Co-translational trimerization of the reovirus cell attachment protein

The reovirus cell attachment protein, σ1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple a-helical coiled-coil) is located at the N-terminal one-third of σ1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of σ1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) σ1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated σ1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of σ1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL σ1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent σ1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.