Inefficient Codon Usage Impairs mRNA Accumulation: the Case of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus.

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Latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes encode a homolog of cellular FLICE-inhibitory proteins (termed v-FLIP) that activates NF-κB and can trigger important proinflammatory and antiapoptotic changes in latently infected cells. The protein is present at very low levels in infection and has generally been difficult to efficiently express in recombinant vectors. Here we show that codon usage in the v-FLIP gene is strikingly suboptimal. Optimization of codon use in expression vectors, as expected, restores efficient protein expression. Surprisingly, however, it also dramatically increases the steady-state level of v-FLIP mRNA, at least in part by increasing mRNA stability. When codon-optimized v-FLIP sequences are reintroduced into intact KSHV genomes, the resulting virus expresses readily detectable monocistronic v-FLIP mRNAs that are undetectable in wild-type (WT) infection by blot hybridization, suggesting that such RNAs are in fact transcribed in WT infection but fail to accumulate. The overexpression of v-FLIP by codon-optimized latent genomes results in a 5- to 7-fold decrement in virus production following lytic induction, indicating that maximizing NF-κB signaling is deleterious to induction. These studies provide a clear explanation for the evolution of inefficient codon usage in this gene and point to a strong connection between translational efficiency and RNA accumulation in mammalian cells. This study reports that inefficient codon usage in a herpesviral gene is strikingly correlated with the inability of its mRNA to accumulate in cells; correction of efficient translatability restores RNA abundance. A similar correlation has been reported in yeast species, but the mechanisms operating in mammalian cells appear substantially different.