Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus

The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.     

The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging

The genomes of influenza A viruses consist of eight negative-strand RNA segments. Recent studies suggest that influenza viruses are able to specifically package their segmented genomes into the progeny virions. Segment-specific packaging signals of influenza virus RNAs (vRNAs) are located in the 5' and 3' noncoding regions, as well as in the terminal regions, of the open reading frames. How these packaging signals function during genome packaging remains unclear. Previously, we generated a 7-segmented virus in which the hemagglutinin (HA) and neuraminidase (NA) segments of the influenza A/Puerto Rico/8/34 virus were replaced by a chimeric influenza C virus hemagglutinin/esterase/fusion (HEF) segment carrying the HA packaging sequences. The robust growth of the HEF virus suggested that the NA segment is not required for the packaging of other segments. In this study, in order to determine the roles of the other seven segments during influenza A virus genome assembly, we continued to use this HEF virus as a tool and analyzed the effects of replacing the packaging sequences of other segments with those of the NA segment. Our results showed that deleting the packaging signals of the PB1, HA, or NS segment had no effect on the growth of the HEF virus, while growth was greatly impaired when the packaging sequence of the PB2, PA, nucleoprotein (NP), or matrix (M) segment was removed. These results indicate that the PB2, PA, NP, and M segments play a more important role than the remaining four vRNAs during the genome-packaging process.     

The Evolutionary Dynamics of Human Influenza B Virus

Despite their close phylogenetic relationship, type A and B influenza viruses exhibit major epidemiological differences in humans, with the latter both less common and less often associated with severe disease. However, it is unclear what processes determine the evolutionary dynamics of influenza B virus, and how influenza viruses A and B interact at the evolutionary scale. To address these questions we inferred the phylogenetic history of human influenza B virus using complete genome sequences for which the date (day) of isolation was available. By comparing the phylogenetic patterns of all eight viral segments we determined the occurrence of segment reassortment over a 30-year sampling period. An analysis of rates of nucleotide substitution and selection pressures revealed sporadic occurrences of adaptive evolution, most notably in the viral hemagglutinin and compatible with the action of antigenic drift, yet lower rates of overall and nonsynonymous nucleotide substitution compared to influenza A virus. Overall, these results led us to propose a model in which evolutionary changes within and between the antigenically distinct 'Yam88' and 'Vic87' lineages of influenza B virus are the result of changes in herd immunity, with reassortment continuously generating novel genetic variation. Additionally, we suggest that the interaction with influenza A virus may be central in shaping the evolutionary dynamics of influenza B virus, facilitating the shift of dominance between the Vic87 and the Yam88 lineages.     

cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.     

In vitro vRNA–vRNA interactions in the H1N1 influenza A virus genome

The genome of influenza A virus consists of eight-segmented, single-stranded, negative-sense viral RNAs (vRNAs). Each vRNA contains a central coding region that is flanked by noncoding regions. It has been shown that upon virion formation, the eight vRNAs are selectively packaged into progeny virions through segment-specific packaging signals that are located in both the terminal coding regions and adjacent noncoding regions of each vRNA. Although recent studies using next-generation sequencing suggest that multiple intersegment interactions are involved in genome packaging, contributions of the packaging signals to the intersegment interactions are not fully understood. Herein, using synthesized full-length vRNAs of H1N1 WSN (A/WSN/33 [H1N1]) virus and short vRNAs containing the packaging signal sequences, we performed in vitro RNA binding assays and identified 15 intersegment interactions among eight vRNAs, most of which were mediated by the 3′- and 5′-terminal regions. Interestingly, all eight vRNAs interacted with multiple other vRNAs, in that some bound to different vRNAs through their respective 3′- and 5′-terminal regions. These in vitro findings would be of use in future studies of in vivo vRNA–vRNA interactions during selective genome packaging.     

Mutational analyses of packaging signals in influenza virus PA, PB1, and PB2 genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3′ and 5′ termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5′ sites in the PA or PB1 coding regions but extended across the entire (80-base) 5′ coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.     

Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3' coding region are critical for efficient NS vRNA incorporation and that deletion of the 3' segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3' NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3' end) form a structure that is required for efficient influenza A virus vRNA packaging.     

A Nine-Segment Influenza A Virus Carrying Subtype H1 and H3 Hemagglutinins

Influenza virus genomic RNAs possess segment-specific packaging signals that include both noncoding regions (NCRs) and adjacent terminal coding region sequences. Using reverse genetics, an A/Puerto Rico/8/34 (A/PR/8/34) virus was rescued that contained a modified PB1 gene such that the PB1 packaging sequences were exchanged for those of the neuraminidase (NA) gene segment. To accomplish this, the PB1 open reading frame, in which the terminal packaging signals were inactivated by serial synonymous mutations, was flanked by the NA segment-specific packaging sequences including the NCRs and the coding region packaging signals. Next, the ATGs located on the 3′ end of the NA packaging sequences of the resulting PB1 chimeric segment were mutated to allow for correct translation of the full-length PB1 protein. The virus containing this chimeric PB1 segment was viable and able to stably carry a ninth, green fluorescent protein (GFP), segment flanked by PB1 packaging signals. Utilizing this method, we successfully generated an influenza virus that contained the genes coding for both the H1 hemagglutinin (HA) from A/PR/8/34 and the H3 HA from A/Hong Kong/1/68 (A/HK/1/68); both subtypes of HA protein were also incorporated into the viral envelope. Immunization of mice with this recombinant virus conferred complete protection from lethal challenge with recombinant A/PR/8/34 virus and with X31 virus that expresses the A/HK/1/68 HA and NA. Using the described methodology, we show that a ninth segment can also be incorporated by manipulation of the PB2 or PA segment-specific packaging signals. This approach offers a means of generating a bivalent influenza virus vaccine.Influenza viruses possess segmented, negative-sense RNA genomes and belong to the family of Orthomyxoviridae. Three types of influenza viruses have been identified: A, B, and C (24). Based on the two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), type A viruses are further divided into different subtypes; there are now 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) of influenza A viruses (24). Current influenza A viruses circulating in humans include the H1N1 and H3N2 subtypes.The genomes of influenza A and B viruses consist of eight RNAs, while C viruses have only seven segments. Influenza virus genomic RNAs associate with nucleoprotein (NP) and three viral polymerase subunits (PB2, PB1, and PA), to form the ribonucleoprotein (RNP) complexes within virions (24). Previous data indicated that each segment of the influenza A/WSN/33 (H1N1) virus possesses segment-specific RNA packaging signals that include both the 3′ and 5′ noncoding regions (NCRs), as well as coding sequences at the two ends of each open reading frame (ORF) (4, 5, 10, 11, 13, 15, 22, 23, 28; and see Fig. 47.23 in reference 24). In addition, an electron microscopy study showed that the wild-type influenza A virus contains exactly eight RNPs within the virions, with seven RNPs surrounding a central one (19). These results suggest that influenza virus genome packaging is a specific process, with each particle containing eight unique RNA segments. Additional evidence supporting a specific packaging theory came from studies of defective interfering (DI) RNAs which contain internal deletions in the coding sequences. These short RNAs can be incorporated into the virus particles despite the fact that they do not encode full-length functional proteins. The finding that incorporation of DI RNAs interferes with the parent full-length RNAs in a segment-specific manner (1, 16, 17) also suggests that influenza virus genome packaging is a specific process.However, there are also data arguing that influenza virus RNA packaging can be nonspecific. First, studies showed that the two different RNA segments of influenza virus can be engineered to share the same set of 3′ and 5′ NCRs, which are important components of the influenza virus RNA packaging signals (18, 31). In addition, under specific circumstances, influenza virus is able to contain nine RNA segments, in which two of them share identical NCRs and partially identical coding region sequences (2, 29). Titrations of the nine-segment virus revealed a linear relationship between dilutions and plaque numbers, suggesting an influenza virus virion can incorporate more than eight segments (2).Herein, we describe a novel approach for the generation of nine-segment influenza viruses based on the manipulation of the segment-specific packaging signals. When the packaging sequences of the PB1 (or PB2 or PA) segment were replaced by those of the NA segment, influenza A/PR/8/34 virus was able to stably incorporate a ninth segment flanked by the PB1 (or PB2 or PA) packaging signals. Using this property, we successfully generated influenza viruses encoding two full-length HA glycoproteins: a subtype H1 A/PR/8/34 HA and a subtype H3 A/HK/1/68 HA. Immunization of mice with the virus carrying two HAs protected them from the lethal challenge with either A/PR/8/34 or X31 virus, the latter of which carries the HA and NA genes of A/HK/1/68. This approach can be used to construct live attenuated influenza vaccine viruses targeting two heterologous strains.     

Intragenomic variation in non-adaptive nucleotide biases causes underestimation of selection on synonymous codon usage

Patterns of non-uniform usage of synonymous codons vary across genes in an organism and between species across all domains of life. This codon usage bias (CUB) is due to a combination of non-adaptive (e.g. mutation biases) and adaptive (e.g. natural selection for translation efficiency/accuracy) evolutionary forces. Most models quantify the effects of mutation bias and selection on CUB assuming uniform mutational and other non-adaptive forces across the genome. However, non-adaptive nucleotide biases can vary within a genome due to processes such as biased gene conversion (BGC), potentially obfuscating signals of selection on codon usage. Moreover, genome-wide estimates of non-adaptive nucleotide biases are lacking for non-model organisms. We combine an unsupervised learning method with a population genetics model of synonymous coding sequence evolution to assess the impact of intragenomic variation in non-adaptive nucleotide bias on quantification of natural selection on synonymous codon usage across 49 Saccharomycotina yeasts. We find that in the absence of a priori information, unsupervised learning can be used to identify genes evolving under different non-adaptive nucleotide biases. We find that the impact of intragenomic variation in non-adaptive nucleotide bias varies widely, even among closely-related species. We show that the overall strength and direction of translational selection can be underestimated by failing to account for intragenomic variation in non-adaptive nucleotide biases. Interestingly, genes falling into clusters identified by machine learning are also physically clustered across chromosomes. Our results indicate the need for more nuanced models of sequence evolution that systematically incorporate the effects of variable non-adaptive nucleotide biases on codon frequencies.     

Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions

A final step in the influenza virus replication cycle is the assembly of the viral structural proteins and the packaging of the eight segments of viral RNA (vRNA) into a fully infectious virion. The process by which the RNA genome is packaged efficiently remains poorly understood. In an approach to analyze how vRNA is packaged, we rescued a seven-segmented virus lacking the hemagglutinin (HA) vRNA (deltaHA virus). This virus could be passaged in cells constitutively expressing HA protein, but it was attenuated in comparison to wild-type A/WSN/33 virus. Supplementing the deltaHA virus with an artificial segment containing green fluorescent protein (GFP) or red fluorescent protein (RFP) with HA packaging regions (45 3' and 80 5' nucleotides) partially restored the growth of this virus to wild-type levels. The absence of the HA vRNA in the deltaHA virus resulted in a 40 to 60% reduction in the packaging of the PA, NP, NA, M, and NS vRNAs, as measured by quantitative PCR (qPCR), and the packaging of these vRNAs was partially restored in the presence of GFP/RFP packaging constructs. To further define nucleotides of the HA coding sequence which are important for vRNA packaging, synonymous mutations were introduced into the full-length HA cDNA of influenza A/WSN/33 and A/Puerto Rico/8/34 viruses, and mutant viruses were rescued. qPCR analysis of vRNAs packaged in these mutant viruses identified a key region of the open reading frame (nucleotides 1659 to 1671) that is critical for the efficient packaging of an influenza virus H1 HA segment.     

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.     

Bridging two scholarly islands enriches both: COI DNA barcodes for species identification versus human mitochondrial variation for the study of migrations and pathologies

DNA barcodes for species identification and the analysis of human mitochondrial variation have developed as independent fields even though both are based on sequences from animal mitochondria. This study finds questions within each field that can be addressed by reference to the other. DNA barcodes are based on a 648‐bp segment of the mitochondrially encoded cytochrome oxidase I. From most species, this segment is the only sequence available. It is impossible to know whether it fairly represents overall mitochondrial variation. For modern humans, the entire mitochondrial genome is available from thousands of healthy individuals. SNPs in the human mitochondrial genome are evenly distributed across all protein‐encoding regions arguing that COI DNA barcode is representative. Barcode variation among related species is largely based on synonymous codons. Data on human mitochondrial variation support the interpretation that most – possibly all – synonymous substitutions in mitochondria are selectively neutral. DNA barcodes confirm reports of a low variance in modern humans compared to nonhuman primates. In addition, DNA barcodes allow the comparison of modern human variance to many other extant animal species. Birds are a well‐curated group in which DNA barcodes are coupled with census and geographic data. Putting modern human variation in the context of intraspecies variation among birds shows humans to be a single breeding population of average variance.     

Concerted evolution between duplicated genetic elements in Helicobacter pylori

The Helicobacter pylori genome includes a family of outer membrane proteins (OMPs) with substantial N and C-terminal identity. To better understand their evolution, the nucleotide sequences for two members, babA and babB, were determined from a worldwide group of 23 strains. The geographic origin of each strain was found to be the major determinant of phylogenetic structure, with strains of Eastern and Western origin showing greatest divergence. For strains 96-10 (Japan) and 96-74 (USA), the 5' regions of babB are replaced with babA sequences, demonstrating that recombination occurs between the two loci. babA and babB have nearly equivalent variation in nucleotide and amino acid identity, and frequencies of synonymous and non-synonymous substitutions. Both genes have segmental conservation but within the 3' segment, substitution patterns are nearly identical. Although babA and babB 5' and midregion segment phylogenies show strong interstrain similarity, the 3' segments show strong intrastrain similarity, indicative of concerted evolution. Within these 3' segments, the lower intrastrain than interstrain frequencies of nucleotide substitutions, which are below mean background H. pylori substitution frequencies, indicate selection against intrastrain diversification. Since babA/babB gene conversions likely underlie the concerted evolution of the 3' segments, in an experimental system, we demonstrate that gene conversions can frequently (10(-3)) occur in H. pylori. That these events are recA-dependent and DNase-resistant indicates their likely cause is intragenomic recombination.     

An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus

The genome of influenza A viruses (IAV) is split into eight viral RNAs (vRNAs) that are encapsidated as viral ribonucleoproteins. The existence of a segment-specific packaging mechanism is well established, but the molecular basis of this mechanism remains to be deciphered. Selective packaging could be mediated by direct interaction between the vRNA packaging regions, but such interactions have never been demonstrated in virions. Recently, we showed that the eight vRNAs of a human H3N2 IAV form a single interaction network in vitro that involves regions of the vRNAs known to contain packaging signals in the case of H1N1 IAV strains. Here, we show that the eight vRNAs of an avian H5N2 IAV also form a single network of interactions in vitro, but, interestingly, the interactions and the regions of the vRNAs they involve differ from those described for the human H3N2 virus. We identified the vRNA sequences involved in five of these interactions at the nucleotide level, and in two cases, we validated the existence of the interaction using compensatory mutations in the interacting sequences. Electron tomography also revealed significant differences in the interactions taking place between viral ribonucleoproteins in H5N2 and H3N2 virions, despite their canonical ‘7 + 1’ arrangement.     

One-Step Lipophilization of Proteins Using Fatty Acyl Anhydride

Nucleotide sequences of mRNAs were compared between major calcium-sensitive caseins of cow (α_s1-casein) and rat (α-casein). A best fit alignment of the two sequences showed homology of 81% and 69% for the 5′- and 3′-untranslated regions, respectively. Homology in the comparable coding region of the mature asl-casein (76% of total codons) was remarkably lower at amino acid level (46%) than at nucleotide level (69%). The low conservation at amino acid level is explained by the unusual nucleotide substitution pattern (random at all three positions of codons) in contrast to synonymous substitutions at the third position revealed on comparison of other related proteins. The evolutionary distances among the number of the casein family were estimated by comparing known nucleotide sequences of the signal peptides which were the most conserved coding regions in the family. The divergence time for most distantly related caseins (both rat α-casein/rat β-casein and rat α-casein/mouse ε-casein) was estimated to be about 170 million years.     

A seven-segmented influenza A virus expressing the influenza C virus glycoprotein HEF

Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.     

RNA Sequence Features Are at the Core of Influenza A Virus Genome Packaging

The influenza A virus (IAV), a respiratory pathogen for humans, poses serious medical and economic challenges to global healthcare systems. The IAV genome, consisting of eight single-stranded viral RNA segments, is incorporated into virions by a complex process known as genome packaging. Specific RNA sequences within the viral RNA segments serve as signals that are necessary for genome packaging. Although efficient packaging is a prerequisite for viral infectivity, many of the mechanistic details about this process are still missing. In this review, we discuss the recent advances toward the understanding of IAV genome packaging and focus on the RNA features that play a role in this process.     

A likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome

A model of DNA sequence evolution applicable to coding regions is presented. This represents the first evolutionary model that accounts for dependencies among nucleotides within a codon. The model uses the codon, as opposed to the nucleotide, as the unit of evolution, and is parameterized in terms of synonymous and nonsynonymous nucleotide substitution rates. One of the model's advantages over those used in methods for estimating synonymous and nonsynonymous substitution rates is that it completely corrects for multiple hits at a codon, rather than taking a parsimony approach and considering only pathways of minimum change between homologous codons. Likelihood-ratio versions of the relative-rate test are constructed and applied to data from the complete chloroplast DNA sequences of Oryza sativa, Nicotiana tabacum, and Marchantia polymorpha. Results of these tests confirm previous findings that substitution rates in the chloroplast genome are subject to both lineage-specific and locus-specific effects. Additionally, the new tests suggest tha the rate heterogeneity is due primarily to differences in nonsynonymous substitution rates. Simulations help confirm previous suggestions that silent sites are saturated, leaving no evidence of heterogeneity in synonymous substitution rates.