Homology modeling and analysis of structure predictions of the bovine rhinitis B virus RNA dependent RNA polymerase (RdRp).

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D(pol)). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D(pol) using a combination of different computational tools. Model structures of BRBV 3D(pol) were verified for their stereochemical quality and accuracy. The BRBV 3D(pol) structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3D(pol) (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3D(pol) as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3D(pols) is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3D(pol) compared to FMDV 3D(pol), indicate that despite a very high homology to FMDV 3D(pol), BRBV 3D(pol) may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.

Kinetic analysis of the catalysis of strand transfer from internal regions of heteropolymeric RNA templates by human immunodeficiency virus reverse transcriptase.

The kinetic mechanism of HIV reverse transcriptase catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142 nucleotide RNA template (donor), primed with a specific 20 nucleotide DNA oligonucleotide that was used to initiate synthesis. An RNA with homology to an internal region of the donor was used as acceptor template. Using 32P-labeled DNA oligonucleotide, the primer-extension products made from full-length synthesis on the donor (108 bases in length) or homologous transfer to and extension on the acceptor (155 bases) were monitored. Results indicated that the maximum efficiency of transfer (the ratio of transfer products to donor-directed+transfer products x 100) in this particular system was about 25% while the theoretical Vmax for the rate of appearance of transfer products at infinite acceptor concentration was about 20-fold lower than the measured rate for full-length donor-directed products. The Km for acceptor template in the transfer reaction was about 8 nM. Experiments using the above donor template hybridized to a specific DNA that has been shown to transfer to the acceptor indicated that RNase H-mediated rapid release of this DNA from the donor while subsequent association with the acceptor was relatively slow.

Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain.

Reverse transcriptase (RT) serves as the replicative polymerase for retroviruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV-1 reverse transcriptase: tRNA(Lys,3) activation.

HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.

Selective inactivation of M-MuLV RT RNase H activity by site-directed PEGylation: an improved ability to synthesize long cDNA molecules.

Moloney murine leukemia virus reverse transcriptase (M-MuLV RT) is a domain structured enzyme that has the N-terminally located DNA polymerization activity and C-terminally located RNase H activity, which interferes with the efficient synthesis of long cDNA molecules. Here we present the PEGylation as a tool for engineering the M-MuLV RT derivative deficient in RNase H activity. We demonstrate that site-directed chemical modification (SDCM) of the RNase H domain by selectively PEGylating C635, one of the eight cysteine residues present in the reverse transcriptase (RT), specifically inactivated its ribonucleolytic activity. As a consequence, the efficiency of long cDNA molecules synthesis by modified enzyme was greatly increased.

Synthetic genetic polymers capable of heredity and evolution.

Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.

Avian myeloblastosis virus reverse transcriptase. Effect of glycerol on its hydrodynamic properties.

Although reverse transcriptase has been the subject of intensive investigation, minimal information is available regarding the physical properties of the enzyme. The basic hydrodynamic properties of avian myeloblastosis virus reverse transcriptase in solution were measured by both sedimentation velocity and equilibrium measurements in two buffer systems. In a 0.3 M potassium phosphate buffer system, pH 7.8, the enzyme sedimented as a homogenous particle with a sedimentation coefficient of (7.1 +/- 0.3) S with a weight-average molecular weight, Mw, of (1.52 +/- 0.05) x 10(5). Since the enzyme consists of an alpha and beta subunit of equal molar ratio with Mw of 6.3 x 10(4) and 9.4 x 10(4), respectively, it was concluded that the enzyme exists as an alpha beta heterodimer in this buffer system. In a Tris buffer system, pH 7.9, containing 0.46 M NaCl and 4% glycerol, the native enzyme also sedimented as a homogeneous particle with an apparent sedimentation coefficient of (10.1 +/- 0.5) S, without considering the effect of glycerol on solvent-protein interaction. Based on the results of Gekko and Timasheff (Gekko, K., and Timasheff, S. N. (1981) Biochemistry 20, 4667-4676) and the polarity of the enzyme, it was estimated that there is significant solvent-protein interaction even at 4% glycerol leading to a value of -0.06 g/g in the preferential solvent interaction parameter. When the solvent effect was taken into consideration, the value for s020,w increased from 10.1 to 11.9 S, implying that the native enzyme dimerizes in the presence of 4% glycerol. The combined results of gel filtration and sedimentation velocity showed that the dimerization of the enzyme to form (alpha beta)2 is favored at 20 degrees C with the alpha beta form predominating at 4 degrees C. The secondary structure of the reverse transcriptase was measured by circular dichroism. Results showed that the enzyme consists of (16 +/- 2)% alpha-helix, (24 +/- 2)% beta-sheet, (24 +/- 2)% beta-turn, and (36 +/- 4)% undefined structures.

Interface peptides as structure-based human immunodeficiency virus reverse transcriptase inhibitors.

Reverse transcriptases from both human immunodeficiency viruses type 1 and 2 are obligatory dimers. A tryptophan-rich repeat motif that is highly conserved between these proteins, as well as in the reverse transcriptase from simian immunodeficiency virus, has been postulated to be involved in hydrophobic subunit interactions. A synthetic 19-mer peptide covering part of this tryptophan repeat motif was recently shown to inhibit human immunodeficiency viruses type 1 reverse transcriptase subunit dimerization (Divita, G., Restle, T., Goody, R. S., Chermann, J.-C., and Baillon, J. G. (1994) J. Biol. Chem. 269, 13080-13083). In the present study, we show that the same peptide can also inhibit human immunodeficiency virus type 2 reverse transcriptase subunit dimerization, suggesting that the same inhibitors might be used as agents against both viruses as well as against variants of human immunodeficiency virus type 1 that differ from the variant against which they were developed. Under appropriate experimental conditions, e.g. at acidic pH, this peptide is also able to induce the dissociation of the enzyme from human immunodeficiency virus type 1.

Analysis of the polymerization kinetics of homodimeric EIAV p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric EIAV p66/51 reverse transcriptase.

Homodimeric EIAV p51/51 and heterodimeric EIAV p66/51 reverse transcriptase were purified in order to compare the different modes of DNA synthesis supported by the enzymes. Analysis of the dimerization behavior of the EIAV enzymes indicates that the dimer stability of EIAV reverse transcriptase enzymes is higher than that of their HIV-1 reverse transcriptase counterparts. EIAV p51/51 polymerizes DNA distributively whereas DNA synthesis by EIAV p66/51 is processive. Steady-state and pre-steady-state kinetic analyses of primer/template binding and nucleotide incorporation were performed with both enzymes to determine the reasons for the different polymerization behavior. Equilibrium fluorescence titrations demonstrated that the Kd values of EIAV p51/51 for binding of DNA/DNA and DNA/RNA substrates are increased 10-fold and 28-fold, respectively, as compared to EIAV p66/51. Stopped-flow measurements with DNA/DNA show that the increase in the Kd is in part due to a 17. 4-fold higher dissociation rate constant (k-1) for EIAV p51/51. Additionally, with EIAV p51/51, kdiss is increased 7-fold for DNA/DNA and 14-fold for DNA/RNA primer/template substrates, respectively. The lack of the RNase H domain in EIAV p51/51 leads to differences in the pre-steady-state kinetics of nucleotide incorporation on DNA/DNA and DNA/RNA templates. The burst of both enzymes is composed of two phases for both substrates, and the values for the corresponding pre-steady-state burst rates, kpol1 and kpol2, are similar for both enzymes, implying the formation of identical polymerase active sites. However, the amplitudes of the two phases differ with DNA/DNA templates, indicating a different distribution between two states varying greatly in their kinetic competence.

Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse transcriptase gene to prevent dimerization of the expressed chimaeric protein: purification and characterization of a monomeric HIV-1 reverse transcriptase.

We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant reverse transcriptase (RT) engineered to contain a 26-amino-acid linker insertion from the tether domain of feline leukaemia virus (FLV) RT. The chimaeric protein was expressed in Escherichia coli and migrated on SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT has been prepared by the coordinated applications of immobilized-metal-affinity chromatography and gel filtration on Superose 12 columns. The monomeric nature of this chimaeric HIV-I RT was further characterized by cross-linking studies using disuccinimidyl suberate. The RNA-dependent DNA polymerase activity of the monomeric chimaeric HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These results support our recent studies on the monomeric polymerase domain (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy, Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The inability of the monomeric chimaeric HIV-1 RT to display polymerase activity like that of the heterodimeric HIV-1 RT is attributed to a decrease in the processive rate of DNA synthesis (75%) and DNA binding (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT. These results suggest that the linker insertion from FLV RT does not interfere with the RNAase H activity associated with the monomeric HIV-1 RT.

Recombinant reverse transcriptase of Rous sarcoma virus: characterization of DNA polymerase and RNAase H activities.

Enzyme preparations of Rous sarcoma virus (RSV) reverse transcriptase have been isolated from a culture of E. coli HB101(pMF14). The enzyme has been purified to homogeneity and been shown to consist of two subunits, of molecular mass 97.4 and 61.3 kDa, respectively. The optimum conditions for the DNA polymerase and RNAase H activities, fidelity of DNA synthesis on a homogeneous RNA template, and the inhibitory effect of azidothymidine triphosphate have been determined. Data on the use of RSV recombinant reverse transcriptase for cDNA synthesis are given.