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. 2010 May;20(5):664-74.
doi: 10.1101/gr.102582.109. Epub 2010 Apr 1.

mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus

Matthieu Legendre  1 , Stéphane Audic, Olivier Poirot, Pascal Hingamp, Virginie Seltzer, Deborah Byrne, Audrey Lartigue, Magali Lescot, Alain Bernadac, Julie Poulain, Chantal Abergel, Jean-Michel Claverie
Affiliations

mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus

Matthieu Legendre et al. Genome Res. 2010 May.

Abstract

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

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Figures

Figure 1.
Figure 1.
Host versus Mimivirus reads over infection time. Relative number of reads confidently mapped on Mimivirus and A. castellanii (nuclear and mitochondrial) genomes during the infection.
Figure 2.
Figure 2.
Progression of A. castellanii infection by Mimivirus over time. (A) T = −15 min: Some virus particles are inside vacuoles in the cytoplasm (arrows). (Left inset) Virus particles are sparsely found in contact with the cytoplasmic membrane. (Right inset) Phagocytosis of a virus particle. (B) T = 0: After 30 min of incubation with a large excess of virus (multiplicity of infection = 1000), the phagocytic vacuoles contain a mixture of empty (arrowheads) and intact (arrows) virus particles (probably not contributing to the measured viral transcripts). (Inset) Several particles can be gathered in the same vacuole. (C) T = 1.5 h. No major change is observed compared with T = 0. Both empty and intact viruses are still visible. (D) T = 3 h: The early virion factory appears as a gray structure, with a fibrous-like aspect, surrounding darker areas. A circular structure (the “seed”) is visible in one of these areas. (Left inset) In some cells, the “seed” is surrounded by the fibrous-like structure only. (Right inset) Higher magnification of the “seed” surrounded by dark matter. (E) T = 6 h: The fully mature virion factory now dominates the picture. Numerous particles are budding from its surface; most capsids are still empty. (F) T = 9 h: A large number of mature (hairy + DNA) virus particles are filling the cytoplasm. New particles are still produced by the virion factory. (Inset) T = 12 h: Ultimate stage of virion production. Panels: bar = 1 μm; insets: bar = 0.2 μm. (N) Nucleus; (VF) virion factory.
Figure 3.
Figure 3.
Pairwise comparison of expression profiles between successive time points. Gene expression is measured in normalized read counts. The scatter plots show the expression of genes (blue points) between successive time points during the infection from early phases (top left) to late phases (bottom right). Linear regressions on the log-transformed values, as well as the correlation coefficients and P-values, are shown on each graph.
Figure 4.
Figure 4.
Main Mimivirus gene expression classes. (A) Heat map of Mimivirus gene expression profiles. Rows correspond to the 841 analyzed genes and columns to the seven infection time points. Expression intensities are displayed from green (low expression) to red (high expression). Expression profiles are clustered using hierarchical clustering (see Methods for details). A dendrogram of the clustering is shown on the left. (B) Heat map of the same expression profiles partitioned into three main classes, “early” (top), “intermediate” (center), and “late” (bottom), by k-means clustering algorithm (see Methods for details). (C) Presence (blue lines) of the AAAATTGA “early” promoter element in the 5′ gene regulatory region; (D) presence (purple lines) of the “late” promoter element (see main text); (E) transcripts corresponding to gene products previously identified in the virus particle proteome (black lines).
Figure 5.
Figure 5.
Variability of individual expression profiles. Normalized gene expression profiles are plotted for representative genes from the three main transcript classes (from top to bottom): genes belonging to the “early” class, the “intermediate” class, and the “late” class. The area of highest expression for each class is highlighted in gray (Fig. 4B, red). The expression data obtained from Agilent tiling array experiments are in good agreement with the RNA-seq expression profiles. The absolute number or reads for RNA-seq experiment are shown in Supplemental Fig. S8.
Figure 6.
Figure 6.
Mimivirus late promoter element in Sputnik genome. Late promoter element locations are depicted by bent arrows in intergenic (yellow and green) and coding regions (gray).
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