Identification of giant Mimivirus protein functions using RNA interference

Abstract

Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases.

Keywords: Megavirales; Mimivirus; RNA interference; fiber; giant virus; nucleocytoplasmic large DNA virus; short interfering RNA.

FIGURE 1

Flow chart of the strategy implemented to characterize Mimivirus gene function using siRNA.

FIGURE 2

Electron micrographs of control Mimivirus and Mimivirus after knocking down genes encoding fiber associated proteins (FAPs). (A) Control, (B) Control si-L425 Mimivirus (meaning Mimvirus treated with siRNA targeting the L425 gene); (C) si-R856 Mimivirus, (D) si-L725 Mimivirus, (E) si-L829 Mimivirus, and (F) si-R135 Mimivirus. The pictures were taken for virions within Acanthamoeba polyphaga host. See Supplementary Figures S1–S6 for additional figures.

FIGURE 3

Electron microscopy with immunogold targeting of Mimivirus fiber proteins using anti-fiber antibodies (1:100). (A) Positive control, (B) Positive control (view from one of the capsid vertices), (C). Negative control with only secondary, not primary, antibodies, (D) si-L425 Mimivirus (meaning Mimvirus treated with siRNA targeting the L425 gene), (E) si-L725 Mimivirus, (F) si-L829 Mimivirus, and (G) si-R856 Mimivirus. The pictures were taken for virions outside A. polyphaga host.

FIGURE 4

Western blot analyses of fibers from silenced Mimivirus and control Mimivirus. (A) Reduction in anti-Mimivirus fiber antibodies (1:1000) binding to fiber proteins of si-R135 Mimivirus and (B) Reduction in anti-fiber antibodies (1:1000) binding to fiber proteins of si-L725, si-L829, si-R856, and si-L425 Mimivirus, (C) Reduction in anti-Mimivirus antibodies (1:5000) binding to si-R135 Mimivirus, and (D) Reduction in anti-Mimivirus antibodies (1:5000) binding to si-L725, si-L829, si-R856, and si-L425 Mimivirus, (E) Reduction in anti-L725 antibodies (1:1000) binding to si-R135 Mimivirus, and (F) Reduction in anti-L725 antibodies (1:1000) binding to si-L725, si-L829, si-R856, and si-L425 Mimivirus. Figures correspond to different experiments. Ctrl indicates control Mimivirus; si- indicates the virus after silencing a target gene; molecular masses are indicated on the left; molecular masses for FAPs are as follows: L725: 27 kDa; R856: 40 kDa; L829: 50 kDa; and R135: 77 kDa.

FIGURE 5

Mimivirus protein profiles as shown by 2D-gel electrophoresis and western blot with anti-fiber antibodies (1:5000). (A) Silver stained and western blotted gel electrophoresis of Mimivirus in the absence of siRNA. (B) Silver stained and western blotted gel electrophoresis of si-L829 Mimivirus. (C) Silver stained and western blotted gel electrophoresis of si-R856 Mimivirus. The decrease in spot intensity indicates the depletion of fiber proteins. Immunoreactive protein spots are shown using arrows and the locus names; the spots were previously identified in (Renesto et al., 2006; Boyer et al., 2011).