The decreased fitness of our codon replacement constructs appears to involve the interaction of several different biological mechanisms. Fitness showed strong inverse correlations with the number and location of CpG and UpA dinucleotides, weaker inverse correlations with the total numbers of nucleotide substitutions and codon changes, and no correlation with NC
. Positive correlations were observed between fitness and the CAI and the CPB score. There appear to be some synergistic effects when high CpG and UpA frequencies are coupled with low NC
and CAI values, as suggested by the very low fitness of construct ABc12
, in which CpG and UpA dinucleotides were maximized within cassette C
codons. Localized increases in %G+C (without increased CpG) to levels well above those found in naturally occurring poliovirus and human enterovirus genomes (43% to 47%) (6
) had little effect on fitness. Replacement in cassette C
of MEF-1 capsid codons with Lansing codons or our randomization of the natural MEF-1 synonymous codons (both replacements incorporated only small changes in CpG and UpA frequencies) also had minimal biological effects.
Decreased translational efficiency does not appear to be the main physiological defect in our codon replacement constructs, despite the large-scale use of unpreferred codons. In our previous (8
) and current experiments, virus constructs with widely varying infectivities produced similar levels of virus-specific proteins in infected cells. Although it could be argued that translation is so tightly coupled to critical steps in the poliovirus replication cycle that small deficits in translation could cause disproportionately large decreases in virus infectivities, other observations counter this view. For example, an approximately four- to eightfold decrease in translational efficiency in type 1 poliovirus was associated with only a proportionate reduction in plaque yields (21
Suppression of CpG is strong in vertebrates (4
) and their single-stranded RNA viruses (19
), including poliovirus (47
), and suppression of UpA is virtually universal (4
). The initial evidence pointing to the negative effects of increased CpG and UpA on poliovirus fitness was the observation that revertants selected during serial passage of codon-deoptimized constructs of Sabin 2 (8
) and MEF-1 (R. Campagnoli, unpublished results) had high frequencies of mutations that eliminated CpG and UpA. Many of these reversions involved fixation of amino acid replacements in normally conserved capsid sites (8
). The properties of the MEF-1 constructs described here provide direct confirmation of fitness reductions associated with increased frequencies of CpG and UpA dinucleotides.
The importance of CpG and UpA frequencies to poliovirus fitness is reinforced by the recent report of Coleman et al. (13
), who replaced the natural capsid region codon pairs of poliovirus type 1 (Mahoney strain) with synonymous codon pairs rarely found in the open reading frames of human genes. The biochemical properties of their constructs, especially the low specific infectivities, were similar to those described here for MEF-1 and previously for Sabin 2 (8
) and strain Mahoney (38
). Their constructs were attenuated for Tg21 transgenic mice expressing the CD155 PVR and could induce protective immunity against subsequent challenge with neurovirulent wild-type Mahoney virus. A prominent feature of the most disfavored codon pairs is the presence of CpG or UpA across codons (13
). Thus, the observed CPB in poliovirus and in humans and higher eukaryotes may be driven primarily by CpG and UpA dinucleotide suppression (4
). In this context, it is notable that in cassette C
of the construct with the lowest fitness, ABc12
, within-codon CpG and UpA frequencies were maximized but the CPB score was similar to those of higher-fitness constructs, including ABC
The underlying drivers of suppression appear to differ between CpG and UpA, although in both instances selection appears to act primarily through genome-wide mutational processes, and only secondarily on translated sequences (4
). In vertebrate genomes, most CpG dinucleotides are methylated at position 5 of cytosine, and spontaneous deamination of 5-methylcytosine results in a TpG mutation (and a CpA mutation on the complementary strand). Suppression of CpG in vertebrate DNA has been attributed to selection against potential mutational hot spots (4
), but this explanation does not account for suppression of CpG in the genomes of single-stranded RNA viruses where cytosine is not 5-methylated (28
). Another possible mechanism for CpG suppression is that unmethylated CpG in DNA stimulates the innate immune system via the Toll-like receptor TLR9 (62
), and RNA viruses may have evolved to evade the innate immune system by mimicking the dinucleotide patterns of host cell genes and mRNAs (19
). In addition, some RNA oligonucleotide motifs containing unmethylated CpGs are directly immunostimulatory via pathways distinct from TLR9 (58
) and therefore may be subject to negative selection.
UpA dinucleotides, in contrast, are direct substrates for cleavage of single-stranded RNA by endoribonucleases (4
), including the antiviral RNase L (22
). Rates of RNA degradation may increase in templates rich in UpA and decrease in homologous templates rich in CpG (17
). The differing biochemical mechanisms underlying CpG and UpA suppression may shape the strategy for optimal design of codon-deoptimized virus constructs.
As previously suggested (8
), codon deoptimization may offer a systematic general approach to the development of RNA virus vaccines with very high genetic stabilities. Moreover, because all substitutions are synonymous, surface antigens remain unaltered. The basic strategy is to modulate fitness along a shallow fitness gradient, thereby maximizing the number of substitutions contributing to the desired phenotypes. If selection coefficients at individual sites are low, full phenotypic reversion would require many incremental steps occurring over many rounds of replication. The distribution of attenuating substitutions over numerous sites contrasts with the properties of the Sabin OPV strains, where the key attenuating substitutions are localized to two to six sites (5
). For poliovirus, the most immediate need is for safer IPV seed strains suitable for posteradication vaccine production in developing countries (12
). Genetic inactivation of infectivity by incorporation of CpG and UpA dinucleotides into synonymous capsid region codons may lead to improved poliovirus vaccine strains having antigenic properties identical to those of the current vaccine strains.