Analysis of a Marseillevirus Transcriptome Reveals Temporal Gene Expression Profile and Host Transcriptional Shift

Abstract

Marseilleviruses comprise a family of large double-stranded DNA viruses belonging to the proposed order "Megavirales." These viruses have a circular genome of ∼370 kbp, coding hundreds of genes. Over a half of their genes are associated with AT-rich putative promoter motifs, which have been demonstrated to be important for gene regulation. However, the transcriptional profile of Marseilleviruses is currently unknown. Here we used RNA sequencing technology to get a general transcriptional profile of Marseilleviruses. Eight million 75-bp-long nucleotide sequences were robustly mapped to all 457 genes initially predicted for Marseillevirus isolate T19, the prototype strain of the family, and we were able to assemble 359 viral contigs using a genomeguided approach with stringent parameters. These reads were differentially mapped to the genes according to the replicative cycle time point from which they were obtained. Cluster analysis indicated the existence of three main temporal categories of gene expression, early, intermediate and late, which were validated by quantitative reverse transcription polymerase chain reaction assays targeting several genes. Genes belonging to different functional groups exhibited distinct expression levels throughout the infection cycle. We observed that the previously predicted promoter motif, AAATATTT, as well as new predicted motifs, were not specifically related to any of the temporal or functional classes of genes, suggesting that other components are involved in temporally regulating virus transcription. Moreover, the host transcription machinery is heavily altered, and many genes are down regulated, including those related to translation process. This study provides an overview of the transcriptional landscape of Marseilleviruses.

Comparative Analysis of the Circular and Highly Asymmetrical Marseilleviridae Genomes

“…We next focused on the marseillevirus virion-associated protein-coding genes to check whether they were expressed in a time-dependent manner. Confirming the results previously obtained [18], we found an enrichment in virion-associated genes in the late expression class. In addition, we also found that marseillevirus genes orthologous to melbournevirus and noumeavirus virion-associated genes were enriched in that category ( Figure S5).…”

“…Starting from the raw sequence data, we mapped the reads to the A. castellanii and marseillevirus genomes, and computed a normalized expression value for each gene (see Materials and Methods). As previously observed [18], we found the marseillevirus' genes expressions to be clustered into three main classes: early, intermediate and late (Figure S5). Early genes are expressed from the beginning of the cycle, with a peak of transcriptional activity between 1 h pi and 2 h pi, intermediate genes are mostly expressed between 1 h pi and 4 h pi, and late genes from 4 h pi until the end of the cycle.…”

“…Raw RNA-seq data from the PRJEB34467 sequencing project [18] of the A. castellanii infection by marseillevirus T19 were obtained from the SRA database. The dataset covers the marseillevirus infection cycle through 9 time points: 0 h pi (ERR3528397), 1 h pi (ERR3528398), 2 h pi (ERR3528399), 4 h pi (ERR3528400), 5 h pi (ERR3528401), 6 h pi (ERR3528402), 8 h pi (ERR3528403), 10 h pi (ERR3528404) and 12 h pi (ERR3528405).…”

“…The global transcriptome of A. castellanii infection by marseillevirus has been studied by RNA-seq through a replicative cycle [18]. We used this dataset to test whether the marseillevirus' transcriptional activity was also regionalized along the genome.…”

“…This not only revealed the proteins that build the structure of the Marseilleviridae virions, but also those packaged within it that could be essential for initiating the viral replication. In addition, the marseillevirus' transcriptional activity during an infection cycle in A. castellanii was recently surveyed by RNA sequencing (RNA-seq), showing that the host translation apparatus is downregulated during the infection [18]. This now provides us with a sufficient body of data to conduct an in-depth comparative genomics study of the Marseilleviridae family.…”