Modulation of the expression of mimivirus-encoded translation-related genes in response to nutrient availability during Acanthamoeba castellanii infection

Abstract

The complexity of giant virus genomes is intriguing, especially the presence of genes encoding components of the protein translation machinery such as transfer RNAs and aminoacyl-tRNA-synthetases; these features are uncommon among other viruses. Although orthologs of these genes are codified by their hosts, one can hypothesize that having these translation-related genes might represent a gain of fitness during infection. Therefore, the aim of this study was to evaluate the expression of translation-related genes by mimivirus during infection of Acanthamoeba castellanii under different nutritional conditions. In silico analysis of amino acid usage revealed remarkable differences between the mimivirus isolates and the A. castellanii host. Relative expression analysis by quantitative PCR revealed that mimivirus was able to modulate the expression of eight viral translation-related genes according to the amoebal growth condition, with a higher induction of gene expression under starvation. Some mimivirus isolates presented differences in translation-related gene expression; notably, polymorphisms in the promoter regions correlated with these differences. Two mimivirus isolates did not encode the tryptophanyl-tRNA in their genomes, which may be linked with low conservation pressure based on amino acid usage analysis. Taken together, our data suggest that mimivirus can modulate the expression of translation-related genes in response to nutrient availability in the host cell, allowing the mimivirus to adapt to different hosts growing under different nutritional conditions.

Keywords: aminoacyl-tRNA-synthetases; gene expression; mimivirus; tRNA; translation.

FIGURE 1

Comparative amino acid usage analysis of Acanthamoeba castellanii and mimivirus strains. The amino acid usage for protein sequences of Brazilian mimivirus strains, A. polyphaga mimivirus (APMV) and APMV M4 strains, and the A. castellanii amoeba were calculated using the CGUA (General Codon Usage Analysis) tool.

FIGURE 2

tRNA expression by different mimivirus strains. Cells of A. castellanii were plated in 24-well plates (1 × 10⁵ cells per well), infected for 8 h with different mimivirus strains in different culture medium, subjected to total RNA extraction, and reverse transcription and the resulting cDNA was used as a template for quantitative PCR. (A) Histidyl-tRNA. (B) Cysteinyl-tRNA. (C) Leucyl-tRNA. (D) Tryptophanyl-tRNA. The quantitative PCR results were expressed in arbitrary units, fitted to a standard curve, normalized to levels of the constitutive amoebal 18S rDNA gene and presented as the mean ± SD from a representative experiment conducted in duplicate. The values were subjected in different combinations to one-way ANOVA tests and Bonferroni post-tests (95% confidence intervals). Differences between groups were considered significant when the p-values were smaller than 0.05. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 3

Aminoacyl-tRNA-synthetase (aaRS), messenger RNA (mRNA) expression by different mimivirus strains. Cells of A. castellanii were plated in 24-well plates (1 × 10⁵ cells per well), infected for 8 h with different mimivirus strains in different culture medium, subjected to total RNA extraction and reverse transcription and the resulting cDNA used as a template for quantitative PCR. (A) Arginyl-RS mRNA levels. (B) Tyrosyl-RS mRNA levels. (C) Cysteinyl-RS mRNA. (D) Methionyl-RS mRNA levels. The real-time PCR results were expressed arbitrary units, fitted to a standard curve, corrected using normalization with amoebal 18S rDNA gene expression levels and presented as the mean ± SD from a representative experiment conducted in duplicate. The values were subjected in different combinations to one-way ANOVA tests and Bonferroni post-tests (95% confidence intervals). Differences between groups were considered significant when the p-values were smaller than 0.05. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 4

Replication of different mimivirus strains in A. castellanii. Cells of A. castellanii were plated in 24-well plates (1 × 10⁵ cells per well) and infected with APMV, APMV M4, Kroon virus, Oyster virus, and Samba virus at an multiplicity of infection (MOI) of 10 for the 8 h in three different culture media: (A) peptone-yeast extract-glucose (PYG) 0% FCS; (B) PYG 7% FCS and (C) Page’s amoeba saline (PAS). The infectivity of viral particles was evaluated by titration in amoebas by TCID₅₀. The results are the mean ± SD of a representative experiment conducted in duplicate.