Duplex structural differences and not 2′-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA

Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA–DNA versus DNA–DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3′ recessed DNA base) are required for stable binding to RNA–DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA–DNA. Replacing 2′- hydroxyls on pivotal RNA bases with 2′-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. RT’s Kd (koff/kon) for DNA–DNA and RNA–DNA were similar, although koff differed almost 40-fold, suggesting a faster kon for DNA–DNA. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA–DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA–DNA and DNA–DNA, respectively, allow the former to conform more easily to HIV-RT’s binding cleft, leading to more stable binding. Biologically, the ability of RT to form a more stable complex on RNA–DNA may aid in degradation of RNA fragments that remain after DNA synthesis

Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity

A SELEX (systematic evolution of ligands by exponential enrichment)-based approach was developed to determine whether HIV-RT showed preference for particular primer-template sequences. A 70 nt duplex DNA was designed with 20 nt fixed flanking sequences at the 3′ and 5′ ends and a randomized 30 nt internal sequence. The fixed sequence at the 5′ end contained a BbsI site six bases removed from the randomized region. BbsI cuts downstream of its recognition site generating four base 5′ overhangs with recessed 3′ termini. Cleavage produced a 50 nt template and 46 nt primer with the 3′ terminus within the randomized region. HIV-RT was incubated with this substrate and material that bound RT was isolated by gel-shift. The recovered material was treated to regenerate the BbsI site, amplified by PCR, cleaved with BbsI and selected with HIV-RT again. This was repeated for 12 rounds. Material from round 12 bound approximately 10-fold more tightly than starting material. All selected round 12 primer-templates had similar sequence configuration with a 6–8 base G run at the 3′ primer terminus, similar to the HIV polypurine tract. Further modifications indicate that the Gs were necessary and sufficient for strong binding

Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity

Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg2+ is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn2+, Co2+, and Zn2+ can also support catalysis. Although Zn2+ supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn2+ is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn2+. We used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn2+, Co2+, and Zn2+. The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn2+ when compared to Mg2+ at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3′ nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn2+ than Mg2+. In agreement with previous literature, we observed that Mn2+ and Co2+ dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn2+ and Co2+ remained similar to Mg2+ at lower concentrations that are optimal for catalysis. This study shows that Zn2+, at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.https://doi.org/10.1186/s12858-015-0041-

998-61 Population Prevalence of Wolff-Parkinson-White Syndrome

Little is known about the epidemiology of Wolff-Parkinson-White (WPW) syndrome in the general population. Virtually all previous studies have been either case series from tertiary care centers or limited to young adult males screened for military training. To date, there are no detailed studies of the prevalence of WPW in the general population. To determine the prevalence of WPW in the general population, we used the Marshfield Epidemiologic Study Area (MESA), a population laboratory of 50,000 people residing in 12 contiguous zip codes in central Wisconsin. Prevalence was determined as of 7/1/91 among MESA residents who had a diagnosis of WPW between 1/1/79 and 6/30/91. Cases were identified by reviewing the medical records and electrocardiograms of: a) all 32 MESA residents with the WPW diagnosis identified by International Classification of Diseases, 9th Revision (ICD-9) Code 426.7 as a hospital discharge or outpatient clinic diagnosis, b) 600 patients with suspected supraventricular arrhythmias identified by three ICD 9 codes, and c) all patients who had an invasive electrophysiology study for overt WPW syndrome in our institution over the last 10 years.ResultsWe identified 25 prevalent cases of WPW resulting in an overall population prevalence of 5.1/10,000 (95% C.I., 3.1–7.1).Age specific-prevalence rates per 10,000 were: 0–19 years –2.0; 20–39 years –5.5; 40–59 years –9.6; > 60 years –4.8. There was no significant difference in males versus females. Al1 25 verified cases were identified from the 32 potential cases with ICD-9 Code 426.7, indicating that this code is 100% sensitive and has a 78% positive predictive value for WPW syndrome.Conclusions1) The prevalence of WPW in the general population is lower than that reported in selected populations and appears to be highest in those of late middle-age. 2) Based on the findings of our study, we estimate that there are approximately 130,000 individuals in the United States with electrocardiographic documentation of WPW

Conformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs. pyrophosphorolysis – binding of foscarnet

HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3′-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg2+ ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg2+ (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors

Changes in Brain Activity Immediately Post-Exercise Indicate a Role for Central Fatigue in the Volitional Termination of Exercise

International Journal of Exercise Science 17(1): 220-234, 2024. Electroencephalography (EEG) allows for the evaluation of real time changes in brain (electrocortical) activity during exercise. A few studies have examined changes in electrocortical activity using stationary cycling, but the findings have been mixed. Some of these studies have found increases in brain activity following exercise, while others have found decreases in brain activity following exercise. Hence, it is of importance to identify post-exercise changes in brain activity. Sixteen healthy, untrained subjects (8 males; 8 females) participated in the study. All 16 participants performed a graded exercise test (GXT) to volitional exhaustion on an upright cycle ergometer. Continuous EEG recordings were sampled before (PRE) and immediately following (IP) the GXT. Regions of interest were primarily the dorsolateral prefrontal cortex (DLPFC), ventrolateral prefrontal cortex (VLPFC), and left and right motor cortex (MC). In the DLPFC, a frontal asymmetry index was also identified. There was a statistically significant increase in theta power in the DLPFC, VLPFC, and left and right MC from PRE to IP (all p \u3c 0.05). There was also a shift towards right hemisphere asymmetry at the IP time point in the DLPFC (p \u3c 0.05). Finally, there was an increase in alpha power from PRE to IP in the right MC (p \u3c 0.05). EEG could prove to be an important way to measure the effects of central fatigue on brain activity before and immediately following exercise

The Status of Masked Bobwhite Recovery in the United States and Mexico

The masked bobwhite (Colinus virginianus ridgwayi) is an endangered species currently numbering \u3c1500 individuals and restricted to 2 locales in southeastern Arizona and northcentral Sonora, Mexico. The subspecies\u27 endangered status is attributed to overgrazing of Sonora savanna grassland that began during the late 1880\u27s and continued well into the 20th century. This overgrazing resulted in the conversion of many native grass pastures to the exotic bufflegrass (Cenchrus ciliaris). The Arizona masked bobwhite population was extirpated around the turn of the century, and the Sonoran population was thought to have disappeared during the 1940\u27s until a small remnant population was discovered on a ranch near Benjamin Hill, Sonora , in 1964 . Masked bobwhite recovery efforts have a dynamic, long history of nearly six decades. Current masked bobwhite recovery efforts focus on reestablishing a self-sustaining population on the Buenos Aires National Wildlife Refuge (BANWR) in the United States, as well as 2 remnant wild populations located on privately owned ranches in northcentral Sonora

Trans-lesion synthesis and RNaseH activity by reverse transcriptases on a true abasic RNA template

While much is known about abasic DNA, the biological impact of abasic RNA is largely unexplored. To test the mutagenic potential of this RNA lesion in the context of retroviruses, we synthesized a 31-mer oligoribonucleotide containing an abasic (rAS) site and used it as a template for studying DNA primer extension by HIV-1, avian myeloblastosis virus (AMV) and moloney murine leukemia virus (MMLV) reversed transcriptases (RT). We found that trans-lesion synthesis readily takes place with HIV-1 RT and to a lesser extent with AMV RT while MMLV RT aborts DNA synthesis. The preference of dNTP incorporation follows the order A∼G > C∼T and thus obeys to the ‘A-rule’. In the case of HIV-1 RT, we measured the kinetic data of dNTP incorporation and compared it to abasic DNA. We found that A-incorporation is only 2-fold slower relative to a matched (undamaged) RNA template while it is 7-fold slower in the case of DNA. Furthermore, there is less discrimination in incorporation between the four dNTPs in the case of abasic RNA compared to abasic DNA. These experiments clearly point to a higher promiscuity of lesion bypass on abasic RNA. Given their known higher chemical stability, such rAS sites can clearly contribute to (retro)viral evolution