Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5–10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT’s activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3′ end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase.

Characterization of the mitochondrial genomes of three powdery mildew pathogens reveals remarkable variation in size and nucleotide composition

Powdery mildews comprise a large group of economically important phytopathogenic fungi. However, limited information exists on their mitochondrial genomes. Here, we assembled and compared the mitochondrial genomes of the powdery mildew pathogens Blumeria graminis f. sp. tritici, Erysiphe pisi, and Golovinomyces cichoracearum. Included in the comparative analysis was also the mitochondrial genome of Erysiphe necator that was previously analysed. The mitochondrial genomes of the four Erysiphales exhibit a similar gene content and organization but a large variation in size, with sizes ranging from 109800 bp in B. graminis f. sp. tritici to 332165 bp in G. cichoracearum, which is the largest mitochondrial genome of a fungal pathogen reported to date. Further comparative analysis revealed an unusual bimodal GC distribution in the mitochondrial genomes of B. graminis f. sp. tritici and G. cichoracearum that was not previously observed in fungi. The cytochrome b (cob) genes of E. necator, E. pisi, and G. cichoracearum were also exceptionally rich in introns, which in turn harboured rare open reading frames encoding reverse transcriptases that were likely acquired horizontally. Golovinomyces cichoracearum had also the longest cob gene (45 kb) among 703 fungal cob genes analysed. Collectively, these results provide novel insights into the organization of mitochondrial genomes of powdery mildew pathogens and represent valuable resources for population genetics and evolutionary studies.

UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

A Combination of Amino Acid Mutations Leads to Resistance to Multiple Nucleoside Analogs in Reverse Transcriptases from HIV-1 Subtypes B and C

Resistance to anti-Human Immunodeficiency Virus (HIV) drugs has been a problem from the beginning of antiviral drug treatments. The recent expansion of combination antiretroviral therapy worldwide has led to an increase in resistance to antiretrovirals; understanding the mechanisms of resistance is increasingly important. In this study, we analyzed reverse transcriptase (RT) variants based on sequences derived from an individual who had a low-level rebound viremia while undergoing therapy with abacavir, azidothymidine (AZT or Zidovudine), and (−)-L-2′,3′-dideoxy-3′-thiacytidine (Lamivudine or 3TC). The RT had mutations at positions 64, 67, 70, 184, 219, and a threonine insertion after amino acid 69 in RT. The virus remained partially susceptible to the nucleoside reverse transcriptase inhibitor (NRTI) regimen. We show how these mutations affect the ability of NRTIs to inhibit DNA synthesis by RT. The presence of the inserted threonine reduced the susceptibility of the RT mutant to inhibition by Tenofovir.

Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale. Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3′ ends of single- and/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.

Novel prokaryotic sensing and regulatory system employing previously unknown nucleic acids-based receptors

The present study describes a previously unknown universal signaling and regulatory system, which we named “TETZ-receptor system”. This system is responsible for sensing, remembering, and regulating cell responses to various chemical, physical or biological stimuli. It controls cell survival, variability, reproduction, adaptation, genome changes, and gene transfer. Importantly, the TETZ-receptor system is responsible for the formation and maintenance of cell memory, as well the ability to “forget” preceding events. The system is composed of DNA- and RNA-based receptors located outside the membrane named “TezRs”, as well as reverse transcriptases and integrases. The sensory and regulatory functions of TezRs enable the TETZ-receptor system to control all major aspects of bacterial behavior, such as growth, biofilm formation and dispersal, utilization of nutrients including xenobiotics, virulence, chemo- and magnetoreception, response to external factors (e.g., temperature, UV, light and gas content), mutation events, phage-host interaction and recombination activity. Additionally, it supervises the function of other receptor-mediated signaling pathways.HIGHLIGHTSThe TETZ-receptor system regulates bacterial sensing and response to various stimuli.The TETZ-receptor system is responsible for maintenance and loss of cell memory.The TETZ-receptor system comprises DNA- and RNA-based “TezRs” receptors.The TETZ-receptor system relies on reverse transcriptases and recombinases.The TETZ-receptor system oversees other receptor-mediated signaling pathways.TezRs are implicated in cell mutation and recombination events.

Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations (~ 5-10 mM) that are several-fold higher than physiological cellular free Mg2+ (~ 0.5 mM). Analysis of fidelity using lacZα-based α-complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated ≈ equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZα. Unlike the α-complementation assay, which is dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. A ~ 4-fold increase in substitution mutations was observed in high Mg2+. The general trend was an exacerbation in high Mg2+ of more common mutation in low Mg2+, rather than the creation of new mutation hotspots. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable.

Structures of substrate complexes of foamy viral protease-reverse transcriptase

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA, a crucial process for the replication of retroviruses. Foamy viruses (FV) possess a unique RT which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of full-length MFV PR-RT - dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting ssRNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their lifecycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates have been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both monomeric configuration, which is observed in our structures in the presence of RNA/DNA hybrid, and an asymmetric dimer arrangement which we observed in the presence of dsDNA.

Funktion rätselhafter Retrons entschlüsselt

VAAM-Forschungspreis 2021Bacterial retrons are operons containing reverse transcriptases. Despite three decades of research, their physiological role remained mysterious. Recently, retrons have been uncovered as a widespread novel class of antiphage defense systems. We here discuss how high-throughput reverse genetics have enabled this discovery, illustrating the power such approaches have to illuminate the function of uncharacterized genes.