scholarly journals Determinants of Adenine-mutagenesis in Diversity-Generating Retroelements

2020 ◽  
Author(s):  
Sumit Handa ◽  
Andres Reyna ◽  
Timothy Wiryaman ◽  
Partho Ghosh
Keyword(s):  
Amino Acids ◽  
Dark Matter ◽  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Genetic Information ◽  
Human Microbiome ◽  
Protein Sequences ◽  
Catalytic Efficiency ◽  
Natural World

ABSTRACTDiversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial ‘dark matter’. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.

scholarly journals Determinants of adenine-mutagenesis in diversity-generating retroelements

2020 ◽  
Author(s):  
Sumit Handa ◽  
Andres Reyna ◽  
Timothy Wiryaman ◽  
Partho Ghosh
Keyword(s):  
Amino Acids ◽  
Dark Matter ◽  
Reverse Transcription ◽  
Genetic Information ◽  
Human Microbiome ◽  
Protein Sequences ◽  
Catalytic Efficiency ◽  
Natural World ◽  
In Vitro System

Abstract Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial ‘dark matter’. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.

The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA

1992 ◽  
Vol 12 (11) ◽  
pp. 5131-5144
Author(s):  
H Wang ◽  
J C Kennell ◽  
M T Kuiper ◽  
J R Sabourin ◽  
R Saldanha ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Full Length ◽  
Minus Strand ◽  
Ribonucleoprotein Particle ◽  
Cdna Synthesis ◽  
Reverse Transcriptases ◽  
In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

scholarly journals A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

2015 ◽  
Vol 89 (16) ◽  
pp. 8119-8129 ◽  
Author(s):  
Eytan Herzig ◽  
Nickolay Voronin ◽  
Nataly Kucherenko ◽  
Amnon Hizi
Keyword(s):  
Life Cycle ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Reverse Transcription ◽  
Polymerase Activity ◽  
Rnase H ◽  
Strand Transfer ◽  
Hiv 1

ABSTRACTThe process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting thein vitrodata. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis.IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting thein vitrodata. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

scholarly journals Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

2000 ◽  
Vol 74 (22) ◽  
pp. 10349-10358 ◽  
Author(s):  
Elias K. Halvas ◽  
Evguenia S. Svarovskaia ◽  
Vinay K. Pathak
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Deoxyribonucleoside Triphosphate ◽  
Murine Leukemia

ABSTRACT Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

scholarly journals The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA.

1992 ◽  
Vol 12 (11) ◽  
pp. 5131-5144 ◽  
Author(s):  
H Wang ◽  
J C Kennell ◽  
M T Kuiper ◽  
J R Sabourin ◽  
R Saldanha ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Full Length ◽  
Minus Strand ◽  
Ribonucleoprotein Particle ◽  
Cdna Synthesis ◽  
Reverse Transcriptases ◽  
In Vitro Transcripts

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

scholarly journals The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

Retrovirology ◽  
2010 ◽  
Vol 7 (1) ◽  
pp. 40 ◽  
Author(s):  
Jinwoo Ahn ◽  
In-Ja L Byeon ◽  
Sanjeewa Dharmasena ◽  
Kelly Huber ◽  
Jason Concel ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Transcription In Vitro ◽  
Hiv 1

scholarly journals Computational approach for selection of epitope-based dengue vaccine targets

2018 ◽  
Vol 14 (4) ◽  
pp. 605-618
Author(s):  
Phuc Nguyen ◽  
Ly Le
Keyword(s):  
Amino Acids ◽  
T Cell ◽  
Dengue Virus ◽  
Rational Design ◽  
Protein Sequences ◽  
Computational Approach ◽  
Amino Acid Sequences ◽  
Universal Vaccine ◽  
E Protein

High antigenic variability in the envelope (E) protein of different virus strains has been a major obstacle in designing effective vaccines for Dengue virus (DENV). To maintain their biological function, some parts of viral proteins remain stable during evolution thus one possible approach to solve this problem is to recognize specific regions within different protein sequences of E that have the tendency to stay constant through evolution. These regions may possess some special attributes to become a vaccine candidate against dengue virus. In this study, a computational approach was utilized to identify and analyze highly conserved amino acid sequences of the DENV E protein. Sequences of 9 amino acids or more were specifically focused due to their immune-relevant as T-cell determinants. Different bioinformatics tools were responsible for revealing conserved regions in the DENV E protein and constructing the phylogenetic tree from the sequence database. The tools also predicted immunogenicity of the identified vaccine targets. Ultimately, two peptide regions of at least 9 amino acids were chosen due to their high conserved attribute in more than 95% of all collected DENV sequences. Moreover, both of them was found to be immune-relevant by their correspondence to known or putative HLA-restricted T cell determinants. The conserved attribute of these sequences through the entire analysis of this study supports their potential as candidates for further in vitro experiments for rational design a universal vaccine which has longer and broader impact.

scholarly journals Reduced Susceptibility of Human Immunodeficiency Virus Type 1 (HIV-1) from Patients with Primary HIV Infection to Nonnucleoside Reverse Transcriptase Inhibitors Is Associated with Variation at Novel Amino Acid Sites

2000 ◽  
Vol 74 (22) ◽  
pp. 10269-10273 ◽  
Author(s):  
Andrew J. Leigh Brown ◽  
Heather M. Precious ◽  
Jeannette M. Whitcomb ◽  
Joseph K. Wong ◽  
Marlynne Quigg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Hiv Infection ◽  
Reverse Transcriptase ◽  
Acid Sites ◽  
Antiretroviral Drugs ◽  
In Vitro Mutagenesis ◽  
Immunodeficiency Virus

ABSTRACT Recently, significant numbers of individuals with primary human immunodeficiency virus (HIV) infection have been found to harbor viral strains with reduced susceptibility to antiretroviral drugs. In one study, HIV from 16% of such antiretroviral-naive individuals was shown to have a susceptibility to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) between 2.5- and 10-fold lower than that of a wild-type control. Mutations in the RT domain that had previously been associated with antiretroviral resistance were not shared by these strains. We have analyzed by logistic regression 46 variable amino acid sites in RT for their effect on susceptibility and have identified two novel sites influencing susceptibility to NNRTIs: amino acids 135 and 283 in RT. Eight different combinations of amino acids at these sites were observed among these patients. These combinations showed a 14-fold range in mean susceptibility to both nevirapine and delavirdine. In vitro mutagenesis of the control strain combined with a phenotypic assay confirmed the significance of amino acid variation at these sites for susceptibility to NNRTIs.

scholarly journals Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination

2006 ◽  
Vol 80 (5) ◽  
pp. 2483-2494 ◽  
Author(s):  
Christian Lanciault ◽  
James J. Champoux
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Reverse Transcription ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Primer Binding Site ◽  
Infection Assay ◽  
Green Fluorescent

ABSTRACT Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3′ terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.

scholarly journals Human Immunodeficiency Virus Type 1 Central DNA Flap: Dynamic Terminal Product of Plus-Strand Displacement DNA Synthesis Catalyzed by Reverse Transcriptase Assisted by Nucleocapsid Protein

2001 ◽  
Vol 75 (7) ◽  
pp. 3301-3313 ◽  
Author(s):  
Laurence Hameau ◽  
Josette Jeusset ◽  
Sophie Lafosse ◽  
Dominique Coulaud ◽  
Etienne Delain ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Strand Displacement ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the proviral DNA generating a 99-nucleotide DNA flap (6). This step, catalyzed by reverse transcriptase (RT), is defined as a discrete strand displacement (SD) synthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Using recombinant HIV-1 RT and a circular single-stranded DNA template harboring the cPPT-CTS sequence, we have developed an SD synthesis-directed in vitro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), the main accessory protein of the reverse transcription complex. A full-length HIV-1 central DNA flap was then carried out in vitro. Its synthesis appears faster in the presence of the HIV-1 NCp or the T4-encoded SSB protein (gp32). Finally, a high frequency of strand transfer was shown during the SD synthesis along the cPPT-CTS site with RT alone. This reveals a local and efficient 3′-5′ branch migration which emphasizes some important structural fluctuations within the flap. These fluctuations may be stabilized by the NCp chaperone activity. The biological implications of the RT-directed NCp-assisted flap synthesis are discussed within the context of reverse transcription complexes, assembly of the preintegration complexes, and nuclear import of the HIV-1 proviral DNA to the nucleus toward their chromatin targets.

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