Tyrosine Residues Direct the Ubiquitination and Degradation of the NY-1 Hantavirus G1 Cytoplasmic Tail

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Abstract
The hantavirus G1 protein contains a long C-terminal cytoplasmic tail of 142 residues. Hantavirus pulmonary syndrome-associated hantaviruses contain conserved tyrosine residues near the C terminus of G1 which form an immunoreceptor tyrosine activation motif (ITAM) and interact with Src and Syk family kinases. During studies of the G1 ITAM we observed that fusion proteins containing the G1 cytoplasmic tail were poorly expressed. Expression of G1 cytoplasmic tail constructs were dramatically enhanced by treating cells with the proteasome inhibitor ALLN, suggesting that the protein is ubiquitinated and degraded via the 26S proteasome. By using a 6-His-tagged ubiquitin, we demonstrated that the G1 cytoplasmic tail is polyubiquitinated and degraded in the absence of proteasome inhibitors. Expression of only the ITAM-containing domain also directed protein ubiquitination and degradation in the absence of upstream residues. Deleting the C-terminal 51 residues of G1, including the ITAM, stabilized G1 and blocked polyubiquitination and degradation of the protein. Site-directed mutagenesis of both ITAM tyrosines (Y619 and Y632) to phenylalanine also blocked polyubiquitination of G1 proteins and dramatically enhanced G1 protein stability. In contrast, the presence of Y627, which is not part of the ITAM motif, had no effect on G1 stability. Mutagenesis of just Y619 enhanced G1 stability, inhibited G1 ubiquitination, and increased the half-life of G1 by threefold. Mutating only Y632 had less of an effect on G1 protein stability, although Y619 and Y632 synergistically contributed to G1 instability. These findings suggest that Y619, which is conserved in all hantaviruses, is the primary signal for directing G1 ubiquitination and degradation. Collectively these findings indicate that specific conserved tyrosines within the G1 cytoplasmic tail direct the polyubiquitination and degradation of expressed G1 proteins and provide a potential means for down-regulating hantavirus G1 surface glycoproteins and cellular proteins that interact with G1.