Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis

Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5′ to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5′ terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G→A or A→G substitution at PPT positions −2, −4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions −2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting −4G with an analog containing a 6-NH2 moiety

Probing Retroviral and Retrotransposon Genome Structures: The “SHAPE” of Things to Come

Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100–500 nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide “connectivity map,” conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges

Revisiting Plus-Strand DNA Synthesis in Retroviruses and Long Terminal Repeat Retrotransposons: Dynamics of Enzyme: Substrate Interactions

Although polypurine tract (PPT)-primed initiation of plus-strand DNA synthesis in retroviruses and LTR-containing retrotransposons can be accurately duplicated, the molecular details underlying this concerted series of events remain largely unknown. Importantly, the PPT 3′ terminus must be accommodated by ribonuclease H (RNase H) and DNA polymerase catalytic centers situated at either terminus of the cognate reverse transcriptase (RT), and in the case of the HIV-1 enzyme, ∼70Å apart. Communication between RT and the RNA/DNA hybrid therefore appears necessary to promote these events. The crystal structure of the HIV-1 RT/PPT complex, while informative, positions the RNase H active site several bases pairs from the PPT/U3 junction, and thus provides limited information on cleavage specificity. To fill the gap between biochemical and crystallographic approaches, we review a multidisciplinary approach combining chemical probing, mass spectrometry, NMR spectroscopy and single molecule spectroscopy. Our studies also indicate that nonnucleoside RT inhibitors affect enzyme orientation, suggesting initiation of plus-strand DNA synthesis as a potential therapeutic target

Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase

Aspartic acid residues comprising the -D-(aa)(n)-Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151→Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity. Asp151→Asn and Asp213→Asn substitutions eliminated both polymerase activities. However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity. Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis. Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers. Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis. In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role. Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition

3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a cis-acting regulatory signal located at the 5’ terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs)

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives β-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). β-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC(50) of 0.2 μM, while the IC(50) for Escherichia coli and human RNases H was 50 μM and 5.7 μM, respectively. In contrast, the related tropolone analog β-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC(50) = 1.5 μM and 40 μM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both β-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 μM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between β-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted

Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors

The natural product α-hydroxytropolones manicol and β-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an “RNase H-like” fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein–nucleic acid contacts over a region of ∼14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme–substrate complex to the enzyme–product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site

Genome-Wide Expression Analysis of a Spinal Muscular Atrophy Model: Towards Discovery of New Drug Targets

Spinal Muscular Atrophy is a recessive genetic disease and affects lower motor neurones and muscle tissue. A single gene is disrupted in SMA: SMN1 activity is abolished but a second copy of the gene (SMN2) provides limited activity. While the SMN protein has been shown to function in the assembly of RNA-protein complexes, it is unclear how the overall reduction in SMN activity specifically results in the neuromuscular phenotypes. Similar to humans, reduced smn activity in the fly causes earliest phenotypes in neuromuscular tissues. To uncover the effects of reduced SMN activity, we have studied gene expression in control and diseased fly tissues using whole genome micro-arrays. A number of gene expression changes are recovered and independently validated. Identified genes show trends in their predicted function: several are consistent with the function of SMN, in addition some uncover novel pathways. This and subsequent genetic analysis in the fly indicates some of the identified genes could be taken for further studies as potential drug targets for SMA and other neuromuscular disorders

SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem–loops, extensive long-range interactions (LRIs) and a small, palindromic stem–loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem–loops are static structures, the 5′ and 3′ regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem–loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs