucidating the possible involvement of a retrovirus in MS. For an extended period electron microscopical analysis (EM) of T-cell lines, derived from MS patients and controls and cultured for 4 weeks was performed. In two cultures obtained 8 months apart from a patient with progressive MS, retrovirus-like particles were observed in 1-2% of the cells examined. Recently a B-lymphoblastoid cell line (LCL) producing retrovirus-like particles and EBV was established from a 30-year-old male patient with a chronic progressive myelopathy, clinically resembling multiple sclerosis. Similar cell lines have now been established from two MS patients. The retrovirus-like particles produced by the LCL have been purified by gradient ultracentrifugation. In the purified material reverse transcriptase assays are clearly positive in the gradients where EM shows retrovirus-like particles. Antigen characterization, nucleic acid sequence analysis and antibody studies are now being performed. The retrovirus found is definitively different from other known human retroviruses. It has previously been found that 100% of patients with MS have antibodies against EBV, in contrast to controls where only 86-95% have antibodies against this virus. Previous epidemiological studies have pointed toward a post-pubertal primary EBV infection as an important event in the induction of MS disease. These studies have now been substantiated by our group. Though it is still unknown whether EBV infection is a prerequisite for development of MS or whether the 100% EBV seropositivity is a consequence of the MS disease, we have put forward the hypothesis that the etiological agent for development of MS and MS-like diseases is a new hitherto uncharacterized retrovirus, whereas development of neurologic disease is related to or even dependent on a delayed infection with a virus from the herpes group, most likely EBV. This dual infection hypothesis has been analyzed and was found to be in accordance with the most consistent epidemiological characteristics of MS. We have previously, also from epidemiological data, negated retroviruses, behaving as the known human retroviruses, as an independent cause of MS.

Small-world solutions

Highlighted this week — a handy device for replica plating microbial colonies on solid growth media, a kit for the direct colorimetric detection of hepatitis B DNA and a nonradioactive reverse transcriptase assay for the detection of HIV-1.

Re-examination of the McCoy cell line for confirmation of its mouse origin: karyotyping, electron microscopy and reverse transcriptase assay for endogenous retrovirus

Background: The McCoy cell line originally derived from human synovial fluid in 1955, has been later found useful for cultivation of Chlamydia trachomatis. This cell line has been subcultured and exchanged between laboratories for many years. In recent years, the McCoy cell line has been widely used in many clinical diagnostic laboratories and has been supplied through commercial companies for the isolation and identification of Chlamydia trachomatis from clinical specimens. Objectives: Since retrovirus-like particles have been observed in McCoy cells and the species of origin of the currently used cell line has not been adequately documented, further characterization of McCoy cell line obtained from commercial sources was carried out. Study design: This study includes karyotypes analysis using G-banding for the confirmation of species origin of McCoy cells, electron microscopy for examination of virus particles associated with the cells and biochemical assay for reverse transcriptase activity for detection of retrovirus. Results: Our results showed by karyotype analysis that McCoy cells are of mouse origin. Electron microscopic examination revealed the presence of endogenous retrovirus type-A and type-C virions. Biochemical assays of culture supernatant fluids from McCoy cells detected reverse transcriptase activity which required Mg 2+ ions. Conclusions: The present study has confirmed that McCoy cells currently used by many laboratories are mouse cells, not the original McCoy cells derived from human cells. Laboratory workers should be aware of the presence of endogenous murine retrovirus in this cell line and appropriate precautions should be taken.

The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures

Drastic inhibition of the human immunodeficiency virus (HIV) reverse transcriptase (RT) by mycoplasma has been noted in many laboratories causing confusion in data interpretation. The mycoplasma-related inhibitor of HIV-1 RT was identified as a soluble protein in the particle-free supernatant of a contaminated culture. Gel filteration studies revealed the molecular mass of this protein to be about 70 kDa. This RT-inhibitor contained a DNase with strong activity on both linear and circular DNAs. Addition of this inhibitor after completion of reverse transcription still reduced the final outcome of the RT assay significantly, implying that the inhibitory mechanism occurred mainly by its DNase activity. Treatment of the culture with an antimycoplasma drug cured the mycoplasma contamination, removed the RT-inhibitor and abolished the DNase activity.

Analysis of long-term viral expression in CEM cells persistently infected with non syncytium-inducing HIV-1 strains.

CEM cells were infected with three HIV-1 non syncytium-inducing (NSI) strains obtained from AIDS patients or seropositive individuals. The surviving cells were followed for several months in the persistently infected cultures designated 65870/CEM, 65871/CEM and 3929/CEM, and analyzed for virus expression using light and electron microscopy, immunofluorescence, reverse transcriptase assay, polymerase chain reaction amplification (PCR), nucleic acid hybridization and flow cytometry. The virus isolates induced relatively few syncytia and other cytopathic effects in the corresponding cell lines and the number of cells positive for virus expression never rose above 44%. Distinct peaks of antigen-positive cells were obtained, coincident with high levels of reverse transcriptase activity. The cultures were strongly resistant to superinfection by laboratory strain Lai, with the exception of 65870/CEM which expressed HIV antigens in up to 15% of the cells for a few days. However, cell lysis was minimal in all cases. After long-term cultivation of the three cultures, no antigen-positive cells were detected and no trace of virus expression could be observed. The remaining cells consisted entirely of CD4-negative cells. PCR analyses indicated that cells harboring a provirus were progressively eliminated from the cultures, leaving only virus-free cells. In this system, cells carrying a latent provirus survive for a limited period of time before virus activation induces cell lysis. These results suggest that at least three types of cells exist in the CEM cell line: CD4-positive cells which are rapidly killed by the virus, a second type harboring a latent viral genome after the infection and which grow normally until activation of the resident genome by external or internal signal(s), and a third type which represents rare CD4-negative cells present in the initial CEM population and which are selected for by the NSI isolates. This is the first study documenting specific interactions between NSI strains of HIV-1 and distinct subpopulations of CEM cells grown as a single cell culture.

55 Optimization of the reverse transcriptase assay for the detection of viral burden in mice infected with rauscher murine leukemia virus: E.L. White, L. Westbrook, M.G. Hollingshead, and W.M. Shannon. Southern Research Institute, Birmingham, AL USA

55 Optimization of the reverse transcriptase assay for the detection of viral burden in mice infected with rauscher murine leukemia virus: E.L. White, L. Westbrook, M.G. Hollingshead, and W.M. Shannon. Southern Research Institute, Birmingham, AL USA

An improved non-radioisotopic reverse transcriptase assay and its evaluation.

We developed an improved, highly sensitive non-radioisotopic (non-RI) reverse transcriptase (RT) assay (RTA). While the original non-RI method previously reported made use of primer immobilization, our improved method was based on a primer-template immobilization procedure. We tested the template specificity, reproducibility and linearity of the new method in assays of human immunodeficiency virus type-1 (HIV-1) RT. The sensitivities of the method previously reported, the improved method and the sensitive radioisotopic (RI-) RTA were compared in assays of recombinant HIV-1 RT, partially purified HIV-1 particles, and the culture supernatant derived from HIV-1-infected cells. For each of these samples except the culture supernatant the improved method was the most sensitive. It appeared that the fetal bovine serum presented in the culture medium interfered with the assay reaction. The curve describing inhibition of the assay reaction by fetal bovine serum showed that the highest degree of sensitivity in the assay was obtained when the culture supernatant sample was diluted four times. With this degree of dilution, the sensitivity of the new method for assay of culture supernatant sample was still half that of the sensitive RI-RTA. Culture supernatants of five peripheral blood mononuclear cell samples obtained from HIV-1-seropositive carriers were assayed by both the improved method and the sensitive RI-RTA; and with each of the methods, however all virus-positive cultures could be detected. The improved non-RI RTA was considered especially useful for assay of culture supernatants for purposes of virus isolation because of its advantages of excellent sensitivity and lack of requirement for radioisotopes.

Characterization of HIV-1 strains isolated from patients treated with TIBO R82913.

The drug sensitivities of human immunodeficiency virus type 1 (HIV-1) isolates from a group of four untreated and seven TIBO R82913-treated patients were determined in a reverse transcriptase (RT) assay. Five of the treated patients harbored HIV-1 isolates with R82913 sensitivity comparable to that of the isolates of untreated patients, ranging from almost 2-fold higher sensitivity to 13-fold lower sensitivity than that of recombinant p66 RT. From one of the seven treated patients, an HIV-1 strain with a 20-fold reduced sensitivity to R82913 could be isolated; and from another patient, a strain with 100-fold reduced sensitivity (resistance) was isolated. The drug-resistant strain in this patient emerged after 3 weeks of treatment and was due to the Y188L mutation in its RT. On passaging the virus in cord blood lymphocytes, but not in CEM cells, the resistant virus was lost in favor of a different HIV-1 strain harboring the wild-type Y188 with a sensitivity to R82913 comparable to that of wild-type p66 RT. In several HIV-1 isolates (from treated and untreated patients), some HIV-2- and CIVgab-specific amino acids were found. One of these substitutions, that is, I/V179D (from an untreated patient), conferred a sevenfold reduced RT sensitivity to R82913.

Human immunodeficiency virus infection of early passage cervical epithelial cultures.

Women are infected with HIV in increasing numbers; the predominant mode of spread is through heterosexual transmission. Little is known regarding the mechanism of HIV transit through the female genital tract. We investigated whether early passage cervical epithelial cells could be directly infected with HIV-1LAI. Virus production was measured using the reverse transcriptase (RT) assay and direct assay for syncytia-forming units. In-situ hybridization was performed on infected cervical cell cultures. Immunostaining was carried out using a monoclonal antibody to leukocyte common antigen (LCA). Virus was recovered in the supernatants of all infected cervical cultures. Localization of HIV infection using in-situ hybridization identified rare cells in the population which gave a strong signal. These infected cells had a lymphoid morphology and were also detected using immunostaining for LAC. Cervical epithelial cells were uninfected in this in vitro model; cells in this population which supported viral replication were most likely of the macrophage/monocyte lineage.

Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase

An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.

Chemiluminescent Enzyme-Linked Immunoassay for Reverse Transcriptase, Illustrated by Detection of HIV Reverse Transcriptase

A chemiluminescent assay for reverse transcriptase (RT) of the human immunodeficiency virus 1 was developed using biotin-labeled oligodeoxythymidylic acid (biotin oligo-dT) and digoxigenin-deoxyuridine triphosphate instead of tritiated thymidine triphosphate. After the RT reaction, the newly polymerized strand from biotin oligo-dT contained digoxigenin labels. This nucleotide was bound to a streptavidin-coated microtiter plate by the reaction to biotin. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the reaction of a chemiluminescent substrate for this enzyme. This method shows very close correlation with the isotopic assay using purified avian myeloblastosis virus reverse transcriptase (RT). This assay was also compared with the isotopic RT assay using lymphocytes infected in vitro with HTLV-IIIB and again demonstrated a close correlation. The total assay time after the RT reaction step was less than 100 min.

The RRE of human immunodeficiency virus type 1 contributes to cell-type-specific viral tropism

As part of a general program investigating the mechanism of the Rev axis of human immunodeficiency virus type 1 (HIV-1) autoregulation, a series of proviral HIV-1 mutants which differ from the parental HXB2 strain at selected positions within the RRE were constructed. All of the mutations were designed to perturb the RRE by introducing local helix disruptions without altering the coding potential of the overlapping envelope open reading frame. Viral replication in various cell types was monitored by a cell supernatant reverse transcriptase assay and Northern (RNA blot) analysis. All proviral RRE mutants displayed at least some impairment in replication. However, the relative impairment varied drastically among the various cell types tested. This suggests that the RRE may contribute to cell-type-specific viral tropism.

Mutational analysis of Moloney murine leukaemia virus surface protein gp70

Ten mutations were generated in the env gene of Moloney murine leukaemia virus DNA. The mutations were made by site-directed mutagenesis to alter basic amino acids (lysine or arginine) in the surface glycoprotein gp70. Mutants were investigated following transfection into NIH/3T3 cells. All 10 mutants released virion particles into the medium, suggesting that none of the mutations affected overall viral gene expression or virion budding. Two mutants were positive in XC plaque assay, reverse transcriptase assay and re-infection experiments, showing that these mutations occurred in parts of the molecule not essential for infection. Three mutants were negative in both the XC plaque assay and re-infection experiments, suggesting that they make non-infectious virus particles. The results indicate a defect in the early phase of infection, perhaps in receptor binding or in the fusion of virion and host membranes. The other mutations resulted in reduced infectivity of released virion particles.