CpG Usage in RNA Viruses: Data and Hypotheses

<div><p>CpG repression in RNA viruses has been known for decades, but a reasonable explanation has not yet been proposed to explain this phenomenon. In this study, we calculated the CpG odds ratio of all RNA viruses that have available genome sequences and analyzed the correlation with their genome polarity, base composition, synonymous codon usage, phylogenetic relationship, and host. The results indicated that the viral base composition, synonymous codon usage and host selection were the dominant factors that determined the CpG bias in RNA viruses. CpG usage variation between the different viral groups was caused by different combinations of these pressures, which also differed from each other in strength. The consistent under-representation of CpG usage in −ssRNA viruses is determined predominantly by base composition, which may be a consequence of the U/A preferred mutation bias of −ssRNA viruses, whereas the CpG usage of +ssRNA viruses is affected greatly by their hosts. As a result, most +ssRNA viruses mimic their hosts' CpG usage. Unbiased CpG usage in dsRNA viruses is most likely a result of their dsRNA genome, which allows the viruses to escape from the host-driven CpG elimination pressure. CpG was under-represented in all reverse-transcribing viruses (RT viruses), suggesting that DNA methylation is an important factor affecting the CpG usage of retroviruses. However, vertebrate-infecting RT viruses may also suffer host' CpG elimination pressure that also acts on +ssRNA viruses, which results in further under-representation of CpG in the vertebrate-infecting RT viruses.</p></div

CpG usage pattern of RNA viruses within coding region.

<p>(<b>A–D</b>) Distribution of CpG at the three locations in the coding regions of −ssRNA, RT, dsRNA, and +ssRNA viruses, respectively.</p

CpG usage of RNA viruses.

<p>CpG usage of RNA viruses.</p

Pair-wised variance analysis the GC contents between viral groups.

<p>Note: <i>F</i> statistic of one-way ANOVA analysis is 33.343 (<i>P</i><0.001). The pair-wised comparisons were performed based on the GC contents of each viral group and the resulting T values of the independent <i>T</i>-test are shown. * indicates <i>P</i><0.05, ** indicates <i>P</i><0.0001.</p

CpG usage pattern of RNA viruses.

<p>The <i>y</i>-axis depicts the number of viruses with the specific CpG<i>_O/E</i> values given on the <i>x</i>-axis.</p

CpG usages of flaviviruses that infect different types of hosts.

<p>CpG usages of flaviviruses that infect different types of hosts.</p

+ssRNA viruses mimic the CpG usage of their respective host.

<p>Correlation between +ssRNA viral and host's mean CpG<i>_O/E</i> (<b>A</b>), mean CpG<i>_O/E</i>_{_CDS_12} (<b>B</b>), mean CpG<i>_O/E</i>_{_CDS_23} (<b>C</b>), and mean CpG<i>_O/E</i>_{_CDS_31} (<b>D</b>). The abbreviations at the bottom of each chart (B, F, I, P, and V) represent bacteria or bacterial-infecting +ssRNA viruses, fungi or fungus-infecting +ssRNA, invertebrates or invertebrate-infecting +ssRNA viruses, plants or plant-infecting +ssRNA viruses, and vertebrates or vertebrate-infecting +ssRNA viruses, respectively.</p

Pair-wised variance analysis CpG usage between viral groups.

<p>Note: <i>F</i> statistic of one-way ANOVA analysis is 239.252 (<i>P</i><0.001). The pair-wised comparisons were performed based on the CpG<i>_O/E</i> values of each viral group, and the resulting <i>T</i> values of the independent <i>T</i>-test are shown. ** indicates <i>P</i><0.0001. For the detailed analysis procedure, please refer to the Materials and Methods section.</p

The influence of GC content on viral CpG usage.

<p>(<b>A</b>) Correlation between CpG odds ratios and GC contents of RNA viruses. (B) CpG usage variation between the four groups of RNA viruses. CpG<i>_O/E</i> distribution range of each viral group is shown by yellow dots, and the mean CpG<i>_O/E</i> value of each viral group is indicated by the purple bar. The standard deviations of the mean CpG<i>_O/E</i> values are also indicated. (<b>C</b>) Correlation between the mean CpG<i>_O/E</i> and mean GC content values. (<b>D</b>) Correlation between the CpG<i>_O/E</i> and CpG<i>_O/E</i>_{_CDS} values.</p

HCPro2 interacts with CI and CP <i>in planta</i>.

(A) Y2H tests the interactions of HCPro2 with P3N-PIPO, CI and CP. The coding sequences of HCPro2, P3N-PIPO, CI and CP were cloned into pGBKT7-DEST or pGADT7-DEST for the expression of these proteins fused with GAL4 BD or AD domain. Yeast competent cells (Y2H Gold) were co-transformed to express the indicated pairs of proteins. The transformed cells were subjected to 10-fold serial dilutions and plated on the SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade mediums. The plates were cultured at 28°C for four to six days before photographing. Co-transformation of a pair of plasmids for simultaneous expression of AD-T7-T and BD-T7-53 was included as the positive control. (B) BiFC tests the interactions of HCPro2 with CI, CP and P3N-PIPO. The coding sequences of HCPro2, CI, CP and P3N-PIPO were individually engineered into pEarleyGate201-YN and pEarleyGate202-YC for the expression of these proteins fused with the YN or YC part of YFP. N. benthamiana leaves were co-inoculated for the expression of the indicated pairs of proteins. YFP signals (shown in green) were observed by fluorescence microscope at 72 hpi. Bars, 50 μm. (C, D) Co-IP tests the interactions of HCPro2 with CI and CP. The inoculated leaves of N. benthamiana plants for co-expression of GFP-HCPro2 / Myc-CI (C) or GFP-HCPro2 / Myc-CP (D) were sampled at 72 hpi for Co-IP assays using GFP-Trap Agarose. Total protein extracts prior to (Input) and after immunoprecipitation (IP) were analyzed by immunoblotting using anti-Myc and anti-GFP antibodies.</p