Nucleotide composition of the Zika virus RNA genome and its codon usage
© The Author(s). 2016
Received: 1 March 2016
Accepted: 26 May 2016
Published: 8 June 2016
RNA viruses have genomes with a distinct nucleotide composition and codon usage. We present the global characteristics of the RNA genome of Zika virus (ZIKV), an emerging pathogen within the Flavivirus genus. ZIKV was first isolated in 1947 in Uganda, caused a widespread epidemic in South and Central America and the Caribbean in 2015 and has recently been associated with microcephaly in newborns.
The nearly 11 kb positive-stranded RNA genome of ZIKV was analyzed for its nucleotide composition, also in the context of the folded RNA molecule. Nucleotide trends were investigated along the genome length by skew analyses and we analyzed the codons used for translation of the ZIKV proteins.
ZIKV RNA has a biased nucleotide composition in being purine-rich and pyrimidine-poor. This preference for purines is a general characteristic of the mosquito-borne and tick-borne flaviviruses. The virus-specific nucleotide bias is further enriched in the unpaired, single-stranded regions of the structured ZIKV RNA genome, thus further imposing this ZIKV-specific signature. The codons used for translation of the ZIKV proteins is also unusual, but we show that it is the underlying bias in nucleotide composition of the viral RNA that largely dictates these codon preferences.
The ZIKV RNA genome has a biased nucleotide composition that dictates the codon usage of this flavivirus. We discuss the evolutionary scenarios and molecular mechanisms that may be responsible for these distinctive ZIKV RNA genome features.
Several members of the Flavivirus genus are the causative agents of significant diseases in humans, livestock and wildlife. These include dengue virus that affects more than 50 million people worldwide each year, West Nile virus and Japanese encephalitis virus that caused outbreaks in North America and Asia, respectively . Zika virus (ZIKV) is an emerging pathogen in the family Flaviviridae that was first isolated in 1947 from a sentinel rhesus monkey placed in the Zika Forest near Lake Victoria in Uganda . ZIKV is transmitted by mosquitoes, especially Aedes africanus, but the virus was also isolated from other Aedes species (reviewed in ). ZIKV infections of humans was first described in 1964 , causing a febrile illness with dengue fever like symptoms . Sporadic cases were reported in sub-Saharan Africa and Southeast Asia, followed by an outbreak in Micronesia in 2007 and major epidemics that started around 2013 in New Caledonia, the Cook Islands, French Polynesia and Easter Island [6, 7]. A rather dramatic increase in the number of ZIKV cases was reported from the Americas starting in 2015, Brazil being the most affected country with around 1 million cases reported at the end of 2015 [8–10]. Here, also cases of neurological manifestations and the Guillain-Barré syndrome were described. Recent reports indicate a significant increase in the number of microcephaly cases among newborns in northeast Brazil, suggesting that ZIKV infection in pregnancy may trigger fetal malformations . Neural progenitor cells can be infected by this virus, leading to attenuation of their growth .
Given the clinical relevance, we performed a detailed analysis of several features of the ZIKV RNA genome, including the nucleotide (nt) composition, also in the context of the structured RNA genome, and the viral codon usage. This insight can be central to the understanding of factors that govern virus evolution. Mutation pressure has been shown to be the dominant factor shaping the nucleotide composition and codon usage in mammalian genomes [13–15]. The ZIKV genome of almost 11,000 nts encodes a single polyprotein of 3419 amino acids that is cleaved by the viral serine and cellular furin proteases into the functional domains: the Capsid (C), Precursor of membrane (prM), Envelope (E) and 7 non-structural proteins (NS) . We report that the nucleotide composition of the ZIKV virus genome is strongly biased and this bias directly influences the codons used for translation of the viral proteins.
Viral RNA genome sequences were obtained from GenBank. The MR-766 prototype ZIKV strain originates from the index case: a monkey infected in 1947 in Uganda (Genbank entry NC_012532). Other ZIKV isolates used: KU497555 (Brazil), KU509998 (Haiti), KU501215 (Puerto Rico), KU312312 (Suriname), KU647676 (Martinique), KJ776791 (French Polynesia), KU701217 (Guatamala), KU681082 (Philippines) and KF268950 (Central African Republic). The full genome sequences were manually curated into bona fide open reading frames.
Maximum Likelihood (ML) phylogenetic analysis
Phylogenetic and molecular evolutionary analyses were conducted with MEGA v6 . The open reading frames (ORFs) of the different ZIKV strains were translated into amino acid sequences, which were aligned by means of the MUSCLE tool. The JTT + G model for assessing amino acid replacements during ZIKV evolution turned out to be the best fitting model judged by BIC score (Bayesian Information Criterion, 22469.52863) and AICc value (corrected Akaike Information Criterion, 22317.61634). Non-uniformity of evolutionary rates among sites was modeled by a discrete Gamma distribution (+G = 0.554328239) with 5 rate categories. The ML value for model selection was logL = −11140.79817. All sites were used for phylogenetic analysis. A bootstrap test (1000 replicates) indicated robustness of the analysis. The hypothetical ancestral sequence (MRCA, Most Recent Common Ancestor) was constructed by means of the FastML server with the advanced options activated . The MLtree was rooted on the MRCA branch to show the evolutionary course of events.
RNA structure prediction
RNA secondary structure prediction was performed by the MFold v3.6 algorithm with default settings . The MFold output file provided the ss-count, a frequency value that indicates whether an individual nucleotide (nt) is unpaired in the collection of folded structures (maximally 50). We scored an unpaired nt (single-stranded, "ss") if present in at least half of the RNA structures. Nts with a lower ss-count were scored double-stranded ("ds"). Excel was used for ss/ds discrimination and we generated fasta files to determine the nucleotide composition in MEGA v6 . Because the size limit for submission to the MFold server is 9000 nts, the ZIKV RNA genome was partitioned into two fragments with 1000 nts overlap. We arithmetically averaged the ss-count data in the overlap before ss/ds discrimination was performed.
Base composition along the complete RNA genome length and the accompanying ss and ds fasta files was analyzed by cumulative skew diagrams using overlapping windows [19, 20]. Overlapping windows were defined around 1 % of the sequence length with a step size of 20 % of the window size, which generated about 500 data points per analysis irrespective of sequence length. A skew between nts N1 and N2 is defined as (N1 − N2)/(N1 + N2). A positive value indicates that N1 exceeds N2.
The single ZIKV reading frame was analyzed using the “Nc-plot”, which plots the effective number of codons (ENC-values) versus the GC-content at the 3rd codon position (GC3) . A continuous line indicates ENC values expected (ENCexp) for random codon usage at that particular GC3 value. Deviation from this line in the direction of lower ENC-values (observed ENC values, ENCobs) points to the selection of a preferred set of codons as described for highly expressed genes in yeast  and Escherichia coli . The ratio ENCobs/ENCexp provides an easy measure of this deviation. A ratio value of 1 indicates zero codon bias. Values close to 1 (0.8 to 1.0) indicate very weak or virtually absent codon bias. ENC and GC3 values of sequence data were determined by means of Simmonic 2005 v1.5 software . ENC and GC3 values human and Aedes genes were derived from codon usage tables . All calculations were performed in Excel v14.0.7128.5000.
Nucleotide composition of the ZIKV RNA
ZIKV RNA composition
2991 (27.7 %)
2305 (21.3 %)
2359 (21.9 %)
3139 (29.1 %)
5’ + 3’UTR
142 (26.6 %)
103 (19.3 %)
134 (25.1 %)
155 (29.0 %)
This nt bias is likely to influence the codon usage in the single ORF that encodes the ZIKV polyprotein, but in fact the same nt trends are apparent for segments of the RNA genome that are not translated into protein: the short 5’-untranslated region (5’UTR) and 3’UTR of the ZIKV genome (Table 1). These regions are very short, 106 and 428 nts respectively for the MR-766 prototype genome, making a statistically sound analysis of the nt-count precarious, but when combined these domains exhibit exactly the same nt ranking order as the full-length RNA: G (29.0 %) > A (26.6 %) > C (25.1 %) > U (19.3 %). Local fluctuations may occur especially near the 5’ and 3’ termini of the genome due to the presence of essential sequence elements that are involved in viral genome replication. For instance, the U-count of the 5’UTR is elevated, in part due to the presence of four U3 stretches in this 106-nt segment. Conservation of these specific molecular signals among different ZIKV strains argues for such a biological role, e.g. the 107-nts 5’UTR of the Natal RGN isolate encodes three U3 and two U4 stretches. In addition, the genome ends may encode specific RNA structures with a replicative function .
Composition of the structured RNA genome
Nucleotide composition in ss/ds segments
2991 (27.7 %)
2305 (21.3 %)
2359 (21.9 %)
3139 (29.1 %)
1154 (17.2 %)
1604 (24.0 %)
1758 (26.3 %)
2171 (32.5 %)
1837 (44.7 %)
701 (17.1 %)
601 (14.6 %)
968 (23.6 %)
Genome skew analysis
We subsequently analyzed the ds and ss positions separately (Fig. 2, ds in middle panel, ss in right panel). The skew lines show more divergence in both the ds and ss segments compared with the all-nt skew analysis. It is also immediately apparent that the ds and ss positions act as communicating vessels. The gain of the A-count in the ss segment (strongly declining lines for GA, UA and especially CA) is mirrored by a loss in the ds compartment (strongly rising line for UA, CA and GA). G seems the second best option in the ss compartment (declining line for UG and CG). In fact, these trends do not mimic any of the virus-specific trends that we described previously, e.g. for retroviruses and coronaviruses [20, 33–35].
ZIKV codon usage
C > A > U > G
C > U
A > C > U > G
U > C
C > U
G = A
G > A
A > G
C = U
C > G > U > A
A > G > C > U
C = U
C > U
A > U > C > G
A > C > U > G
G > A
G > A
G > C > U > A
G > C > U > A
C > U
A second dinucleotide motif that is discriminated against is UpA with an ApU/UpA ratio of 1.86. This explains most cases where A-ending codons are losing (marked in bold and by underling in Table 3), e.g. within the Leu 6-codon set it clarifies the choices made for both the 2-codon and 4-codon sets. The complete dinucleotide analysis (not shown) indicates that the all purine (GpA/ApG) and all pyrimidine (CpU/UpC) choices are unbiased compared to the four purine/pyrimidine combinations (results not shown). Both CpG and UpA discrimination have been reported for other flaviviruses [39, 40], but surprisingly little attention was given to the purine/pyrimidine composition as the potential unifying signature.
Overall, the purines G or A seem to dominate the codon choices made, and U/C choices seem relatively balanced. The G-bias is also apparent in the 2-codon groups (G/A column in Table 3), where G wins over A in 3 of 5 case, with a draw for the Gln group and a unique A-win for the 2-codon set within the outlier Arg 6-codon group. Only modest effects were scored for the U/C choice in the 2-codon groups, but C wins in 4 out of 7 cases with two draws, consistent with the overall nt count. Thus, codon usage in ZIKV RNA seems to follow the nt compositional trend with regard to the purine/pyrimidine bias that is present across the viral genome.
This survey of the ZIKV RNA genome indicates a preference for purines over pyrimidines that is enhanced in the unpaired domains of the viral RNA and that influences the codons used for translation of the viral proteins. No significant differences were found among the highly related ZIKV strains concerning these basic properties, ranging from the prototype Uganda strain isolated in 1947 to recent south American isolates. The strong correlation between nucleotide composition and codon usage bias suggests that mutation pressure in ZIKV is an important determinant of the codon bias observed. This finding is consistent with previous findings for other viruses, which demonstrate a wide variation in codon usage that usually correlates with the viral RNA-specific nucleotide composition . The ZIKV nt characteristics are quite distinctive from those of other viruses and may thus help to comprehend virus evolution and to provide an additional tool for virus classification purposes or the development of diagnostic reagents for improved surveillance of this class of emerging pathogens. A quick comparison of ZIKV to other members of the family Flaviviridae, including the major pathogens dengue virus , yellow fever virus, Japanese encephalitis virus, West Nile virus  and hepatitis C virus, indicated a similar purine-preference for the mosquito-borne and tick-borne flaviviruses and the pestivirus genus, but not for the hepacivirus group (results not shown). Intriguingly, these purine-loving viruses seem to favor either A or G, similar to the “communicating” pyrimidine vessels described for coronaviruses . Although quite extensive codon analyses have been conducted for flaviviruses [42–50], as far as we know the typical purine/pyrimidine pattern has not yet been described previously.
We previously presented two possible causes for the presence of viral RNA genomes with a biased nt-composition . One frequently entertained possibility is that this is due – over evolutionary times - to mutational bias of the viral polymerase . Alternatively, we suggested a specific genome composition may be selected for a specific function, e.g. to facilitate RNA packaging in the virion particle or to prevent recognition by innate immune sensors in the infected cell. For HIV-1, there are recent indications to support both functional scenario’s [30, 53]. For ZIKV, one cannot formally exclude that functions are executed by the viral minus-RNA strand, the critical replication intermediate that in terms of nt-composition is the mirror image of the viral plus-strand RNA genome. In addition, the double-stranded RNA replication intermediate and its nt-composition and structure may be screened by several innate immune sensors in the human, monkey and Aedes hosts.
We previously reported that virus-specific compositional signatures are commonly enhanced in the unpaired domains of a structured RNA genome. This is true for HIV-1, with an average A-count of 36.2 % that increases to 47.5 % in the unpaired genome segments, and also for other retroviruses with a distinct nt bias [28, 34]. We recently analyzed coronaviruses and reported some common characteristics (high U, low C count), but also species-specific signatures that differentiate the pathogenic MERS/SARS strains from other coronaviruses . Again, nucleotide biases were boosted in the unpaired domains of the viral RNA genome. The concentration of nt-bias in certain genome domains may have been selected for a certain function.
Many reports dwell on the exotic codon usage employed by viruses. For instance, a recent report documented codon usage adaptation in pandemic ZIKV virus strains . However, a sobering finding is that this bias usually coincides with a bias in nt-composition of the viral RNA genome, and therefore does not represent translational selection of certain codons and/or the matching tRNA species. This also seems true for ZIKV, which preferentially uses A- or G-rich codons, but this seems to be a direct consequence of having a purine-rich RNA genome (56.8 %). HIV is the only virus in this collection that combines A-accumulation with weak translational selection by means of codon bias. Previously, we have even documented a tendency in HIV proteins for selection of amino acids encoded by A-rich codons . Codon usage can become an important aspect when it comes to optimization of protein production, e.g. in the context of ZIKV vaccine development where efficient viral gene expression may be required to generate immunity . Given the biased codon usage of this virus, it is important to change towards synonymous codons that are more favored in the relevant production platform, e.g. human cell lines.
We thank Alexander Pasternak and Tonja van der Kuyl for critical reading of the manuscript. RNA research in the BB laboratory is sponsored by NWO-Chemical Sciences (TOP grant).
BB conceived the study. FvH and BB participated in the study design. FvH performed the analyses. BB wrote the manuscript and FvH helped to draft the manuscript and produced all Figures and Tables. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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