Modulation of Poliovirus Replicative Fitness in HeLa Cells by Deoptimization of Synonymous Codon Usage in the Capsid Region

Abstract

We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (N C ) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G؉C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an ϳ10-fold range, and virus yields decreased over an ϳ65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced ϳ3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased ϳ18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (ϳ50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.The use of synonymous codons at unequal frequencies, the codon usage bias, is characteristic of all biological systems (26,27). The strength and direction of codon usage bias are related to the genomic GϩC content and the relative abundance of different isoaccepting tRNAs (reviewed in references 1, 16, and 53). Codon usage can affect the efficiency of gene expression. In bacteria (Escherichia coli) (26, 75), yeast (Saccharomyces cerevisiae) (5, 27), plants (Arabidopsis thaliana) (12), nematodes (Caenorhabditis elegans) (16), and insects (Drosophila melanogaster) (50), the most highly expressed genes use codons matched to the most abundant tRNAs (2). In contrast, in humans and other vertebrates, codon usage bias is much more strongly correlated with the GϩC content of the isochore where the gene is located (51, 71) than with the breadth or level of gene expression (16) or the number of corresponding tRNA genes (28, 30). Despite the weak correlation between codon usage and the levels of gene expression in mammalian cells (16,71), imbalances between codon usage and tRNA abundance can sharply reduce the levels of gene expression.Optimization of codon composition is frequently required for the efficient expression of genes in heterologous host systems (3,31,67,76). For example, the expression of human immunodeficiency virus type 1 ...

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

“…To date, genetic stability studies of large-scale deoptimized viruses have shown that deattenuation, indeed, seems to be low, suggesting that these viruses are genetically stable (6,8,(15)(16)(17)(18)(19)(20)(21). However, an important limitation of these studies is that the deoptimized viruses generally have not been subjected to strong selective pressure that…”

“…In principle, on the background of thousands of attenuating mutations, any single-site reversion should yield only a minuscule selective advantage. The most likely path to reversion imaginable under this model is the progressive accumulation of many individual mutations, providing for a slow progression of deattenuation (12)(13)(14).To date, genetic stability studies of large-scale deoptimized viruses have shown that deattenuation, indeed, seems to be low, suggesting that these viruses are genetically stable (6,8,(15)(16)(17)(18)(19)(20)(21). However, an important limitation of these studies is that the deoptimized viruses generally have not been subjected to strong selective pressure that…”

Characteristics of synonymous codon usage bias in the beginning region of West Nile virus

“…In addition to analyzing pathways of virus infection in hosts, examining the overall codon usage for an RNA virus open reading frame (ORF) is useful for estimating the evolutionary processes and genetic features of viruses that respond or adapt to the host cell environment (Bahir et al, 2009;Lobo et al, 2009;Wong et al, 2010;Zhou et al, 2013a,b). Recent progress has been made in measuring viral protein translation at the level of synonymous codon usage bias (Sanchez et al, 2003;Burns et al, 2006;Mueller et al, 2006;Bahir et al, 2009;Wong et al, 2010;Poyry and Jackson, 2011;Zhou et al, 2011); however, the contribution of the WNV coding sequence to replication efficiency remains unclear. Here, we employed a simple method for analyzing synonymous codon usage bias in the beginning region of the WNV coding sequence to evaluate the role of codon usage in regulating WNV polyprotein translation.…”

Genetic features of a translation initiation system composed of IRES element, nucleotide context surrounding the initiation codon, and translation initiation region of classical swine fever virus RNA

“…These codons likely reduce the probability of abortive translation initiation of the CSFV genome through adaptation of codon usage patterns in this region to the host environment. The adaptation of codon usage patterns in exogenous genes to the overall codon usage pattern of the hosts is an effective approach for producing functional proteins (Gustafsson et al, 2004;Burns et al, 2006;Hershberg and Petrov, 2008;Sharp et al, 2010).…”