A New Method to Obtain the Complete Genome Sequence of Multiple-Component Circular ssDNA Viruses by Transcriptome Analysis

Abstract

Circular single-stranded DNA (ssDNA) viruses are widely distributed globally, infecting diverse hosts ranging from bacteria, archaea, and eukaryotes. Among these, the genome of Banana bunchy top virus (BBTV) comprises at least six circular, ssDNA components that are ∼1 kb in length. Its genome is usually amplified and obtained at the DNA level. However, RNA-based techniques to obtain the genome sequence of such multi-component viruses have not been reported. In this study, transcriptome sequencing analysis showed that the full-length of BBTV each genomic component was transcribed into viral mRNA (vmRNA). Accordingly, the near-complete genome of BBTV B2 isolate was assembled using transcriptome sequencing data from virus-infected banana leaves. Assembly analysis of BBTV-derived reads indicated that the full-length sequences were obtained for DNA-R, DNA-U3, DNA-S, DNA-M, DNA-N, NewS2, and Sat4 components, while two gaps (73 and 25 nt) missing in the DNA-C component which was further filled by reverse transcription-PCR (RT-PCR). The RT-qPCR analysis indicated that the vmRNA levels of coding regions were 3.19-103.53 folds higher than those of non-coding regions, implying that the integrity of genome assembly depended on the transcription level of non-coding region. In conclusion, this study proposes a new approach to obtain the genome of nanovirids, and provides insights for studying the transcriptional mechanism of the family Nanoviridae, Genomoviridae, and Geminiviridae.

Keywords: DNA virus; genome assembly; multi-component; nanovirids; transcriptomic sequencing.

FIGURE 1

BBTV transcripts from the transcriptome sequencing data and mapping profiles of three representative components in BBTV B2 isolate. BBTV-derived reads and mapping profiles for DNA-U3 (A), DNA-M (B), and DNA-C (C). The transcriptional reads were assembled by CodonCode Aligner version 3.7. Gap1 and gap2 represent nucleotide sequences with uncoverage regions.

FIGURE 2

Phylogenetic tree based on the concatenated amino acid sequences of BBTV Rep and CP. Evolutionary analyses of BBTV B2 and BBTV isolates from other parts of the world were conducted in MEGA6. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 15 amino acid sequences (Supplementary Table 2). All positions containing gaps and missing data were eliminated. There were a total of 424 positions in the final dataset. ABTV was used as an outgroup.

FIGURE 3

Detection of DNA-R full-length transcripts using RT-PCR. The schematic map of DNA-R component and primers localization (A). Detection of DNA-R full-length transcripts using RT-PCR (B). Lane M: DL2000 DNA marker; Amplification of DNA-R from total DNA (Lane 1), total RNA (Lane 2), cDNA (Lane 3) using R-F1/R-R1; and Amplification of DNA-R from total DNA (Lane 4), total RNA (Lane 5), and cDNA (Lane 6) using R-F2/R-R2.

FIGURE 4

Transcriptional levels of coding and non-coding regions of BBTV each component. The transcriptional levels of non-coding and coding regions of DNA-R (A), DNA-U3 (B), DNA-S (C), DNA-M (D), DNA-C (E), DNA-N (F), NewS2 (G), and Sat4 (H) by RT-qPCR analysis. Student’s t-test was used to evaluate the differences.