Faustovirus E12 Transcriptome Analysis Reveals Complex Splicing in Capsid Gene

Abstract

Faustoviruses are the first giant viruses of amoebae isolated on Vermamoeba vermiformis. They are distantly related to African swine fever virus, the causative agent of lethal hemorrhagic fever in domestic pigs. Structural studies have shown the presence of a double protein layer encapsidating the double-stranded DNA genome of Faustovirus E12, the prototype strain. The major capsid protein (MCP) forming the external layer has been shown to be 645-amino acid-long. Unexpectedly, its encoding sequence has been found to be scattered along a 17 kbp-large genomic region. Using RNA-seq, we studied expression of Faustovirus E12 genes at nine time points over its entire replicative cycle. Paired-end 250 bp-long read sequencing on MiSeq instrument and double-round spliced alignment enabled the identification of 26 different splice-junctions. Reads corresponding to junctions represented 2% of mapped reads and mostly matched with the predicted MCP encoding sequences. Moreover, our study enabled describing a 1,939 bp-long transcript that corresponds to the MCP, delineating 13 exons. At least two types of introns coexist in the MCP gene: group I introns that can self-splice (n = 5) and spliceosome-like introns with non-canonical splice sites (n = 7). All splice-sites were non-canonical with five types of donor/acceptor splice-sites among which AA/TG was the most frequent association.

Comparative Genomics Unveils Regionalized Evolution of the Faustovirus Genomes

“…Furthermore, we found between 12 and 17 introns in the MCP gene (see below), the size of which ranged from 343 to 4635 bp. We also found a single intron of 452 to 507 bp in the DNA-directed RNA polymerase subunit 1 gene of FVs from clades E9 and D. No robust evidence of additional genes containing introns was found specifically in F-E12 by mapping previously generated transcriptome data [17], or in all strains by aligning homologs of FV proteins back to the FV genomes. The predicted proteins of all FVs were assembled into 767 orthologous protein families containing two or more members, including a core set of 282 protein families that were found present in all sequenced FVs ( Figure 1, Table S1).…”

“…A unique feature of FVs compared to other viruses is the large number of introns found in their MCP gene [7,8,17]. Overall, we identified between 13 and 18 introns in this gene across the 17 FV genomes.…”

“…Protein-coding genes were predicted with the GeneMarkS program using the -virus option [22], and potential tRNA genes were searched on the RNAscan-SE On-line server [23]. The structure of the FV MCP gene was determined manually by using the MCP protein sequence of F-E12 (whose coding sequence has been validated by transcriptomics [17]) as a query in TBLASTN searches against the contig sequences. The boundaries between exons and introns were determined based on the returned alignments.…”

“…Moreover, cryo-electron microscopy has demonstrated the existence of a double protein layer encapsulating its genome [16]. Using RNA-seq, Cherif Louazani et al [17] studied gene expression in F-E12 at nine time points over its entire replicative cycle and identified 26 putative splice-site junctions. The combination of genomic, transcriptomic and proteomic data revealed the unique structure of the F-E12 gene encoding the major capsid protein (MCP), one of the most ubiquitously conserved core proteins among NCLDVs [18].…”

“…The combination of genomic, transcriptomic and proteomic data revealed the unique structure of the F-E12 gene encoding the major capsid protein (MCP), one of the most ubiquitously conserved core proteins among NCLDVs [18]. This MCP gene extended over 17 Kb and was riddled with 13 introns [7,16,17], whereas this gene is generally lacking introns in other NCLDVs, excepted in KV [10]. The origin and biological significance of the invasion of introns in the MCP gene of FVs and KV remain unknown.…”