A giant virus creates a subcellular environment to overcome the codon-tRNA mismatch [tRNA-seq]

A codon usage consonant with the cellular tRNA pool is important for efficient translation. However, Mimivirus, a giant DNA virus, relies on the host translation system of amoeba yet exhibits a codon usage distinct from its host. How this virus copes with the mismatch between the demand and supply of tRNAs remains unknown. Here, we found that the virus generates a subcellular compartment to translate virus mRNAs efficiently. The combination of genome-wide Ribo-Seq and RNA-Seq reveals that ribosomes traversed along viral mRNAs without pausing, despite the codon usage incompatibility with the host mRNAs. Although the virus encodes tRNA genes in the genome, mim-tRNA-Seq shows that the expression does not impact the composition of the cellular tRNA pool during infection. Instead, we observed that through in situ labeling of newly synthesized proteins, viral protein synthesis locally occurs around the periphery region of the viral factory, likely creating a unique environment to facilitate viral translation. Our data provide a perspective on how local translation shapes viral replication overcoming the mismatch of tRNA supply and codon demand. Overall design: To study the changes that occur in viral and host translation over the course of Acanthamoeba polyphaga mimivirus (APMV) infection, we applied ribosome profiling, and RNA-seq to Acanthamoeba castellanii str. Neff cells infected with APMV as well as mock infected cells. The cells were infected with APMV at a multiplicity of infection (MOI) of 10 and harvested at 2, 4, and 8 hours post infection (hpi). For each time point, ribosome-protected footprints as well as total RNA were sequenced. Two biological replicates were prepared for each time point. To characterize the changes in tRNA library composition during the course of APMV infection, we applied mim-tRNA-seq on A. castellanii cells infected with APMV as well as mock infected cells. The cells were infected at a MOI of 10 and harvested at 2, 4, and 8 hpi. Two biological replicates were prepared for each time point.