Synonymous genome recoding : a tool to explore microbial biology and new therapeutic strategies

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies

Clinical, virological and biochemical evidence supporting the association of HIV-1 reverse transcriptase polymorphism R284K and thymidine analogue resistance mutations M41L, L210W and T215Y in patients failing tenofovir/emtricitabine therapy

Background: Thymidine analogue resistance mutations (TAMs) selected under treatment with nucleoside analogues generate two distinct genotypic profiles in the HIV-1 reverse transcriptase (RT): (i) TAM1: M41L, L210W and T215Y, and (ii) TAM2: D67N, K70R and K219E/Q, and sometimes T215F. Secondary mutations, including thumb subdomain polymorphisms (e.g. R284K) have been identified in association with TAMs. We have identified mutational clusters associated with virological failure during salvage therapy with tenofovir/emtricitabine-based regimens. In this context, we have studied the role of R284K as a secondary mutation associated with mutations of the TAM1 complex. Results: The cross-sectional study carried out with >200 HIV-1 genotypes showed that virological failure to tenofovir/emtricitabine was strongly associated with the presence of M184V (P < 10-10) and TAMs (P < 10-3), while K65R was relatively uncommon in previously-treated patients failing antiretroviral therapy. Clusters of mutations were identified, and among them, the TAM1 complex showed the highest correlation coefficients. Covariation of TAM1 mutations and V118I, V179I, M184V and R284K was observed. Virological studies showed that the combination of R284K with TAM1 mutations confers a fitness advantage in the presence of zidovudine or tenofovir. Studies with recombinant HIV-1 RTs showed that when associated with TAM1 mutations, R284K had a minimal impact on zidovudine or tenofovir inhibition, and in their ability to excise the inhibitors from blocked DNA primers. However, the mutant RT M41L/L210W/T215Y/R284K showed an increased catalytic rate for nucleotide incorporation and a higher RNase H activity in comparison with WT and mutant M41L/L210W/T215Y RTs. These effects were consistent with its enhanced chain-terminated primer rescue on DNA/DNA template-primers, but not on RNA/DNA complexes, and can explain the higher fitness of HIV-1 having TAM1/R284K mutations. Conclusions: Our study shows the association of R284K and TAM1 mutations in individuals failing therapy with tenofovir/emtricitabine, and unveils a novel mechanism by which secondary mutations are selected in the context of drug-resistance mutations

Changes in codon-pair bias of human immunodeficiency virus type 1 have profound effects on virus replication in cell culture

[spa]El virus de la immunodeficiència humana 1 (VIH-1) conté una composició de nucleòtids diferent de la existent en el gens humans. Aquest fet planteja les qüestions de com la evolució ha triat la seqüència nucleotídica del VIH1 observada avui en dia, i de fins a quin punt aquesta seqüència actual contribueix a la capacitat replicativa, evolució i patogènesis virals. S’ha descrit que canvis en el ús de parelles de codons són eficaços per tal de generar virus atenuats de Poliovirus i Influenza. En aquesta tesi, hem aplicat la tecnologia prèviament descrita, “synthetic attenuated virus engineering” (SAVE) al VIH-1. Emprant parelles de codó sinònimes de manera racional, hem recodificat reoptimizant i desoptimizant per parelles de codó diferents fragments dels gens gag i pol del VIH-1. Les estructures de ARN i el us de codó dels nous fragments recodificats no es van veure afectades per la recodificació. Els virus desoptimizats van mostrar una replicació viral significativament inferior al virus control en cèl·lules MT-4 i en cèl·lules mononuclears de sang perifèrica (PBMCs). Depenent de la regió específica desoptimizada i del número de codons desoptimizats, es van obtenir diversos nivells d’atenuació ex vivo. Una reducció significant en la producció proteica es va observar quan la replicació viral va ser restringida a un sol cicle de replicació emprant un vector VIH-1 d’un sol cicle de replicació. La menor producció proteica no va correlacionar amb una reducció en el número de còpies del transcrit diana. Aquest fet suggereix que la transcripció, i no la traducció, es troba implicada en la generació dels fenotips atenuats produïts per la tecnologia de SAVE. El virus de proteasa reoptimizat que contenia 38 mutacions sinònimes, no es va mostrar atenuat, ans el contrari, mostrava una capacitat replicativa similar a la del virus control en cèl·lules MT-4 i en PBMCs. Encara que l’atenuació dels virus desoptimizats es basava en varies desenes de canvis nucleotídics, després de varis passis seriats en cèl·lules MT-4s, els virus desoptimizats de les regions de gag i proteasa van revertir a la virulència del virus control en cèl·lules MT-4. Alguns virus desoptimizats passats encara van mantenir un cert grau d’atenuació en PBMCs. Els anàlisis de quasiespècies de les seqüències dels virus passats en cultiu van mostrar que els virus atenuats acumulaven o bé mutacions sinònimes (reversions a la seqüència control o noves mutacions) o bé mutacions no-sinònimes. Els virus recodificats per la tecnologia de SAVE exploren diferents espais de seqüència. Singularment, no es va observar cap reversió important al virus reoptimizat passat en cultiu. Per tant, totes aquestes dades demostren que la tecnologia de SAVE és una estratègia útil per a afectar gradualment fenotípicament la capacitat replicativa del VIH-1, mitjançant un mecanisme que implica la traducció. El VIH-1 amb diferents nivells d’atenuació pot ser una eina utilitzable per al desenvolupament d’una vacuna segura i efectiva, així com pel desenvolupament de vectors lenvirals per a teràpia gènica més segurs[eng]Human immunodeficiency virus type 1 (HIV-1) has a biased nucleotide composition different from human genes. This raises the question of how evolution has chosen the nucleotide sequence HIV-1 observed today, or to what extent the actual encoding contributes to virus replication capacity, evolvability and pathogenesis. Prior work has documented the effectiveness of making changes to the codon-pair bias of viral genomes in order to generate attenuated poliovirus and influenza virus. In this thesis, we applied the previously described synthetic attenuated virus engineering (SAVE) approach to HIV-1. Using synonymous codon pairs, we rationally recoded and codon pair–reoptimized and deoptimized different moieties of the HIV-1 gag and pol genes. RNA structures and codon usage of new recoded fragments were not affected by recoding. Deoptimized viruses had significantly lower viral replication capacity in MT-4 cells and peripheral blood mononuclear cells (PBMCs). Various degrees of ex vivo attenuation were obtained depending upon the specific deoptimized region and the number of deoptimized codons. After restricting viral replication to a single cycle by using a single-cycle HIV-1 vector, a significant reduction in protein production was observed in the vector carrying an attenuated virus variant. This reduction in protein synthesis was not accompanied by a reduction in the targeted transcript copy number, which strongly suggests that translation, and not transcription, is implicated in the generation of the attenuated phenotype by SAVE technology. A protease reoptimized virus carrying 38 synonymous mutations was not attenuated and displayed a replication capacity similar to that of the wild type virus in MT-4 cells and PBMCs. Although attenuation is based on several tens of nucleotide changes, after serial passages in MT-4 cells, both gag and protease deoptimized HIV-1 reverted to wild-type virulence in MT-4 cells while some maintain a certain attenuation degree in PBMCs. Quasispecies analysis of viral passaged sequences showed that attenuated viruses accumulated either synonymous mutations (reversions to wild-type sequences or novel mutations) or non-synonymous mutations. Recoded viruses explored different space sequences. Remarkably, no important reversion was observed in the reoptimized virus. Thus, these data demonstrate that SAVE is a useful strategy to gradually affect the replicative properties of HIV-1 by a mechanism that involves translation. HIV-1 with different degrees of attenuation can be a useful tool for the development of a safe and effective vaccine as well as the development of safer gene-therapy lentiviral vector

Synonymous genome recoding : a tool to explore microbial biology and new therapeutic strategies

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies