Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. and Harrison, S.C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These 'selectively deleted' heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Δ13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from pSI had little effect on synthesis-dependent and synthesis- independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Δ13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis

Investigation of previously implicated genetic variants in chronic tic disorders: a transmission disequilibrium test approach

Genetic studies in Tourette syndrome (TS) are characterized by scattered and poorly replicated findings. We aimed to replicate findings from candidate gene and genome-wide association studies (GWAS). Our cohort included 465 probands with chronic tic disorder (93% TS) and both parents from 412 families (some probands were siblings). We assessed 75 single nucleotide polymorphisms (SNPs) in 465 parent–child trios; 117 additional SNPs in 211 trios; and 4 additional SNPs in 254 trios. We performed SNP and gene-based transmission disequilibrium tests and compared nominally significant SNP results with those from a large independent case–control cohort. After quality control 71 SNPs were available in 371 trios; 112 SNPs in 179 trios; and 3 SNPs in 192 trios. 17 were candidate SNPs implicated in TS and 2 were implicated in obsessive–compulsive disorder (OCD) or autism spectrum disorder (ASD); 142 were tagging SNPs from eight monoamine neurotransmitter-related genes (including dopamine and serotonin); 10 were top SNPs from TS GWAS; and 13 top SNPs from attention-deficit/hyperactivity disorder, OCD, or ASD GWAS. None of the SNPs or genes reached significance after adjustment for multiple testing. We observed nominal significance for the candidate SNPs rs3744161 (TBCD) and rs4565946 (TPH2) and for five tagging SNPs; none of these showed significance in the independent cohort. Also, SLC1A1 in our gene-based analysis and two TS GWAS SNPs showed nominal significance, rs11603305 (intergenic) and rs621942 (PICALM). We found no convincing support for previously implicated genetic polymorphisms. Targeted re-sequencing should fully appreciate the relevance of candidate genes

Truncating α-Helix E′ of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer

The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed α-helix E′ of the RNase H domain (p66Δ8/p51 and p66Δ16/p51, respectively), while mutant p66Δ23/p51 lacked αE′ and the β5′-αE′ connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66Δ16/p51 and p66Δ23/ p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66Δ8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66Δ8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5′ and 3′ ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication

Therapeutische Ansaetze gegen HIV: Die Nukleinsaeurekomponenten im reversen Transkriptionskomplex. Schlussbericht

Our study deals with the analysis of the enzymatic activities of HIV-RT, an enzyme which is, due to its key position in the HIV life cycle, a preferred target in the treatment of AIDS. We analyzed the RT-associated RNaseH activity in vitro. We show that RT has also double-strand RNase activity which cleaves 18 nucleotides upstream of the 3'-terminus (EMBOJ, 1995). Kinetic analysis shows that cleavage of double stranded RNA is 30 times slower than that of DNA/RNA. In the context of this study we have developed a model of the translocation mechanism of HIV-RT which takes into account the different processivities of RT observed at different templates, such as DNA and RNA (Eur.J.Biochem, 1996). A patent about the possible use of RT-associated RNaseH activity and modified tRNALys,3 is pending. In order to analyze conformational changes of tRNALys,3 with RT and template nuclei acids, we have introduced two new probes: a: Copper phenanthrolin (OPCu). We show that this probe can be used for quantitative determination of structural changes in RNA (RNA, 1995). b: Peroxinitrous acid (PeNA): We show that PeNA-generated OH. can be used as an alternative for Fe-generated OH. for analyzing RNA-protein complexes (FEBSL, 1996). (orig.)SIGLEAvailable from TIB Hannover: DtF QN1(71,23) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

N-Substituted Quinolinonyl Diketo Acid Derivatives as HIV Integrase Strand Transfer Inhibitors and Their Activity against RNase H Function of Reverse Transcriptase

Bifunctional quinolinonyl DKA derivatives were first described as nonselective inhibitors of 3′-processing (3′-P) and strand transfer (ST) functions of HIV-1 integrase (IN), while 7-aminosubstituted quinolinonyl derivatives were proven IN strand transfer inhibitors (INSTIs) that also displayed activity against ribonuclease H (RNase H). In this study, we describe the design, synthesis, and biological evaluation of new quinolinonyl diketo acid (DKA) derivatives characterized by variously substituted alkylating groups on the nitrogen atom of the quinolinone ring. Removal of the second DKA branch of bifunctional DKAs, and the amino group in position 7 of quinolinone ring combined with a fine-tuning of the substituents on the benzyl group in position 1 of the quinolinone, increased selectivity for IN ST activity. In vitro, the most potent compound was 11j (IC50 = 10 nM), while the most active compounds against HIV infected cells were ester derivatives 10j and 10l. In general, the activity against RNase H was negligible, with only a few compounds active at concentrations higher than 10 μM. The binding mode of the most potent IN inhibitor 11j within the IN catalytic core domain (CCD) is described as well as its binding mode within the RNase H catalytic site to rationalize its selectivity. (Chemical Presented)

Pre- and perinatal complications in relation to Tourette syndrome and co-occurring obsessive-compulsive disorder and attention-deficit/hyperactivity disorder

Pre- and perinatal complications have been implicated in the onset and clinical expression of Tourette syndrome albeit with considerable inconsistencies across studies. Also, little is known about their role in co-occurring obsessive-compulsive disorder (OCD) and attention–deficit/hyperactivity disorder (ADHD) in individuals with a tic disorder. Therefore, we aimed to investigate the role of pre- and perinatal complications in relation to the presence and symptom severity of chronic tic disorder and co-occurring OCD and ADHD using data of 1113 participants from the Tourette International Collaborative Genetics study. This study included 586 participants with a chronic tic disorder and 527 unaffected family controls. We controlled for age and sex differences by creating propensity score matched subsamples for both case-control and within-case analyses. We found that premature birth (OR = 1.72) and morning sickness requiring medical attention (OR = 2.57) were associated with the presence of a chronic tic disorder. Also, the total number of pre- and perinatal complications was higher in those with a tic disorder (OR = 1.07). Furthermore, neonatal complications were related to the presence (OR = 1.46) and severity (b = 2.27) of co-occurring OCD and also to ADHD severity (b = 1.09). Delivery complications were only related to co-occurring OCD (OR = 1.49). We conclude that early exposure to adverse situations during pregnancy is related to the presence of chronic tic disorders. Exposure at a later stage, at birth or during the first weeks of life, appears to be associated with co-occurring OCD and ADHD