Ribosome customization and functional diversification among P-stalk proteins regulate late poxvirus protein synthesis

Growing evidence suggests that ribosomes selectively regulate translation of specific mRNA subsets. Here, quantitative proteomics and cryoelectron microscopy demonstrate that poxvirus infection does not alter ribosomal subunit protein (RP) composition but skews 40S rotation states and displaces the 40S head domain. Genetic knockout screens employing metabolic assays and a dual-reporter virus further identified two RPs that selectively regulate non-canonical translation of late poxvirus mRNAs, which contain unusual 5' poly(A) leaders: receptor of activated C kinase 1 (RACK1) and RPLP2. RACK1 is a component of the altered 40S head domain, while RPLP2 is a subunit of the P-stalk, wherein RPLP0 anchors two heterodimers of RPLP1 and RPLP2 to the large 60S subunit. RPLP0 was required for global translation, yet RPLP1 was dispensable, while RPLP2 was specifically required for non-canonical poxvirus protein synthesis. From these combined results, we demonstrate that poxviruses structurally customize ribosomes and become reliant upon traditionally non-essential RPs from both ribosomal subunits for efficient initiation on their late mRNAs.