While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37°C but displays full activity in cells grown at 31°C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37°C but exits the ER and accumulates in endosomal structures in cells grown at 31°C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37°C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37°C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.

Distinct cytoplasmic sorting signals target integral membrane proteins to late endosomal compartments, but it is not known whether different signals direct targeting by different pathways. The availability of multiple pathways may permit some cell types to divert proteins to specialized compartments, such as the melanosome of pigmented cells. To address this issue, we characterized sorting determinants of tyrosinase, a tissue-specific resident protein of the melanosome. The cytoplasmic domain of tyrosinase was both necessary and sufficient for internalization and steady state localization to late endosomes and lysosomes in HeLa cells. Mutagenesis of two leucine residues within a conventional di-leucine motif ablated late endosomal localization. However, the properties of this di-leucine-based signal were distinguished from that of CD3␥ by overexpression studies; overexpression of the tyrosinase signal, but not the well characterized CD3␥ signal, induced a 4-fold enlargement of late endosomes and lysosomes and interfered with endosomal sorting mediated by both tyrosine-and other di-leucine-based signals. These properties suggest that the tyrosinase and CD3␥ dileucine signals are distinctly recognized and sorted by distinct pathways to late endosomes in non-pigmented cells. We speculate that melanocytic cells utilize the second pathway to divert proteins to the melanosome.

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