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

ABSTRACT: BACKGROUND: 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 of HIV-1 that is observed today, or to what extent the actual encoding contributes to virus replication capacity, evolvability and pathogenesis. Here, we applied the previously described synthetic attenuated virus engineering (SAVE) approach to HIV-1. RESULTS: Using synonymous codon pairs, we rationally recoded and codon pair-optimized and deoptimized different moieties of the HIV-1 gag and pol genes. Deoptimized viruses had significantly lower viral replication capacity in MT-4 and peripheral blood mononuclear cells (PBMCs). Varying degrees of ex vivo attenuation were obtained, depending upon both the specific deoptimized region and the number of deoptimized codons. A protease optimized 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, deoptimized HIV-1 reverted to wild-type virulence after serial passages in MT-4 cells. Remarkably, no reversion was observed in the optimized virus. CONCLUSION: These data demonstrate that SAVE is a useful strategy to phenotypically affect the replicative properties of HIV-1.

SUBMITTER: Martrus G

PROVIDER: S-EPMC3726367 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Martrus Gloria G Nevot Maria M Andres Cristina C Clotet Bonaventura B Martinez Miguel Angel MA

Retrovirology 20130725

<h4>Background</h4>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 of HIV-1 that is observed today, or to what extent the actual encoding contributes to virus replication capacity, evolvability and pathogenesis. Here, we applied the previously described synthetic attenuated virus engineering (SAVE) approach to HIV-1.<h4>Results</h4>Using synonymous codon pairs, ...[more]

PMID: 23885919

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Project description:BackgroundCodon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias.ResultsIn this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris.ConclusionsCodon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

Project description:BackgroundSegment 6 of the ISA virus codes for hemoagglutinin-esterase (HE). This segment is highly variable, with more than 26 variants identified. The major variation is observed in what is called the high polymorphism region (HPR). The role of the different HPR zones in the viral cycle or evolution remains unknown. However viruses that present the HPR0 are avirulent, while viruses with important deletions in this region have been responsible for outbreaks with high mortality rates. In this work, using bioinformatic tools, we examined the influence of different HPRs on the adaptation of HE genes to the host translational machinery and the relationship to observed virulence.MethodsTranslational efficiency of HE genes and their HPR were estimated analyzing codon-pair bias (CPB), adaptation to host codon use (codon adaptation index-CAI) and the adaptation to available tRNAs (tAI). These values were correlated with reported mortality for the respective ISA virus and the ΔG of RNA folding. tRNA abundance was inferred from tRNA gene numbers identified in the Salmo salar genome using tRNAScan-SE. Statistical correlation between data was performed using a non-parametric test.ResultsWe found that HPR0 contains zones with codon pairs of low frequency and low availability of tRNA with respect to salmon codon-pair usage, suggesting that HPR modifies HE translational efficiency. Although calculating tAI was impossible because one third of tRNAs (~60.000) were tRNA-ala, translational efficiency measured by CPB shows that as HPR size increases, the CPB value of the HE gene decreases (P = 2x10⁻⁷, ρ = -0.675, n = 63) and that these values correlate positively with the mortality rates caused by the virus (ρ = 0.829, P = 2x10⁻⁷, n = 11). The mortality associated with different virus isolates or their corresponding HPR sizes were not related with the ΔG of HPR RNA folding, suggesting that the secondary structure of HPR RNA does not modify virulence.ConclusionsOur results suggest that HPR size affects the efficiency of gene translation, which modulates the virulence of the virus by a mechanism similar to that observed in production of live attenuated vaccines through deoptimization of codon-pair usage.

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

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

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