A Codon-Pair Bias Associated With Network Interactions in Influenza A, B, and C Genomes

A new codon-pair bias present in the genomes of different types of influenza virus is described. Codons with fewer network interactions are more frequency paired together than other codon-pairs in influenza A, B, and C genomes. A shared feature among three different influenza types suggests an evolutionary bias. Codon-pair preference can affect both speed of protein translation and RNA structure. This newly identified bias may provide insight into drivers of virus evolution.

Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria

The speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain. Peptide bond formation is especially slow with proline and two adjacent prolines can even cause ribosome stalling. While previous studies focused on how the amino acid context of a Pro-Pro motif determines the stalling strength, we extend this question to the mRNA level. Bioinformatics analysis of the Escherichia coli genome revealed significantly differing codon usage between single and consecutive prolines. We therefore developed a luminescence reporter to detect ribosome pausing in living cells, enabling us to dissect the roles of codon choice and tRNA selection as well as to explain the genome scale observations. Specifically, we found a strong selective pressure against CCC/U-C, a sequon causing ribosomal frameshifting even under wild-type conditions. On the other hand, translation efficiency as positive evolutionary driving force led to an overrepresentation of CCG. This codon is not only translated the fastest, but the corresponding prolyl-tRNA reaches almost saturating levels. By contrast, CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength. Thus, codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers.

Comparative analysis of the codon usage patterns in two closely related Marsupenaeus species based on comparative transcriptomics

Background: Marsupenaeus japonicus, a major commercial shrimp species in the world, has two cryptic or sibling species, Marsupenaeus japonicus and Marsupenaeus pulchricaudatus. Due to the lack of genomic information, little is known about the correlations among codon usage bias, gene expression, and evolutionary trends in Marsupenaeus orthologs.Results: Using the CodonW 1.4.2 software, we performed the codon bias analysis of two Marsupenaeus species transcriptomes. The average contents of GC and ENc were 51.61% and 52.1 for VI (M. japonicus), 51.54% and 52.22 for VII (M. pulchricaudatus), respectively. Parity Rule 2 (PR2) plot analysis showed that purines (A and G) were used more frequently than pyrimidines (C and T) in two Marsupenaeus species. The average ENc value was 52.1 and 52.22 for M. japonicus and M. pulchricaudatus, respectively. Overall, orthologous genes that underwent positive selection (ω > 1) had a higher correlation coefficient than that experienced purifying selection (ω < 1). In M. japonicus, the relationships were highly significant positive about Axis 1 and A3, T3 and ENc (p < 0.01). However, all relationships in M. pulchricaudatus were the opposite. We determined 12 and 14 optimal codons for M. japonicus and M. pulchricaudatus, respectively. Two Marsupenaeus species had 31 different codon pairs. The results of multi-species clustering based on codon preference were consistent with traditional classification. Conclusions: We characterized the codon usage patterns of the two Marsupenaeus species and the evolutionary trends in Marsupenaeus orthologs, which provides new insights into the genetic divergence and the phylogenetic relationships of two Marsupenaeus species.

Evidence for Strong Mutation Bias toward, and Selection against, U Content in SARS-CoV-2: Implications for Vaccine Design

Large-scale re-engineering of synonymous sites is a promising strategy to generate vaccines either through synthesis of attenuated viruses or via codon-optimized genes in DNA vaccines. Attenuation typically relies on deoptimization of codon pairs and maximization of CpG dinucleotide frequencies. So as to formulate evolutionarily informed attenuation strategies that aim to force nucleotide usage against the direction favored by selection, here, we examine available whole-genome sequences of SARS-CoV-2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias toward U. In turn, analysis of observed synonymous site composition implicates selection against U. Accounting for dinucleotide effects reinforces this conclusion, observed UU content being a quarter of that expected under neutrality. Possible mechanisms of selection against U mutations include selection for higher expression, for high mRNA stability or lower immunogenicity of viral genes. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV-2 genes. We propose an evolutionarily informed approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.

Deoptimized influenza virus for treatment of TNBC and induction of anticancer immunity in the EMT6 mouse model.

e13109 Background: Triple negative breast cancer (TNBC) is resistant to classical hormone-based therapy, prone to relapse and aggressive metastasis. Oncolytic viruses (OVs) when injected into the tumor, can promote local lysis of tumor cells and drive presentation of tumor antigens, thereby making tumors visible to the immune system. CodaLytic is a re-coded influenza A virus that has a high frequency of disfavored codons and codon pairs which attenuate the virus yet preserve all antigens and immune stimulation properties of the wild type influenza virus. CodaLytic is produced from the same master virus seed as CodaVax, a universal influenza vaccine candidate currently in Phase I clinical trials under a US IND. Methods: BALB/c mice were implanted with EMT6 mouse TNBC cells into a mammary fat pad. Six days post-implantation, when tumors became palpable, treatment was initiated by intratumoral administration of PBS (mock) or CodaLytic (108 PFU in 50 µL). Mice were treated three times a week for a total of three weeks. Tumor growth was monitored daily for 33 days and animals were euthanized if the tumor volume exceeded 500 mm3. Survivors were challenged via flank or intravenous (IV) injection of EMT6 cells. Two weeks after IV challenge, lungs were removed and compared to the lungs of control mice. Results: At the end of the initial experiment (day 33), 80% of CodaLytic-treated mice were alive with 70% of survivors being tumor-free as compared to mock, of which none survived. Survivors on day 33 had a median decrease in tumor size of 75%. CodaLytic stimulated lasting anti-tumor immunity as 100% of CodaLytic-treated survivors failed to establish tumors following challenge with EMT6 cells via flank injection. To model metastasis, surviving treated mice were also challenged with EMT6 cells delivered 2x104 cells IV. IV challenged mice had over 10-fold fewer nodules in their lungs as compared to naïve controls and 40% of the animals remained tumor free, whereas 100% of the control animals developed tumors. Additionally, CodaLytic-treated survivors showed no weight loss after IV re-challenge compared to controls which lost an average of 12% body mass. Conclusions: CodaLytic OV treatment of TNBC increased survival, led to complete tumor clearance in the EMT6 mouse model, and induced systemic anti-tumor immunity that has potential for preventing and treating metastatic disease.

Evidence for strong mutation bias towards, and selection against, T/U content in SARS-CoV2: implications for attenuated vaccine design

ABSTRACTLarge-scale re-engineering of synonymous sites is a promising strategy to generate attenuated viruses for vaccines. Attenuation typically relies on de-optimisation of codon pairs and maximization of CpG dinculeotide frequencies. So as to formulate evolutionarily-informed attenuation strategies, that aim to force nucleotide usage against the estimated direction favoured by selection, here we examine available whole-genome sequences of SARS-CoV2 to infer patterns of mutation and selection on synonymous sites. Analysis of mutational profiles indicates a strong mutation bias towards T with concomitant selection against T. Accounting for dinucleotide effects reinforces this conclusion, observed TT content being a quarter of that expected under neutrality. A significantly different mutational profile at CDS sites that are not 4-fold degenerate is consistent with contemporaneous selection against T mutations more widely. Although selection against CpG dinucleotides is expected to drive synonymous site G+C content below mutational equilibrium, observed G+C content is slightly above equilibrium, possibly because of selection for higher expression. Consistent with gene-specific selection against CpG dinucleotides, we observe systematic differences of CpG content between SARS-CoV2 genes. We propose an evolutionarily informed gene-bespoke approach to attenuation that, unusually, seeks to increase usage of the already most common synonymous codons. Comparable analysis of H1N1 and Ebola finds that GC3 deviated from neutral equilibrium is not a universal feature, cautioning against generalization of results.

Optimization of the Codon Pair Usage of Human Respiratory Syncytial Virus Paradoxically Resulted in Reduced Viral Replication In Vivo and Reduced Immunogenicity

ABSTRACT We subjected various open reading frames (ORFs) in the genome of respiratory syncytial virus (RSV) to codon pair optimization (CPO) by increasing the content of codon pairs that are overrepresented in the human genome without changing overall codon usage and amino acid sequences. CPO has the potential to increase the expression of the encoded protein(s). Four viruses were made: Max A (with CPO of NS1, NS2, N, P, M, and SH ORFs), Max B (with CPO of G and F), Max L (with CPO of L), and Max FLC (with CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon deletion mutation (Δ1313) and a missense mutation (I1314L) in the L polymerase. CPO had no effect on multicycle virus replication in vitro, temperature sensitivity, or specific infectivity. Max A and L, which in common had CPO of one or more ORFs of proteins of the polymerase complex, exhibited global increases in viral protein synthesis. Max B alone exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro. All CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response. IMPORTANCE Using computer algorithms and large-scale DNA synthesis, one or more ORFs of a microbial pathogen can be recoded by different strategies that involve the introduction of up to thousands of nucleotide changes without affecting amino acid coding. This approach has been used mostly to generate deoptimized viruses used as vaccine candidates. However, the effects of the converse approach of generating optimized viruses are still largely unknown. Here, various ORFs in the genome of respiratory syncytial virus (RSV) were codon pair optimized (CPO) by increasing the content of codon pairs that are overrepresented in the human genome. CPO did not affect RSV replication in multicycle replication experiments in vitro. However, replication was marginally reduced in two rodents models. In hamsters, CPO RSVs induced lower levels of serum RSV-neutralizing antibodies. Thus, CPO of an RNA virus for a mammalian host has paradoxical effects on virus replication and the adaptive humoral immune response.

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination ofin vitrobiochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use anin vitroreconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

Codon Pairs are Phylogenetically Conserved: Codon pairing as a new class of phylogenetic characters

Identical codon pairing and co-tRNA codon pairing increase translational efficiency within genes when two codons that encode the same amino acid are located within a ribosomal window. By examining both identical and co-tRNA codon pairing across 23 423 species, we determined that both pairing techniques are phylogenetically informative across all domains of life using either an alignment-free or parsimony framework. We also determined that conserved codon pairing typically has a smaller window size than the length of a ribosome. We also analyzed frequencies of codon pairing for each codon to determine which codons are most likely to pair. The alignment-free method does not require orthologous gene annotations and recovers species relationships that are comparable to other alignment-free techniques. Parsimony generally recovers phylogenies that are more congruent with the established phylogenies than the alignment-free method. However, four of the ten taxonomic groups do not have sufficient ortholog annotations and are therefore recoverable using only the alignment-free methods. Since the recovered phylogenies using only codon pairing largely match established phylogenies and are comparable to other algorithms, we propose that codon pairing biases are phylogenetically conserved and should be considered in conjunction with current techniques in future phylogenomic studies. Furthermore, the phylogenetic conservation of codon pairing indicates that codon pairing plays a greater role in the speciation process than previously acknowledged.AvailabilityAll scripts used to recover and compare phylogenies, including documentation and test files, are freely available on GitHub at https://github.com/ridgelab/codon_pairing.