Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga

Abstract

The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis.IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes.

Keywords: R458; gene silencing; giant virus; mimivirus; protein expression; translation.

FIG 1

A schematic representation of putative conserved domains in R458 protein. Prediction of putative conserved domains in R458 protein was performed by analysis of proteins using BLASTp search, Pfam, SMART, Phyre2, and CDD search database analyses. These analyses indicated the location nomenclature of five domains of sequence homology: the DEAD box-like helicase superfamily, superfamily II DNA and RNA helicase (SrmB), helicase conserved C terminus, ATP-dependent DNA helicase (RecQ), and P-loop NTPase superfamily.

FIG 2

Control of siRNA transfection in amoeba revealed by visualization of the green fluorescence of oligonucleotides. The siRNA fluorescence was checked at 3 h postinfection as a control for a good transfection.

FIG 3

Downregulation of R458 in mimivirus using specific siRNA and RT-PCR analysis. Agarose gel electrophoresis showed the siRNA effect on R458 expression. RT-PCR analysis was done with total RNA extracted from wild-type mimivirus and siRNA-transfected mimivirus, showing R458 downregulation at the mRNA level. Amplification of R458 was done using specific primers listed in Table 4. pb, base pairs.

FIG 4

Kinetics of mimivirus DNA replication in both wild-type and silenced mimivirus. Growth analysis of wild-type mimivirus and silenced mimivirus in amoeba-infected cells by qPCR at 0, 8, 16, and 24 h postinfection at an MOI of 0.2. The x axis shows the time points, and the y axis shows the log concentration of mimivirus DNA (all values represent means of results from three independent assays).

FIG 5

Analysis of mimivirus particle accumulation based on endpoint dilution assays demonstrating an evaluation of mimivirus multiplication. Titers were determined from supernatants of amoeba cells infected with wild-type mimivirus and silenced mimivirus at the designated time points (h 0 [H0], H8, H16, and H24) in triplicate. The x axis shows the time points, and the y axis shows the mimivirus particle accumulation.

FIG 6

The development cycles of wild-type and silenced mimivirus. The cycles were demonstrated using fluorescence microscopy and mimivirus-specific polyclonal antibodies in three replicates. At H4, during the eclipse phase, mimivirus was not detected in either wild-type or silenced mimivirus. At H7, perinuclear particles become visible only in wild-type mimivirus. At H9, the first appearance of perinuclear particles in silenced mimivirus occurred. At H14, the number of amoebae infected with mimivirus particles was found to have increased progressively.

FIG 7

Quantification of virus factories in both wild-type and silenced mimivirus by randomly selecting 1,000 infected amoeba cells using the immunodetection method. The x axis shows the time points, and the y axis shows the mimivirus factory numbers (all values represent means of results from three replicates).

FIG 8

Representative 2D differential gel electrophoresis (2D-DIGE) analysis for comparative expression proteomics in wild-type and silenced mimivirus. Each individual sample from wild-type and silenced mimivirus and a pooled reference sample were labeled using fluorescent dyes (Cy5, Cy3, and Cy2) and were then separated on the same gel using the 2D-DIGE system. Three images were obtained from each gel, and an overlay of the dye scan images was also obtained. Each scanned fluorescent image was analyzed using SameSpot analysis software. Selected protein spots exhibiting an ANOVA score lower than or close to 0.05 and a fold change value of at least 1.5 are indicated by circles and spot numbers as listed in Table 2.

FIG 9

Putative regulation of R458 mimivirus during initiation of translation. (A) Association of R458 with the noncoding RNA initiates the translation by stimulating the ATP activity of R458 and binds with a complex of other translational factors. (B) The small ribosomal subunit initiation complex scans through the 5′ untranslated region for the first AUG initiator codon to begin translation.