Transcriptase Of Avian Myeloblastosis Virus Research Articles

An unnatural hydrophobic base pair with shape complementarity between pyrrole-2-carbaldehyde and 9-methylimidazo[(4,5)-b]pyridine.

An unnatural hydrophobic base, pyrrole-2-carbaldehyde (denoted as Pa), was developed as a specific pairing partner of 9-methylimidazo[(4,5)-b]pyridine (Q). The Q base is known to pair with 2,4-difluorotoluene (F) as an isostere of the A-T pair, and F also pairs with A efficiently in replication. In contrast, the Q-Pa pair showed specific selectivity in replication, and the five-membered-ring base Pa paired efficiently with Q but paired poorly with A. In addition, the interaction of Pa with DNA polymerases was superior, in comparison to that of F. The aldehyde group of Pa was recognized well by the Klenow fragment of Escherichia coli DNA polymerase I and the reverse transcriptase of Avian myeloblastosis virus. The structural features of the Q-Pa pair in a DNA duplex were analyzed by NMR, showing the shape complementarity of the Pa fitting with Q. The structurally unique base Pa provides valuable information for the development of unnatural base pairs toward the expansion of the genetic alphabet.

A monoclonal antibody that neutralizes Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, and bacteriophage T4 DNA polymerases.

A monoclonal antibody (mAb) designated 55H3 was produced by using chemically induced Epstein-Barr virus genome-positive B95-8 cells. mAb 55H3, which reacted with an 85- to 80-kDa polypeptide, neutralized Epstein-Barr virus-encoded DNA polymerase activity in crude extracts of chemically induced M-ABA, HR-1, and B95-8 cells, as well as the partially purified Epstein-Barr virus DNA polymerase in a dose-dependent manner. The mAb also neutralized the virus-encoded DNA polymerase activity from cells infected with human cytomegalovirus, human herpesvirus 6, and the purified bacteriophage T4 DNA polymerases. However, mAb 55H3 did not neutralize the DNA polymerase activities encoded for by herpes simplex virus types 1 and 2, the reverse transcriptase of avian myeloblastosis virus, or Escherichia coli DNA polymerase 1 (Klenow fragment). These results suggest that mAb 55H3 recognizes an epitope common to some herpesviruses and T4 DNA polymerases and further supports the hypothesis that these organisms are evolutionarily related.

Feline immunodeficiency virus, a model for reverse transcriptase-targeted chemotherapy for acquired immune deficiency syndrome

Feline immunodeficiency virus (FIV), formerly called feline T-lymphotropic lentivirus, causes an immune deficiency in cats that is very similar to the acquired immune deficiency syndrome in humans (N. C. Pedersen, E. M. Ho, M. L. Brown, and J. K. Yamamoto, Science 235:790-793, 1987). We have examined the reverse transcriptase of this virus to determine whether it is similar enough to the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1) to enable its use as a model for chemotherapy for acquired immune deficiency syndrome. The FIV reverse transcriptase is similar to that of HIV-1 in sensitivity to the noncompetitive inhibitor phosphonoformate (Ki, 0.3 microM) and relative insensitivity to phosphonoacetate. This enzyme was also sensitive to two competitive inhibitors, the 5'-triphosphates of 2', 3'-dideoxythymidine (Ki, 3.4 nM) and 3'-azido-3'-deoxythymidine (AZT; Ki, 6.2 nM). The ratios of Ki/Km for these two competitive inhibitors are similar to the ratios calculated from previously reported data for the HIV-1 enzyme assayed under identical conditions. In contrast, the FIV enzyme is different from the reverse transcriptase of avian myeloblastosis virus in sensitivity to those inhibitors. The replication of FIV in Crandell feline kidney cells was inhibited by AZT; virus production was inhibited more than 95% by 1.0 microM AZT.

New antibiotics SF2315A and B produced by an Excellospora sp. I. Taxonomy of the strain, isolation and characterization of antibiotics.

Two new antibiotics SF2315A and B have been isolated from culture filtrate of an actinomycete strain, Excellospora viridilutea SF2315. They are weakly active against Gram-positive bacteria and inhibited reverse transcriptase of avian myeloblastosis virus. Empirical molecular formula of antibiotics SF2315A and B were determined to be C19H16O5 and C19H20O6, respectively.

Purification of a specific inhibitor of reverse transcriptase from human placenta.

Human placental extracts contain a factor which specifically and reversibly inhibits the reverse transcriptase of mammalian retroviruses. This placental inhibitor has been partially purified and characterized. It elutes at 0.1-0.2 M phosphate on hydroxyapatite chromatography and can be further purified by phosphocellulose chromatography where it elutes at 0.4 M KCl. By these purification procedures, specific activities of 40-70,000 units of inhibitor per mg of protein were obtained. The size of the inhibitor is about 60-65,000 daltons as estimated by velocity sedimentation. The inhibitor purified by these techniques selectively inhibits the activity of purified reverse transcriptase from Rauscher murine leukemia virus and baboon endogenous virus. It is substantially less active against the reverse transcriptase of avian myeloblastosis virus. The specificity of this inhibitor for mammalian enzymes and particularly for the human placental reverse transcriptase suggests that it plays a role in the regulation of DNA synthesis in human placental development.

Synthesis and characterization of DNA complementary to carnation mottle virus RNA

DNA complementary to carnation mottle virus-RNA (CarMV RNA) was synthesized using the reverse transcriptase of avian myeloblastosis virus (AMV) as enzyme, and an hydrolysate of calf thymus DNA as primer. After purification steps that included self-annealing and hydroxyapatite chromatography, the DNA transcript was more than 99% sensitive to S 1 nuclease and hybridized to at least 92% of the CarMV-RNA sequences, but not to heterologous RNA. Therefore, the purified transcript was single stranded and represented almost all CarMV-RNA sequences. The cDNA consisted of small pieces that hybridized to CarMV-RNA with a sharp transition corresponding to C r t 1 2 of 2.3 × 102 M sec liter −1. This value is consistent with the complexity expected for CarMV-RNA if there is only one kind of virion RNA. CarMV-cDNA hybridized completely to a crude RNA preparation from CarMV-infected carnation leaves, but not to a comparable RNA preparation from healthy leaves, thus permitting the use of CarMV-cDNA to detect and to quantify the viral nucleotide sequences in total nucleic acid preparations from fragments of carnation cuttings.

RNA primer used in synthesis of anticomplementary DNA by reverse transcriptase of avian myeloblastosis virus.

When either the homologous RNA (avian myeloblastosis virus RNA) or a heterologous RNA (poliovirus RNA) was used as a template, the anticomplementary DNA synthesized in vitro by avian myeloblastosis virus reverse transcriptase (RNA-directed DNA nucleotidyltransferase, EC 2.7.7.7) was primed by fragments of the original RNA template that usually had adenosine at their 3' ends. When we used phage T/ RNA ligase (EC 6.5.1.3) to label the 3' end of the RNA template fragments contained in the RNA . cDNA hybrid intermediate, adenosine was found to be the principal nucleoside carrying the label. We infer from these results that the ribonuclease H (hybrid nuclease) activity of the reverse transcriptase creates fragments of the original RNA template with adenosine as the principal 3' terminus and that these fragments serve as primers for the synthesis of anticomplementary DNA.

Influence of various 2- and 2′-substituted polyadenylic acids on murine leukemia virus reverse transcriptase

Several newly synthesized polyadenylic acid [(A)n] analogues, including poly(2-methyladenylic acid) [(m2A)n], poly(2-ethyladenylic acid) [(e2A)n], poly(2-isopropyladenylic acid) [(i-pro2A)n], poly(2-methylthioadenylic acid) [(ms2A)n], poly(2-ethylthioadenylic acid) [(e2A)n], poly(2'-fluoro-2'-deoxyadenylic acid) [(dAfl)n] and poly(2'-azido-2'-deoxyadenylic acid) [(dAz)n] have been evaluated for their effects on the RNA-directed DNA polymerase (reverse transcriptase) activity of Moloney murine leukemia virus; (m2A)n and (e2A)n did not markedly affect reverse transcriptase activity, (dAfl)n served as an efficient template for the reverse transcriptase reaction, and (i-pro2A)n, (ms2A)n, (es2A)n and (dAz)n strongly inhibited reverse transcriptase activity. (dAfl)n also served as an efficient template (Km : 0.025 micron) for the reverse transcriptase of avian myeloblastosis virus.

Discrimination of DNA polymerase and RNase H activities in reverse transcriptase of avian myeloblastosis virus.

The active sites in reverse transcriptase of avian myeloblastosis virus have been selectively modified by various chemical reagents. The DNA polymerase activity is very sensitive to hydrophobic sulfhydryl reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate but resistant to sulfhydryl reagents with hydrophilic properties. The RNase H activity, on the other hand, is resistant to both hydrophobic and hydrophilic sulfhydryl reagents, indicating the absence of cysteinyl residues essential for RNase H activity. N-Ethylmaleimide (NEM), an amino and sulfhydryl group specific reagent, inactivates both DNA polymerase and RNase H, the later activity being fourfold more stable. Polynucleotides, but not nucleotide triphosphates, protect the two enzymatic activites of reverse transcriptase against NEM. Since pretreatment of the enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) does not prevent N-ethylmaleimide from reacting with a residue necessary for DNA polymerase activity, two different reactive groups are probably involved with this enzymatic activity. The pH profile of reverse transcriptase inhibition by N-ethylmaleimide also suggests the involvement of two reactive groups essential for the DNA polymerase activity with apparent pKas of 5.5 and 6.5. Only one reactive group with a pKa of 7.5 is found associated with the RNase H activity.

Properties of the DNA product of reverse transcription of turnip yellow mosaic virus RNA preparations

The reverse transcriptase of avian myeloblastosis virus catalyzes the synthesis of complementary DNA in the presence of turnip yellow mosaic virus (TYMV) RNA template and oligo(dG) 6 primer. The transcription product (single-stranded cDNA) had an estimated molecular weight of 150,000; it hybridized with TYMV-RNA, eggplant mosaic virus RNA, and Andean potato latent virus RNA, which are all members of the tymovirus group, but not with tobacco mosaic virus (TMV) RNA. It is concluded that all three tymovirus RNA preparations have common nucleotide sequences not shared with TMV-RNA. Furthermore, it appears that reverse transcription of TYMV-RNA preparations occurs in the absence of poly(A) at the 3′-end of genome RNA, when primed with oligo(dG) 6.

TRNATrp (bovine) binding to the reverse transcriptase of avian myeloblastosis virus and function as a heterologous primer.

The primary structures for tTNATrp (bovine) and primer tRNATrp (avian) show only minor differences in nucleotide sequence. The heterologous tRNATrp (bovine) appears to have properties similar to the tRNATrp (avian) in its ability to bind the alphabeta from of RNA-dependent DNA nucleotidyltransferase of avian myeloblastosis virus. A stable enzyme-tRNA complex has been isolated by gel filtration. In addition, tRNATrp (bovine) can hydridize to the avian viral 35S RNA and act as a primer for transcription of the RNA. tRNATrp (bovine) can be obtained in larger amounts than the avian primer and can be used to study the interactions between the primer and the viral enzyme.

Immunological distinction between ribonuclease H activity of α and αβ forms of avian myeloblastosis virus (AMV) DNA polymerase

The reverse transcriptase of avian myeloblastosis virus (AMV) exists either as a dimer of two polypeptides or as a single polypeptide, designated αβ and α, respectively. Both the α and αβ forms of the enzyme possess DNA polymerase and RNase H activities (EC 3.1.4.34). Antisera raised in rabbits against the α and αβ forms inhibit the DNA polymerase activity of both forms but only the RNase H activity of the enzyme form used as immunogen. Antibody specifically inhibitory for αβ RNase H activity can be absorbed by purified α, and antibody specifically inhibitory for α RNase H activity can be absorbed by purified αβ. These data indicate that antibodies against α and αβ forms of AMV reverse transcriptase recognize distinct antigenic determinants present on α and that the α and αβ forms differ in the steric relationship of these determinants to the RNase H active site. These results are consistent with possible functional differences between the RNase H activity of the α and αβ forms of AMV reverse transcriptase.

Studies on the reverse transcriptase of RNA tumor viruses. Structural relatedness of two subunits of avian RNA tumor viruses.

The structural relationship of the small (alpha) and large (beta) subunits of reverse transcriptase isolated from two avian RNA tumor viruses has been examined by tryptic peptide analysis. Comparison of the tryptic hydrolysates of the isolated subunits by two-dimensional separation on thin-layer cellulose plates indicates that (i) the alpha subunit of reverse transcriptase of avian myeloblastosis virus is structurally related to the beta subunit; (ii) the alpha and beta subunits of the enzyme of Rous sarcoma virus also appear to be related; and (iii) there appears to be an extensive amino-acid sequence homology between reverse transcriptases of avian myeloblastosis virus and Rous sarcoma virus. Evidence is also presented that both alpha and beta subunits can be identified in purified avian myeloblastosis virions.