Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As

Nature Microbiology, Journal Year: 2024, Volume and Issue: 9(5), P. 1340 - 1355

Published: April 11, 2024

Abstract Although the significance of chemical modifications on RNA is acknowledged, evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values for fragmented RNAs at low resolution relied indirect analyses phenotypic effects by perturbing host effectors. Here we analysed HIV-1 full-length, single level nucleotide using direct sequencing methods. Our data reveal an unexpectedly simple modification landscape, highlighting three predominant N 6 -methyladenosine (m A) near 3′ end. More densely installed spliced viral messenger than genomic RNAs, these m As play a crucial role maintaining normal levels splicing translation. generates diverse subspecies with distinct A ensembles, multiple its provides additional stability resilience to replication, suggesting unexplored RNA-level strategy.

Language: Английский

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Enhanced NF-κB activation via HIV-1 Tat-TRAF6 cross-talk

Science Advances, Journal Year: 2024, Volume and Issue: 10(3)

Published: Jan. 19, 2024

The Tat proteins of HIV-1 and simian immunodeficiency virus (SIV) are essential for activating viral transcription. In addition, stimulates nuclear factor κB (NF-κB) signaling pathways to regulate gene expression although its molecular mechanism is unclear. Here, we report that directly activates NF-κB through the interaction with TRAF6, which an upstream molecule canonical pathway. This increases TRAF6 oligomerization auto-ubiquitination, as well synthesis K63-linked polyubiquitin chains further activate pathway Moreover, ectopic significantly transcription, whereas knockdown inhibits Furthermore, Tat-mediated activation conserved among HIV-1, HIV-2, SIV isolates. Our study uncovers yet another by subverts host transcriptional enhance own

Language: Английский

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Prolonging genetic circuit stability through adaptive evolution of overlapping genes

Nucleic Acids Research, Journal Year: 2023, Volume and Issue: 51(13), P. 7094 - 7108

Published: June 1, 2023

The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed overlapping sequences in which one gene is encoded or 'entangled' entirely within an alternative reading frame another gene. In this design, the toxin-encoding relE was entangled ilvA, encodes threonine deaminase, enzyme essential for isoleucine biosynthesis. A functional entanglement construct obtained upon modification ribosome-binding site internal Using optimized found selection pressure to IlvA stabilized production burdensome RelE >130 generations, compares favorably with most stable kill-switch developed date. This stabilizing effect achieved through complete alteration allowable landscape mutations such inactivating genes were disfavored. Instead, majority lineages accumulated regulatory region ilvA. By reducing baseline expression, these more 'benign' lowered circuit burden, suppressed accumulation relE-inactivating mutations, thereby prolonging function. Overall, work demonstrates utility sequence paired adaptive laboratory evolution campaign increase evolutionary stability circuits.

Language: Английский

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Functional and structural segregation of overlapping helices in HIV-1

eLife, Journal Year: 2022, Volume and Issue: 11

Published: May 5, 2022

Overlapping coding regions balance selective forces between multiple genes. One possible division of nucleotide sequence is that the predominant force on a particular can be attributed to just one gene. While this arrangement has been observed in which gene structured and other disordered, we sought explore how overlapping genes constraints when both protein products are over same sequence. We use combination analysis, functional assays, selection experiments examine an overlapped region HIV-1 encodes helical Env Rev. find segregation occurs even overlap, with each spacing its residues manner allows mutable non-binding face helix encode important charged helix. Additionally, our reveal novel critical have implications for therapeutic targeting HIV-1.

Language: Английский

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Rev–Rev Response Element Activity Selection Bias at the Human Immunodeficiency Virus Transmission Bottleneck

Open Forum Infectious Diseases, Journal Year: 2023, Volume and Issue: 10(10)

Published: Sept. 29, 2023

Abstract Background Sexual transmission of human immunodeficiency virus (HIV) is inefficient and results in selection viral variants based on incompletely understood factors. Functional variation the Rev–Rev response element (RRE) regulatory axis HIV affect replication kinetics relative expression proteins. We explored whether differences this among isolates fitness. Methods sequences were identified from nine female-to-male pairs. Using a rapid flow cytometric assay, we analyzed Rev-RRE functional activity primary isolates. Results was significantly lower recipient viruses compared with corresponding donor viruses. In most events, clustered at extreme low end range activity. Conclusions These data indicate pressure during sexual transmission. Variation may permit adaptation to different fitness landscapes could play an important role pathogenesis.

Language: Английский

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Zero-shot prediction of mutation effects on protein function with multimodal deep representation learning

Research Square (Research Square), Journal Year: 2023, Volume and Issue: unknown

Published: Oct. 11, 2023

Abstract Mutations in amino acid sequences can disrupt protein structures and functions. Accurate unsupervised prediction of mutation effects is critical biotechnology biomedicine, but remains a fundamental challenge. To resolve this challenge, we first presented multimodal deep representation learning model that comprehensively learns both sequence context structural constraints from ~160 million proteins with reliable structures. Based on the proposed model, developed ProMEP (Protein Mutational Effect Predictor) to predict zero-shot manner. capture multi-scale signatures at atomic-resolution achieves state-of-the-art (SOTA) performance mutational prediction. showed an average accuracy 91.67% during engineering TnpB protein. Remarkably, identified variant triple-mutations approximately 2.4-fold editing efficiency than wild-type. enables efficient exploration gigantic space will greatly facilitate studies

Language: Английский

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Biological factors in the synthetic construction of overlapping genes

BMC Genomics, Journal Year: 2021, Volume and Issue: 22(1)

Published: Dec. 1, 2021

Abstract Background Overlapping genes (OLGs) with long protein-coding overlapping sequences are disallowed by standard genome annotation programs, outside of viruses. Recently however they have been discovered in Archaea, diverse Bacteria, and Mammals. The biological factors underlying life’s ability to create require more study, may important applications understanding evolution biotechnology. A previous study claimed that protein domains from viruses were much better suited forming overlaps than those other cellular organisms - this we assessed claim, order discover what might underlie taxonomic differences the creation gene overlaps. Results After arbitrary Pfam domain pairs evaluating them Hidden Markov Models find OLG construction be less constrained expected. For instance, close 10% constructed cannot distinguished typical their family. Most also indistinguishable natural regarding identity secondary structure. Surprisingly, contrary a virus suitable for designing OLGs bacterial or eukaryotic were. In general, amount amino acid change required force overlap is approximately equal variation observed within resulting high similarity between altered so as mostly due combination redundancy genetic code evolutionary exchangeability many acids. Conclusions Synthetic which closely resemble sequences, measured HMM profiles, remarkably easy construct, most can while retaining original sequences. Future work will need assess not considered such intragenic interactions affect folding. While analysis here sufficient guarantee functional folding proteins, further improve our origin these remarkable elements across life opens up exciting possibilities synthetic biology.

Language: Английский

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Highly Similar Average Collateral Effect of Synonymous Mutations Across Alternative Reading Frames: A Potential Role In Evolvability

BioCosmos, Journal Year: 2023, Volume and Issue: 3(1), P. 1 - 11

Published: Jan. 1, 2023

Abstract Synonymous mutations in a protein coding gene lead to remarkably similar average “collateral” mutation effect size across alternative reading frames (1). Here we quantify the rarity of this feature among possible block structure codes as 0.77%. Then develop simple model evolutionary search with two types mutation. Across different step sizes and ratios types, fitness-maximizing region corresponds using single value. The analogous constant collateral observed for standard genetic code may likewise facilitate frame sequences.

Language: Английский

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Creating overlapping genes by alternate-frame insertion

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 7, 2024

Abstract Overlapping genes–wherein two different proteins are translated from alternative frames of the same DNA sequence–provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that overlapped gene. However, creating overlapping pairs is challenging as it requires redesign both protein products accommodate overlap constraints. Here, we present new “ o verlapping, lternate- f rame insertion” (OAFI) method for genes insertion “inner” gene, encoded in alternate frame, into flexible region “outer” Using OAFI, create bacterial toxins within antibiotic resistance We show inner and outer retain functionality despite redesign, translation influenced position Additionally, selection alters permitted inactivating mutations can restrict horizontal transfer Overall, OAFI offers versatile tool synthetic biology, expanding applications stabilization biocontainment.

Language: Английский

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Prolonging Genetic Circuit Stability through Adaptive Evolution of Overlapping Genes

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Feb. 28, 2023

ABSTRACT The development of synthetic biological circuits that maintain functionality over application relevant timescales remains a significant challenge. Here, we employed overlapping sequences in which one gene is encoded or “entangled” entirely within an alternative reading frame another gene. In this design, the toxin-encoding relE was entangled ilvA , encodes threonine deaminase, enzyme essential for isoleucine biosynthesis. A functional entanglement construct obtained upon modification ribosome binding site internal Using optimized found selection pressure to IlvA stabilized production burdensome RelE 130 generations, compares favorably with most stable kill-switch developed date. This stabilizing effect achieved through complete alteration mutational landscape such mutations inactivating genes were disfavored. Instead, majority lineages accumulated regulatory region . By reducing baseline expression, these more ‘benign’ lowered circuit burden, suppressed accumulation mutations, thereby prolonging function. Overall, work demonstrates utility sequence paired adaptive laboratory evolution campaign increase evolutionary stability circuits.

Language: Английский

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