RTase Activity Research Articles

Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells.

Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.

Unusual subunits are directly involved in binding substrates for natural rubber biosynthesis in multiple plant species

Rubber particles from rubber-producing plant species have many different species-specific proteins bound to their external monolayer biomembranes. To date, identification of those proteins directly involved in enzymatic catalysis of rubber polymerization has not been fully accomplished using solubilization, purification or reconstitution approaches. In an alternative approach, we use several tritiated photoaffinity-labeled benzophenone analogs of the allylic pyrophosphate substrates, required by rubber transferase (RT-ase) to initiate the synthesis of new rubber molecules, to identify the proteins involved in catalysis.Enzymatically-active rubber particles were purified from three phylogenetically-distant rubber producing species, Parthenium argentatum Gray, Hevea brasiliensis Muell. Arg, and Ficus elastica Roxb., each representing a different Superorder of the Dicotyledonae. Geranyl pyrophosphate with the benzophenone in the para position (Bz-GPP(p)) was the most active initiator of rubber biosynthesis in all three species. When rubber particles were exposed to ultra-violet radiation, 95% of RT-ase activity was eliminated in the presence of 50 μΜ Bz-GPP(p), compared to only 50% of activity in the absence of this analog. 3H-Bz-GPP(p) then was used to label and identify the proteins involved in substrate binding and these proteins were characterized electrophoretically.In all three species, three distinct proteins were labeled, one very large protein and two very small proteins, as follows: P. argentatum 287,000, 3,990, and 1,790 Da; H. brasiliensis 241,000, 3,650 and 1,600 Da; F. elastica 360,000, 3,900 and 1,800 Da. The isoelectric points of the P. argentatum proteins were 7.6 for the 287,000 Da, 10.4 for the 3,990 Da and 3.5 for the 1,790 Da proteins, and of the F. elastica proteins were 7.7 for the 360,000 Da, 6,0 for the 3,900 Da, and 11.0 for the 1,800 Da proteins. H. brasiliensis protein pI values were not determined.Additional analysis indicated that the three proteins are components of a membrane-bound complex and that the ratio of each small protein to the large one is 3:1, and the large protein exists as a dimer. Also, the large proteins are membrane bound whereas both small proteins are strongly associated with the large proteins, rather than to the rubber particle proteolipid membrane.

Purification and characterization of small and large rubber particles from Hevea brasiliensis.

Natural rubber (NR) is synthesized by the rubber transferase (RTase) on rubber particles (RPs) in latex. Due to the heterogeneity of the RPs in latex, it is difficult to precisely characterize the RTase activity. In this study, we separated the RPs of Hevea brasiliensis with different particle size distributions, via stepwise centrifugations. Analyses of protein compositions and size distributions of NR in the RPs suggest that RPs in Hevea latex can be categorized into two distinct subclasses, the larger RPs (termed 1kRP, 2kRP, and 8kRP) and the smaller RPs (termed 20kRP and 50kRP). Precise enzymatic assays using the RPs revealed that 50kRP showed the highest RTase activity, whereas the larger RPs, which had been regarded to have quite low activity, also exhibited a comparable activity to the smaller RPs. Immunological detections of cis-prenyltransferases in the RPs showed that the abundance of these enzymes correlates with the extent of RTase activity.

Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis.

Natural rubber (NR) is stored in latex as rubber particles (RPs), rubber molecules surrounded by a lipid monolayer. Rubber transferase (RTase), the enzyme responsible for NR biosynthesis, is believed to be a member of the cis-prenyltransferase (cPT) family. However, none of the recombinant cPTs have shown RTase activity independently. We show that HRT1, a cPT from Heveabrasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed HeveaRPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactucasativa also exhibited RTase activity, indicating proper introduction of cPT on RP is the key to reconstitute active RTase. RP proteomics and interaction network analyses revealed the formation of the protein complex consisting of HRT1, rubber elongation factor (REF) and HRT1-REF BRIDGING PROTEIN. The RTase activity enhancement observed for the complex assembled on WRPs indicates the HRT1-containing complex functions as the NR biosynthetic machinery.

Identification of anti-HIV and anti-Reverse Transcriptase activity from Tetracera scandens

We report here that an ethanol extract of Tetracera scandens, a Vietnamese medicinal plant, has anti-HIV activity and possesses strong inhibitory activity against HIV-1 reverse transcriptase (RTase). Using a MT-4 cell-based assay, we found that the T. scandens extract inhibited effectively HIV virus replication with an IC(50) value in the range of 2.0-2.5 μg/ml while the cellular toxicity value (CC50) was more than 40-50 μg/ml concentration, thus yielding a minimum specificity index of 20-fold. Moreover, the anti-HIV efficacy of the T. scandens extract was determined to be due, in part, to its potent inhibitory activity against HIV-1 RTase activity in vitro. The inhibitory activity against the RTase was further confirmed by probing viral cDNA production, an intermediate of viral reverse transcription, in virus-infected cells using quantitative DNA-PCR analysis. Thus, these results suggest that T. scandens can be a useful source for the isolation and development of new anti- HIV-1 inhibitor(s). [BMB reports 2012; 45(3): 165-170].

A modified single-tube one-step product-enhanced reverse transcriptase (mSTOS-PERT) assay with heparin as DNA polymerase inhibitor for specific detection of RTase activity

Background Current regulations and recommendations proposed for the production of vaccines in continuous cell lines of any origin demand that these be free of exogenous viruses, particularly retroviruses. Recently, the ultra-sensitive product-enhanced reverse transcriptase (PERT) assay can be used to detect minute of reverse transcriptase (RTase) in single retroviral particle and is 10 6 times more sensitive than the conventional RTase assays. However, coincidental with this increase in sensitivity is an increase in false-positive reactions derived from contaminating cellular DNA polymerases, which are known to have RTase-like activities. Objectives To develop a modified single-tube one-step PERT (mSTOS-PERT) assay with improvements on decreasing significantly the level of false-positive reactions, and to evaluate the mSTOS-PERT assay for sensitivity and specificity. Study design Ampliwax™ was used to compartmentalize the reverse transcription (RT) and PCR step in the same micro-tube with more efficiency and reproducibility, while maintaining the high sensitivity. The DNA amplification products were separated by 2% agarose gel electrophoresis, and then analyzed by non-isotopic Southern blot hybridization. A wide variety of cell lines used in biologicals production were detected to validate the improved mSTOS-PERT assay. Results The detection limit for the mSTOS-PERT assay was at least 10 −9 U, when using AMV-RTase as a positive control. Furthermore, heparin involvement in the RT step can eliminate completely the false-positive PERT signals which are exhibited by cellular polymerases such as DNA-dependent DNA polymerase α, γ released by cell death. Most mammalian cells (MRC-5, Vero, WISH, 2BS, RK-13, MDCK, etc.) are PERT-negative in cell supernatants. Some PERT-positive signals in cell lysates were found to be introduced by the cellular DNA polymerases and could be inhibited specifically by heparin. Chick cells derived from either chick embryo fibroblasts (CEF) or allantoic fluid from SPF embryonated eggs, murine hybridoma cell SP2/0, etc., contained authentic RTase activities, which could not be inactivated by heparin. Conclusions The improved mSTOS-PERT assay described here may distinguish the genuine RTase activity from cellular polymerases with high sensitivity and specificity, and is rapid and easy to perform to screen for the possible contamination of minute retroviruses in the cell substrates used in vaccine production.

Assessing the Significance of Reverse Transcriptase Activity in Chick Cell-derived Vaccines

A recent publication reported the detection of low levels of the enzyme reverse transcriptase (RTase) in live vaccines prepared in chick embryo cells. The enzyme was detected using an assay with greatly increased sensitivity compared to more conventional methods. The authors have confirmed the observation of RTase activity and demonstrate that the activity is not dependent on the production of viral vaccines in chick cells but is present ubiquitously in chick embryonic fluids.The authors have also been unable to transmit the RTase activity from chick cells to a wide variety of cells of human, monkey, rabbit and turkey origin, suggesting that the activity is not associated with an avian agent capable of infecting these cells. It is concluded that the data available present no cause for concern over the safety of vaccines derived in chick cells and current WHO requirements for such vaccines remain appropriate.

T-cell lymphoma induction by radiation leukemia virus in athymic nude mice.

We report the development of extrathymic lymphoblastic lymphomas in RadLV-inoculated congenitally athymic nude mice. Thus, a leukemogenic virus which appears to require the presence of a thymus for its replication in normothymic mice can infect and transform target cells in the absence of this organ in the athymic host. The cells of one of these lymphomas have been established in vitro as a permanent cell line, BALB/Nu1. This cell line as well as a lymphoma induced in NIH/Swiss nude mice exhibit several T-cell markers, including terminal deoxynucleotidyl transferase activity, Thy-1.2, and Ly-2.2, but not Ly-1.2 nor TL. Ig determinants were not detected. The characteristics of the tumor cells support the view that cells with T-cell markers may normally exist in nude mice and undergo neoplastic transformation and clonal expansion after infection with a leukemogenic virus. The alternative possibility that virus-induced differentiation of prothymocytes may lead to the expression of Thy-1.2 and Ly-2.2 antigens is also considered. BALB/Nu1 cells release large numbers of type C viral particles. The virus, designated radiation leukemia virus (RadLV)/Nu1, has RTase activity and the protein profile characteristic of murine leukemia virus (MuLV). In radioimmunoassays, it cross-reacts completely with RadLV/VL3, a virus obtained from RadLV-induced C57BL/Ka thymic lymphoma cells in culture, and slightly with a xenotropic virus (BALB:virus-2) and with AKR MuLV. On inoculation into C57BL/Ka mice it has thymotropic and leukemogenic activity. In vitro it is B-tropic, poorly fibrotropic, and has limited xenotropic activity. Thus, RadLV/Nu1 appears to be biologically and serologically similar or identical to its parent virus, RadLV.