Synthesis and biological assessment of 3,7-dihydroxytropolones.

3,7-Dihydroxytropolones (3,7-dHTs) are highly oxygenated troponoids that have been identified as lead compounds for several human diseases. To date, structure-function studies on these molecules have been limited due to a scarcity of synthetic methods for their preparation. New synthetic strategies towards structurally novel 3,7-dHTs would be valuable in further studying their therapeutic potential. Here we describe the successful adaptation of a [5 + 2] oxidopyrilium cycloaddition/ring-opening for 3,7-dHT synthesis, which we apply in the synthesis of a plausible biosynthetic intermediate to the natural products puberulic and puberulonic acid. We have also tested these new compounds in several biological assays related to human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV) in order to gain insight into structure-functional analysis related to antiviral troponoid development.

Synthesis of nucleoside 5'-O-alpha,beta-methylene-beta-triphosphates and evaluation of their potency towards inhibition of HIV-1 reverse transcriptase.

A polymer-bound alpha,beta-methylene-beta-triphosphitylating reagent was synthesized and subjected to reactions with unprotected nucleosides, followed by oxidation, deprotection of cyanoethoxy groups, and acidic cleavage to afford nucleoside 5'-O-alpha,beta-methylene-beta-triphosphates. Among all the compounds, cytidine 5'-O-alpha,beta-methylene-beta-triphosphate inhibited RNase H activity of HIV-1 reverse transcriptase with a K(i) value of 225 microM.

Footprint analysis of replicating murine leukemia virus reverse transcriptase.

Replication complexes that contained either murine leukemia virus reverse transcriptase (MLV RT) or a variant reverse transcriptase without a ribonuclease (RNase) H domain (delta RH MLV RT) were visualized by enzymatic footprinting. Wild-type MLV RT protected template nucleotides +6 to -27, and primer nucleotides -1 to -26 of primers that had first been extended by one or four nucleotides. Although it catalyzed DNA synthesis, delta RH MLV RT stably bound template-primer only under conditions of reduced ionic strength and protected the duplex portion only as far as position -15. Despite altered hydrolysis profiles, both enzymes covered primarily the template-primer duplex, contradicting recent predictions based on the structure of rat DNA polymerase beta.

In vitro evidence for the interaction of tRNA(3)(Lys) with U3 during the first strand transfer of HIV-1 reverse transcription.

Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA(3)(Lys)primer by utilizing it in several steps of reverse transcription. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA(3)(Lys). In order to test its possible role in the first strand transfer reaction, we applied an assay using a donor RNA corresponding to the 5'-part and an acceptor RNA spanning the 3'-part of HIV-1 RNA. In addition, we constructed two acceptor RNAs in which the nonanucleotide sequence complementary to tRNA(3)(Lys)was either substituted (S) or deleted (Delta). We used either natural tRNA(3)(Lys)or an 18 nt DNA as primer and measured the efficiency of (-) strand strong stop DNA transfer in the presence of wild-type, S or Delta acceptor RNA. Mutations in U3 did not decrease the transfer efficiency when reverse transcription was primed with the 18mer DNA. However, they significantly reduced the strand transfer efficiency in the tRNA(3)(Lys)-primed reactions. This reduction was also observed in the presence of nucleocapsid protein. These results suggest that tRNA(3)(Lys)increases (-) strand strong stop transfer by interacting with the U3 region of the genomic RNA. Sequence comparisons suggest that such long range interactions also exist in other lentiviruses.

Interaction of retroviral reverse transcriptase with template-primer duplexes during replication.

Conversion of the single-stranded RNA of an invading retrovirus into double-stranded proviral DNA is catalyzed in a multi-step process by a single virus-coded enzyme, reverse transcriptase (RT). Achieving this requires a combination of DNA polymerase abd ribonuclease H (RNase H) activities, which are located at the amino and carboxy terminus of the enzyme, respectively. Moreover, proviral DNA synthesis requires that three structurally-distinct nucleic acid duplexes are accommodated by this enzyme, namely (a) A-form RNA (initiation of minus strand synthesis), non-A, non-B RNA/DNA hybrid (minus strand synthesis and initiation of plus strand synthesis) and B-form duplex DNA (plus strand synthesis). This review summarizes our current understanding of the manner in which retroviral RT interacts with this diverse array of nucleic acid duplexes, exploiting in many cases mutants unable to catalyze a specific event. These studies illustrate that seemingly 'simple' events such as tRNA-primed initiation of minus strand synthesis are considerably more complex, involving intermolecular tRNA-viral RNA interactions outside the primer binding site. Moreover, RNase H activity, generally thought to catalyze non-specific degradation of the RNA-DNA replicative intermediate, is required for highly specialized events including DNA strand transfer and polypurine selection. Finally, a unique structure near the center of HIV proviral DNA, the central termination sequence, serves to halt the replication machinery in a manner analogous to termination of transcription. As these highly specialized events are better understood at the molecular level, they may open new avenues of therapeutic intervention in the continuing effort to stem the progression of HIV infection and AIDS.

Involvement of C-terminal structural elements of equine infectious anemia virus reverse transcriptase in DNA polymerase and ribonuclease H activities.

In order to investigate the modes of DNA synthesis supported by the 66 and 51 kDa subunits of equine infectious anemia virus reverse transcriptase (EIAV RT), recombinant p66 polypeptides containing a modified ribonuclease H (RNase H) domain were purified and evaluated. Defined heteropolymeric template-primer combinations and high-resolution gel electrophoresis provided a qualitative evaluation of DNA polymerase and RNase H activities, while DNase I footprinting revealed features of replication complexes containing the truncated enzymes. Removal of alpha-helix E' and the conserved beta 5'-alphaE' "His-loop" in p66delta20 RT uncouples the RNase H activities, alters affinity for template-primer and dictates how the replicating enzyme responds to secondary structure on both DNA and RNA templates. Despite these alterations, DNase I footprinting shows no major difference in the overall structure of DNA-directed DNA synthesis complexes. In contrast, removing 47 C-terminal residues, which includes alpha-helix D', beta-strand 5' and alpha-Helix E', yields an enzyme with distributive DNA polymerase properties closely resembling the purified p51 subunit.

Analogs of cyclic AMP that elicit the biochemically defined conformational change in catabolite gene activator protein (CAP) but do not stimulate binding to DNA.

We have measured the effects on catabolite gene activator protein (CAP) of 22 synthetic analogs of cAMP. Each analog was assayed to test three parameters: (1) binding to CAP; (2) induction of the conformational change in CAP; and (3) activation of transcription. Thus we have identified seven cAMP analogs that bind to CAP as well or better than does cAMP, cause the assayed conformational change in CAP, yet exhibit no ability to activate transcription. We designate these analogs class D. The conformational change elicited in CAP by the class D analogs was further investigated by: (1) sensitivity to the proteolytic enzymes chymotrypsin, Staphylococcus aureus V8 protease, subtilisin and trypsin; (2) formation of inter-subunit covalent crosslinks by 5,5'-dithiobis(2-nitrobenzoic acid); and (3) degree of labeling of cysteine by [3H]N-ethylmaleimide. These experiments failed to detect a conformational difference between the CAP-class D and CAP-cAMP complexes. Filter binding and nuclease protection experiments indicate that the class D analogs do not efficiently support the binding of CAP to DNA. From these results, we suggest that there exists a hitherto undetected event dependent on cAMP, and required for CAP to bind to DNA. We suggest that this event involves a change that takes place in proximity to the N6 atom of cAMP. Three possible interpretations are discussed.

Primer selection by HIV-1 reverse transcriptase on RNA-tRNA(3Lys) and DNA-tRNA(3Lys) hybrids.

During reverse transcription of the genomic RNA of human immunodeficiency virus type 1 (HIV-1) into double-stranded DNA, reverse transcriptase (RT) must accommodate RNA-RNA, DNA-RNA, RNA-DNA and DNA-DNA hybrids as primer-template. In this study, we examined extension of RNA-tRNA3Lys, and DNA-tRNA3Lys complexes by HIV-1 RT. When the 3' end of tRNA3Lys is annealed to oligoribonucleotides, tRNA3Lys, but not the complementary RNAs, is extended by HIV-1 RT, indicating that tRNA3Lys is efficiently used as primer and RNA as template. An opposite primer usage is observed when tRNA3Lys is annealed to complementary oligodeoxyribonucleotides. In this case, the oligodeoxyribonucleotides are efficiently used as primer and tRNA3Lys as template. This result indicates that the nature of nucleic acid bound to tRNA3Lys determines which strand of the RNA-tRNA3Lys and DNA-tRNA3Lys hybrids is extended by HIV-1 RT. When an oligoribonucleotide is annealed to an unmodified transcript of tRNA3Lys, both nucleic acids are extended by HIV-1 RT, indicating that specific selection of tRNA3Lys as primer requires the post-transcriptional modifications of tRNA3Lys.

Biosynthesis and analysis of a genetically engineered HIV-1 reverse transcriptase/endonuclease polyprotein in Escherichia coli.

Elimination of the protease domain from the polymerase open reading frame (pol) of the human immunodeficiency virus type 1 (HIV-1) leads, in Escherichia coli, to synthesis and accumulation of a non-processed 98-kDa reverse transcriptase/endonuclease (RT/ENDO) polyprotein. A partially purified preparation of this reverse RT/ENDO polyprotein displays little or no RT activity. Introduction of the pol protease domain as a separate transcriptional unit on the same plasmid restores the processing program, generating correctly sized RT and ENDO polypeptides. Concomitant with restoration of processing is the reappearance of RT activity. These results suggest that for HIV-1 RT to be active, it must be matured from the pol polyprotein.

Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis.

A 2.4-kb DNA fragment from the pol region of the human T-cell lymphotropic virus, encoding the protease, reverse transcriptase and a portion of the endonuclease N-terminus was stably introduced into a high-level Bacillus subtilis expression system under inducible control of the Escherichia coli lac regulatory elements. The major expression plasmid, pRTL11, contains a bacteriophage T5 promoter/lac operator element, which is controlled by lac repressor, supplied by the secondary plasmid, pBL1. Upon IPTG induction, a 90-kDa polyprotein is synthesised and subsequently proteolytically cleaved to reveal 64-kDa and 52-kDa polypeptides. Partial purification reveals that reverse transcriptase activity co-migrates with these two polypeptides.

Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase.

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.

Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNase H function.

Two single site substitutions (E478----Q and H539----F) were introduced into the C-terminal RNase H domain of HIV-1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.

High prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.

The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.

Nucleic Acid, Antibody, and Virus Culture Methods to Detect Xenotropic MLV-Related Virus in Human Blood Samples.

The MLV-related retrovirus, XMRV, was recently identified and reported to be associated with both prostate cancer and chronic fatigue syndrome. At the National Cancer Institute-Frederick, MD (NCI-Frederick), we developed highly sensitive methods to detect XMRV nucleic acids, antibodies, and replication competent virus. Analysis of XMRV-spiked samples and/or specimens from two pigtail macaques experimentally inoculated with 22Rv1 cell-derived XMRV confirmed the ability of the assays used to detect XMRV RNA and DNA, and culture isolatable virus when present, along with XMRV reactive antibody responses. Using these assays, we did not detect evidence of XMRV in blood samples (N = 134) or prostate specimens (N = 19) from two independent cohorts of patients with prostate cancer. Previous studies detected XMRV in prostate tissues. In the present study, we primarily investigated the levels of XMRV in blood plasma samples collected from patients with prostate cancer. These results demonstrate that while XMRV-related assays developed at the NCI-Frederick can readily measure XMRV nucleic acids, antibodies, and replication competent virus, no evidence of XMRV was found in the blood of patients with prostate cancer.