Establishment of a system for finding inhibitors of ε RNA binding with the HBV polymerase.

Although several nucleo(s)tide analogs are available for treatment of HBV infection, long-term treatment with these drugs can lead to the emergence of drug-resistant viruses. Recent HIV-1 studies suggest that combination therapies using nucleo(s)tide reverse transcriptase inhibitors (NRTIs) and non-nucleo(s)tide reverse transcriptase inhibitors (NNRTIs) could drastically inhibit the viral genome replication of NRTI-resistant viruses. In order to carry out such combinational therapy against HBV, several new NRTIs and NNRTIs should be developed. Here, we aimed to identify novel NNRTIs targeting the HBV polymerase terminal protein (TP)-reverse transcriptase (RT) (TP-RT) domain, which is a critical domain for HBV replication. We expressed and purified the HBV TP-RT with high purity using an Escherichia coli expression system and established an in vitro ε RNA-binding assay system. Then, we used TP-RT in cell-free assays to screen candidate inhibitors from a chemical compound library, and identified two compounds, 6-hydroxy-DL-DOPA and N-oleoyldopamine, which inhibited the binding of ε RNA with the HBV polymerase. Furthermore, these drugs reduced HBV DNA levels in cell-based assays as well by inhibiting packaging of pregenome RNA into capsids. The novel screening system developed herein should open a new pathway the discovery of drugs targeting the HBV TP-RT domain to treat HBV infection.

Biochemical characterization of the Lassa virus L protein.

The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthomyxovirus polymerase complex. Although the L protein did not exhibit cap-snatching endonuclease activity, it synthesized RNA in vitro RNA polymerization required manganese rather than magnesium ions, was independent of nucleotide primers, and was inhibited by viral Z protein. Maximum activity was mediated by double-stranded promoter sequences with a minimum length of 17 nucleotides, containing a nontemplated 5'-G overhang, as in the natural genome context, as well as the naturally occurring base mismatches between the complementary promoter strands. Experiments with various short primers revealed the presence of two replication initiation sites at the template strand and evidence for primer translocation as proposed by the prime-and-realign hypothesis. Overall, our findings provide the foundation for a detailed understanding of the mechanistic differences and communalities in the polymerase proteins of segmented negative-strand RNA viruses and for the search for antiviral compounds targeting the RNA polymerase of Lassa virus.

Limited reverse transcriptase activity of phi29 DNA polymerase.

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

[Cloning and analysis of reverse transcriptase(RT) of Ty1-copia retrotransposons in Dendrobium officinale].

Using universal primer Ty1-copia retrotransposon RT,43 Ty1-copia like retrotransposon RT with high heterogeneity, stop codon mutation and frameshift mutation were amplified by PCR from genomic DNA of Zhejiang Lin'an (C15) and Yunnan Guangnan (A39) of Dendrobium officinale. The length of these sequences varied from 260 to 266 bp, and was rich in AT and consistency ranged from 47.1% to 97.7%. Different c/s-acting regulatory elements induced by low temperature, heat, light, all kinds of plant growth regulating substances and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. When being translated into amino acids, ten sequences presented stop codon mutation, five sequences presented frameshift mutation, and thirty-seven sequences presented conserved sequence "SLYGKQ" mutation. Six categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with other plants (eg. Triticum aestivum, Eleocharis quinqueflora) having high homology, which indicated that horizontal transmission of retrotransposon occurred among the plants in the past.

The duck hepatitis B virus reverse transcriptase functions as a full-length monomer.

Hepadnaviral reverse transcription occurs within cytoplasmic capsid particles and is catalyzed by a virally encoded reverse transcriptase, but the primary structure and multimeric state of the polymerase during reverse transcription are poorly understood. We measured these parameters for the duck hepatitis B virus polymerase employing active enzyme translated in vitro and derived from intracellular core particles and mature virions. In vitro-translated polymerase immunoprecipitated as a monomer, and polymerase molecules with complementary defects in the enzymatic active site and tyrosine 96, which primes DNA synthesis, could not complement or inhibit each other in priming assays. Western analysis using antibodies recognizing epitopes throughout the polymerase combined with nuclease digestion of permeabilized virion-derived capsid particles revealed that only full-length polymerase molecules were in virions and that they were all covalently attached to large DNA molecules. Because DNA synthesis is primed by the polymerase itself and only one copy of the viral DNA is in each capsid, the polymerase must function as an uncleaved monomer. Therefore, a single polymerase monomer is encapsidated, primes DNA synthesis, synthesizes both DNA strands, and participates in the three-strand transfers of DNA synthesis, with all steps after DNA priming performed while the polymerase is covalently coupled to the product DNA. Because the N-terminal domain of the polymerase is displaced from the active site on the same molecule by the viral DNA during reverse transcription, P must be structurally dynamic during DNA synthesis. Therefore, non-nucleoside compounds that interfere with this change may be novel antiviral agents.

Inhibition of reverse transcription in vivo by elevated manganese ion concentration.

Mutations in PMR1, a yeast gene encoding a calcium/manganese exporter, dramatically decrease Ty1 retrotransposition. Ty1 cDNA is reduced in pmr1 mutant cells, despite normal levels of Ty1 RNA and proteins. The transposition defect results from Mn(2+) accumulation that inhibits reverse transcription. Cytoplasmic accumulation of Mn(2+) in pmr1 cells may directly affect reverse transcriptase (RT) activity. Trace amounts of Mn(2+) potently inhibit Ty1 RT and HIV-1 RT in vitro when the preferred cation, Mg(2+), is present. Both Mn(2+) and Mg(2+) alone activate Ty1 RT cooperatively with Hill coefficients of 2, providing kinetic evidence for a dual divalent cation requirement at the RT active site. We propose that occupancy of the B site is the major determinant of catalytic activity and that Mn(2+) at this site greatly reduces catalytic activity.

Characterization and splicing in vivo of a Sinorhizobium meliloti group II intron associated with particular insertion sequences of the IS630-Tc1/IS3 retroposon superfamily.

By sequence analysis of Sinorhizobium meliloti strain GR4 plasmid pRmeGR4b, we have identified a group II intron named RmInt1 inserted within the insertion sequence ISRm2011-2 of the IS630-Tc1/IS3 retroposon superfamily. Like some other group II introns, RmInt1 possesses, in addition to the structurally conserved ribozyme core, an open reading frame (ORF) with homology to reverse transcriptases. Using a T7 expression system in Escherichia coli, we show that the intron is active in splicing in vivo and that splicing efficiency requires the intron-encoded ORF, which suggests that the putative intron encoded protein has a maturase function. DNA hybridization studies indicate that intron RmInt1 is widespread within S. meliloti native populations and appears to be mostly located within this IS element. Nevertheless, some S. meliloti strains harbour one copy of RmInt1 at a different location. DNA sequence analysis of the 5' exon of one of these heterologous intron insertion sites revealed the presence of a putative IS element closely related to insertion sequence ISRm2011-2. The intron-binding sites (IBS1 and IBS2 motifs) are conserved, although a transition of a G-->A in the IBS1 has occurred. Our results demonstrate an association of intron RmInt1 with particular insertion sequences of the IS630-Tc1/IS3 retroposon superfamily that may have ensured the spread and maintenance of this group II intron in S. meliloti.

Expression, purification, and characterization of an active RNase H domain of the hepatitis B viral polymerase.

The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate. This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product. The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities. Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems. The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis. We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification. The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture. This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component. The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.

Hybrid selection with cDNA-silica.

A partial length ovalbumin cDNA-silica was produced using primer extension of (dT)18-silica with annealed partial ovalbumin RNA and reverse transcriptase. This cDNA-silica was used to test whether full-length ovalbumin RNA could be selectively purified in the presence of a large excess of other (mouse muscle) RNA. The cDNA-silica synthesized had minimally 60 pmol cDNA per gram silica and had a capacity for full-length ovalbumin RNA of minimally 38 micrograms/g. Even when other RNA was present in greater than 1000-fold excess, ovalbumin RNA was selectively retained by the cDNA-silica and was eluted in yields of 43% with an enrichment which varied over the range of 29-162-fold in various experiments. These results show that even rare RNAs can be selectively purified in high yield using cDNA-silica. The importance of these results to hybrid selection and subtractive library preparation is discussed.

Preparation of cDNA-silica using reverse transcriptase and its DNA sequence determination.

A new method for producing macroporous silica (suitable for high-performance liquid chromatography) with covalently attached DNA is presented. The method uses (dT)18-silica as a primer, annealed to a poly(A)-RNA template, which is then transcribed using reverse transcriptase. The RNA template is eluted and single-stranded cDNA-silica is recovered. The cDNA-silica can be sequenced using the dideoxy method. These methods provide a facile method for producing cDNA-silica of demonstrable authenticity and provide a unique approach to DNA and RNA sequencing.

Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides.

A set of mutations [Ala-62-->Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers on the virus a reduced sensitivity to multiple antiretroviral dideoxynucleosides and has been seen in HIV-1 variants isolated from patients receiving combination chemotherapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddI). The IC50 values of AZT, ddC, ddI, 2',3'-dideoxyguanosine, and 2',3'-didehydro-3'-deoxythymidine against an infectious clone constructed to include the five mutations were significantly higher than those of a wild-type infectious clone. The K1 value for AZT 5'-triphosphate determined for the virus-associated RT from a posttherapy strain was 35-fold higher than that of RT from a pretherapy strain. Detailed analysis of HIV-1 strains isolated at various times during therapy showed that the Q151M mutation developed first in vivo, at the time when the viremia level suddenly increased, followed by the F116Y and F77L mutations. All five mutations ultimately developed, and the viremia level rose even further. Analyses based on the three-dimensional structure of HIV-1 RT suggest that the positions where at least several of the five mutations occur are located in close proximity to the proposed dNTP-binding site of RT and the first nucleotide position of the single-stranded template.

Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase.

Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)