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

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

Synthesis of Nucleoside 5′-O-α,β-methylene-β-triphosphates and Evaluation of Their Potency Towards Inhibition of HIV-1 Reverse Transcriptase

A polymer-bound α,β-methylene-β-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-α,β-methylene-β-triphosphates. Among all the compounds, cytidine 5′-O-α,β-methylene-β-triphosphate inhibited RNase H activity of HIV-1 reverse transcriptase with a Ki value of 225 μM

Synthesis of nucleoside 5′-\u3cem\u3eO\u3c/em\u3e-α,β-methylene-β-triphosphates and evaluation of their potency towards inhibition of HIV-1 reverse transcriptase

A polymer-bound α,β-methylene-β-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-α,β-methylene-β-triphosphates. Among all the compounds, cytidine 5′-O-α,β-methylene-β-triphosphate inhibited RNase H activity of HIV-1 reverse transcriptase with a Ki value of 225 μM

A cis-Acting Element in Retroviral Genomic RNA Links Gag-Pol Ribosomal Frameshifting to Selective Viral RNA Encapsidation

SummaryDuring retroviral RNA encapsidation, two full-length genomic (g) RNAs are selectively incorporated into assembling virions. Packaging involves a cis-acting packaging element (Ψ) within the 5′ untranslated region of unspliced HIV-1 RNA genome. However, the mechanism(s) that selects and limits gRNAs for packaging remains uncertain. Using a dual complementation system involving bipartite HIV-1 gRNA, we observed that gRNA packaging is additionally dependent on a cis-acting RNA element, the genomic RNA packaging enhancer (GRPE), found within the gag p1-p6 domain and overlapping the Gag-Pol ribosomal frameshift signal. Deleting or disrupting the two conserved GRPE stem loops diminished gRNA packaging and infectivity >50-fold, while deleting gag sequences between Ψ and GRPE had no effect. Downregulating the translation termination factor eRF1 produces defective virus particles containing 20 times more gRNA. Thus, only the HIV-1 RNAs employed for Gag-Pol translation may be specifically selected for encapsidation, possibly explaining the limitation of two gRNAs per virion

Examining the ribonuclease H primer grip of HIV-1 reverse transcriptase by charge neutralization of RNA/DNA hybrids

The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bound to an RNA/DNA hybrid reveals an extensive network of contacts with the phosphate backbone of the DNA strand ∼4–9 bp downstream from the ribonuclease H (RNase H) catalytic center. Collectively designated as ‘the RNase H primer grip’, this motif contains a phosphate binding pocket analogous to the human and Bacillus halodurans RNases H. The notion that the RNase H primer grip mediates the trajectory of RNA/DNA hybrids accessing the RNase H active site suggests that locally neutralizing the phosphate backbone may be exploited to manipulate nucleic acid flexibility. To examine this, we introduced single and tandem methylphosphonate substitutions through the region of the DNA primer contacted by the RNase H primer grip and into the RNase H catalytic center. The ability of mutant hybrids to support RNase H and DNA polymerase activity was thereafter examined. In addition, site-specific chemical footprinting was used to evaluate movement of the DNA polymerase and RNase H domains. We show here that minor alteration to the RNase H primer can have a dramatic effect on enzyme positioning, and discuss these findings in light of recent crystallography of human RNase H containing an RNA/DNA hybrid

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

A computational model of open-irrigated radiofrequency catheter ablation accounting for mechanical properties of the cardiac tissue

Radiofrequency catheter ablation (RFCA) is an effective treatment for cardiac arrhythmias. Although generally safe, it is not completely exempt from the risk of complications. The great flexibility of computational models can be a major asset in optimizing interventional strategies, if they can produce sufficiently precise estimations of the generated lesion for a given ablation protocol. This requires an accurate description of the catheter tip and the cardiac tissue. In particular, the deformation of the tissue under the catheter pressure during the ablation is an important aspect that is overlooked in the existing literature, that resorts to a sharp insertion of the catheter into an undeformed geometry. As the lesion size depends on the power dissipated in the tissue, and the latter depends on the percentage of the electrode surface in contact with the tissue itself, the sharp insertion geometry has the tendency to overestimate the lesion obtained, especially when a larger force is applied to the catheter. In this paper we introduce a full 3D computational model that takes into account the tissue elasticity, and is able to capture the tissue deformation and realistic power dissipation in the tissue. Numerical results in FEniCS-HPC are provided to validate the model against experimental data, and to compare the lesions obtained with the new model and with the classical ones featuring a sharp electrode insertion in the tissue.La Caixa 2016 PhD grant to M. Leoni, and Abbott non-conditional grant to J.M. Guerra Ramo