Functional expression of the reverse transcriptase αß heterodimer from avian myeloblastosis virus in Escherichia coli.

The α subunit of avian myeloblastosis virus reverse transcriptase (AMV-RT) is generated from the ß-subunit by proteolysis, and the αß heterodimer represents the active form. The codon-optimized gene was expressed in Escherichia coli, and an active αß heterodimer was generated. The RNA amplification activity of the purified recombinant AMV-RT αß heterodimer was similar to that of the native one.

Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives.

Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18-mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs.

Fluorescent Tricyclic Cytidine Analogues as Substrates for Retroviral Reverse Transcriptases.

We report on the ability of the reverse transcriptases (RTs) from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M-MLV), and human immunodeficiency virus 1 (HIV-1) to generate labeled DNA using the fluorescent tricyclic cytidine analogues d(tC)TP and d(DEA tC)TP as substrates. Michaelis-Menten kinetics for the insertion of these analogues show Vmax /KM from 0.0-5 times that of natural dCTP across from G, depending on the polymerase and whether the template is RNA or DNA. The analogues are prone to misinsertion across from adenosine with both RNA and DNA templates. Elongation after analogue insertion is efficient with RNA templates, but the analogues cause stalling after insertion with DNA templates. A model reverse transcription assay using HIV-1-RT, including RNA-dependent DNA synthesis, degradation of the RNA template by the RT's RNase H activity, and synthesis of a second DNA strand to form fluorescently labeled dsDNA, shows that d(tC)TP and d(DEA tC)TP are compatible with a complete reverse transcription cycle in vitro.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA- amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency;

Microwave-Assisted Rapid Enzymatic Synthesis of Nucleic Acids.

Herein we report microwave-induced enhancement of the reactions catalyzed by Escherichia coli DNA polymerase I and avian myeloblastosis virus-reverse transcriptase. The reactions induced by microwaves result in a highly selective synthesis of nucleic acids in 10-50 seconds. In contrast, same reactions failed to give desired reaction products when carried out in the same time periods, but without microwave irradiation. Each of the reactions was carried out for different duration of microwave exposure time to find the optimum reaction time. The products produced by the respective enzyme upon microwave irradiation of the reaction mixtures were identical to that produced by the conventional procedures. As the microwave-assisted reactions are rapid, microwave could be a useful alternative to the conventional and time consuming procedures of enzymatic synthesis of nucleic acids.

Viral reverse transcriptases show selective high affinity binding to DNA-DNA primer-templates that resemble the polypurine tract.

Previous results using a SELEX (Systematic Evolution of Ligands by Exponential Enrichment)-based approach that selected DNA primer-template duplexes binding with high affinity to HIV reverse transcriptase (RT) showed that primers mimicking the 3' end, and in particular the six nt terminal G tract, of the RNA polypurine tract (PPT; HIV PPT: 5'-AAAAGAAAAGGGGGG-3') were preferentially selected. In this report, two viral (Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV)) and one retrotransposon (Ty3) RTs were used for selection. Like HIV RT, both viral RTs selected duplexes with primer strands mimicking the G tract at the PPT 3' end (AMV PPT: 5'-AGGGAGGGGGA-3'; MuLV PPT: 5'-AGAAAAAGGGGGG-3'). In contrast, Ty3, whose PPT lacks a G tract (5'-GAGAGAGAGGAA-3') showed no selective binding to any duplex sequences. Experiments were also conducted with DNA duplexes (termed DNA PPTs) mimicking the RNA PPT-DNA duplex of each virus and a control duplex with a random DNA sequence. Retroviral RTs bound with high affinity to all viral DNA PPT constructs, with HIV and MuLV RTs showing comparable binding to the counterpart DNA PPT duplexes and reduced affinity to the AMV DNA PPT. AMV RT showed similar behavior with a modest preference for its own DNA PPT. Ty3 RT showed no preferential binding for its own or any other DNA PPT and viral RTs bound the Ty3 DNA PPT with relatively low affinity. In contrast, binding affinity of HIV RT to duplexes containing the HIV RNA PPT was less dependent on the G tract, which is known to be pivotal for efficient extension. We hypothesize that the G tract on the RNA PPT helps shift the binding orientation of RT to the 3' end of the PPT where extension can occur.

Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis.

Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa ß subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299-306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)(15), was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.

Mechanism of HIV reverse transcriptase inhibition by zinc: formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.

Several physiologically relevant cations including Ca(2+), Mn(2+), and Zn(2+) have been shown to inhibit HIV reverse transcriptase (RT), presumably by competitively displacing one or more Mg(2+) ions bound to RT. We analyzed the effects of Zn(2+) on reverse transcription and compared them to Ca(2+) and Mn(2+). Using nucleotide extension efficiency as a readout, Zn(2+) showed significant inhibition of reactions with 2 mM Mg(2+), even when present at only ∼5 µM. Mn(2+) and Ca(2+) were also inhibitory but at higher concentrations. Both Mn(2+) and Zn(2+) (but not Ca(2+)) supported RT incorporation in the absence of Mg(2+) with Mn(2+) being much more efficient. The maximum extension rates with Zn(2+), Mn(2+), and Mg(2+) were ∼0.1, 1, and 3.5 nucleotides per second, respectively. Zinc supported optimal RNase H activity at ∼25 µM, similar to the optimal for nucleotide addition in the presence of low dNTP concentrations. Surprisingly, processivity (average number of nucleotides incorporated in a single binding event with enzyme) during reverse transcription was comparable with Zn(2+) and Mg(2+), and single RT molecules were able to continue extension in the presence of Zn(2+) for several hours on the same template. Consistent with this result, the half-life for RT-Zn(2+)-(primer-template) complexes was 220 ± 60 min and only 1.7 ± 1 min with Mg(2+), indicating ∼130-fold more stable binding with Zn(2+). Essentially, the presence of Zn(2+) promotes the formation of a highly stable slowly progressing RT-(primer-template) complex.

Effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus.

The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). At 42 °C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 °C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 °C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.

Duplex structural differences and not 2'-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA.

Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA-DNA versus DNA-DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3' recessed DNA base) are required for stable binding to RNA-DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA-DNA. Replacing 2'- hydroxyls on pivotal RNA bases with 2'-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. RT's K(d) (k(off)/k(on)) for DNA-DNA and RNA-DNA were similar, although k(off) differed almost 40-fold, suggesting a faster k(on) for DNA-DNA. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, the ability of RT to form a more stable complex on RNA-DNA may aid in degradation of RNA fragments that remain after DNA synthesis.

Characterization of Moloney murine leukaemia virus/avian myeloblastosis virus chimeric reverse transcriptases.

Reverse transcriptases (RTs) from Moloney murine leukaemia virus (MMLV) and avian myeloblastosis virus (AMV) contain all the fingers, palm, thumb, connection and RNase H domains. The fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the RNase H domain is in the RNase H activity. In this study, we characterized four chimeric RTs which comprise one of the fingers, palm, thumb and RNase H domains originated from AMV RT and the other three and connection domains originated from MMLV RT. Unexpectedly, all chimeric RTs exhibited the same characteristics: their specific reverse transcription activities decreased to less than 0.1% of that of MMLV RT, while their specific RNase H activities were approximately 20% of that of MMLV RT. The decreases in the two activities of the chimeric RTs were ascribed to the decreases in k(cat). Based on that the reverse transcription activity of MMLV RT was impaired by substituting its RNase H domain with that from AMV RT, we propose that in MMLV RT, there might be an interaction between the fingers/palm/thumb domain and the RNase H domain.

RNA-dependent DNA polymerases.

Reverse transcriptases (RTs) are multifunctional enzymes, but are mainly used as RNA-directed DNA polymerases in first-strand cDNA synthesis. Specifically, oligodeoxynucleotides are used as primers for extension on RNA templates. The DNA synthesized from an RNA template is referred to as complementary DNA (cDNA) and is often used as a template for PCR or converted to dsDNA for cloning. This unit describes appropriate reaction conditions for RTs from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV), along with applications such as synthesizing cDNA, 3' fill-in reactions, and labeling the 3' terminus of DNA fragments with 5' protruding ends, and DNA sequencing.

Novel FRET-based assay to detect reverse transcriptase activity using modified dUTP analogues.

We have developed a novel continuous assay to measure reverse transcriptase (RT) polymerase activity. The assay uses fluorescence energy transfer measurements to detect the incorporation of complementary pairs of fluorescently labeled deoxyuridine into cDNA product. The fluorescently labeled dUTP substrates were prepared using commercially available reagents with a simple coupling reaction. The fluorescent dye pairs have significant spectral overlap which allows FRET interaction between dyes incorporated into the cDNA. Using a polyA/oligo dT primer/template, the assay can readily detect DNA polymerase activity from any viral reverse transcriptase enzyme. The reaction proceeds linearly over time, and the rate is proportional to the enzyme concentration. We used the assay to compare the thermostability of a number of wild-type and mutant viral RT enzymes. Our results indicate that the wild-type AMV (avian myeloblastosis virus) enzyme is slightly more stable at 43 degrees C than the HIV-1 (human immunodeficiency virus) or MMLV (Moloney murine leukemia virus) enzymes. The thermostability of the RT enzyme was dramatically increased by the presence of primer/template with the enzyme. We also used the assay to study the effects of inhibitors on HIV-1 RT polymerase activity. This assay may be highly useful for the identification and characterization of potent RT inhibitors which could be candidates for development as therapeutic antiviral agents.

Comparison of the thermal stabilities of reverse transcriptases from avian myeloblastosis virus and Moloney murine leukaemia virus.

Reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukaemia virus (MMLV) have been most extensively used as a tool for conversion of RNA to DNA. In this study, we compared the thermal stabilities of AMV RT and MMLV RT by observing their irreversible thermal inactivation. The temperatures reducing initial activity by 50% in 10-min incubation, T(50), of AMV RT were 47 degrees C without the template-primer (T/P), poly(rA)-p(dT)(12-18), and 52 degrees C with the T/P (28 microM). T(50) of MMLV RT were 44 degrees C without the T/P and 47 degrees C with the T/P (28 microM). Unexpectedly, AMV RT was considerably activated when incubated with the T/P at 45 and 48 degrees C. Such activation was not observed in MMLV RT. These results suggest that AMV RT and MMLV RT are different in the following: (i) The intrinsic thermal stability of AMV RT is higher than that of MMLV RT; (ii) AMV RT is activated by thermal treatment with the T/P at 45-48 degrees C; and (iii) AMV RT is stabilized by the T/P more potently than MMLV RT. Thermodynamic analysis indicates that thermal inactivation of AMV RT and MMLV RT is due to the large entropy change of activation for thermal inactivation.

Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template.

While much is known about abasic DNA, the biological impact of abasic RNA is largely unexplored. To test the mutagenic potential of this RNA lesion in the context of retroviruses, we synthesized a 31-mer oligoribonucleotide containing an abasic (rAS) site and used it as a template for studying DNA primer extension by HIV-1, avian myeloblastosis virus (AMV) and moloney murine leukemia virus (MMLV) reversed transcriptases (RT). We found that trans-lesion synthesis readily takes place with HIV-1 RT and to a lesser extent with AMV RT while MMLV RT aborts DNA synthesis. The preference of dNTP incorporation follows the order A approximately G > C approximately T and thus obeys to the 'A-rule'. In the case of HIV-1 RT, we measured the kinetic data of dNTP incorporation and compared it to abasic DNA. We found that A-incorporation is only 2-fold slower relative to a matched (undamaged) RNA template while it is 7-fold slower in the case of DNA. Furthermore, there is less discrimination in incorporation between the four dNTPs in the case of abasic RNA compared to abasic DNA. These experiments clearly point to a higher promiscuity of lesion bypass on abasic RNA. Given their known higher chemical stability, such rAS sites can clearly contribute to (retro)viral evolution.

A chimeric plum pox virus shows reduced spread and cannot compete with its parental wild-type viruses in a mixed infection.

The effect of a recombination event in the genomic 3' end on the biological properties and competitiveness of plum pox virus (PPV) was investigated. Therefore, a fragment spanning the coat protein (CP) coding region and a part of the 3' non-translated region of a non-aphid-transmissible strain of PPV (PPV-NAT) was replaced by the corresponding region of a PPV sour cherry isolate (PPV-SoC). The resulting chimera (PPV-NAT/SoC) caused severe symptoms in Nicotiana benthamiana, resembling those of PPV-NAT. In mixed infections with either of the parental viruses, the chimera PPV-NAT/SoC was less competitive. Labelling experiments with DsRed showed that PPV-NAT/SoC (PPV-NAT/SoC-red) moved more slowly from cell to cell than PPV-NAT (PPV-NAT-red). In mixed infections of PPV-NAT/SoC-red with a green fluorescent protein-expressing PPV-NAT (PPV-NAT-AgfpS), spatial separation of the viruses was observed. These data suggest that, in PPV infections, symptom severity and competitiveness are independent aspects and that spatial separation may contribute to the displacement of a recombinant virus.

Mn2+ suppressor mutations and biochemical communication between Ty1 reverse transcriptase and RNase H domains.

Ty1 reverse transcriptase/RNase H (RT/RH) is exquisitely sensitive to manganese concentrations. Elevated intracellular free Mn(2+) inhibits Ty1 retrotransposition and in vitro Ty1 RT-polymerizing activity. Furthermore, Mn(2+) inhibition is not limited to the Ty1 RT, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 RTs. To further characterize Mn(2+) inhibition, we generated RT/RH suppressor mutants capable of increased Ty1 transposition in pmr1 Delta cells. PMR1 codes for a P-type ATPase that regulates intracellular calcium and manganese ion homeostasis, and pmr1 mutants accumulate elevated intracellular manganese levels and display 100-fold less transposition than PMR1(+) cells. Mapping of these suppressor mutations revealed, surprisingly, that suppressor point mutations localize not to the RT itself but to the RH domain of the protein. Furthermore, Mn(2+) inhibition of in vitro RT activity is greatly reduced in all the suppressor mutants, whereas RH activity and cleavage specificity remain largely unchanged. These intriguing results reveal that the effect of these suppressor mutations is transmitted to the polymerase domain and suggest biochemical communication between these two domains during reverse transcription.

SAR and mode of action of novel non-nucleoside inhibitors of hepatitis C NS5b RNA polymerase.

Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochemical potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were determined by X-ray crystallography, and the inhibitors were found to bind in an allosteric binding site separate from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compounds in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.