👤 Richard Longnecker

Latent membrane protein 2A (LMP2A) of latent Epstein-Barr virus (EBV) specifically associates with HECT domain-containing Nedd4-family ubiquitin-protein ligases (E3s). Here we demonstrate that LMP2A is specifically ubiquitinated by the HECT domains of AIP4 and WWP2. Deletion and site-specific mutation of LMP2A indicates that LMP2A is ubiquitinated at its amino-terminus and is not ubiquitinated on lysine residues. LMP2A and LMP1, also encoded by EBV, are two of only four proteins that have been identified that are ubiquitinated at the amino-terminus, indicating that EBV may specifically target and utilize this host cell protein modification. Show less

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A amino-terminal domain. In this study, LMP2A PY motif mutant viruses were constructed to investigate the role of the LMP2A PY motifs. AIP4 was found to specifically associate with the LMP2A PY motifs in EBV-transformed lymphoblastoid cell lines (LCLs), extending our original observation to EBV-infected cells. Mutation of both of the LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A, which resulted in an increase in the expression of Lyn and the constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa protein. In addition, there was a modest increase in the constitutive phosphorylation of Syk and an unidentified 60-kDa protein. These results indicate that the PY motifs contained within LMP2A are important in regulating phosphorylation in EBV-infected LCLs, likely through the regulation of Lyn activity by specifically targeting the degradation of Lyn by ubiquination by Nedd4 family E3s. Despite differences between PY motif mutant LCLs and wild-type LCLs, the PY motif mutants still exhibited a block in B-cell receptor (BCR) signal transduction as measured by the induction of tyrosine phosphorylation and BZLF1 expression following BCR activation. EBV-transformed LCLs with mutations in the PY motifs were not different from wild-type LCLs in serum-dependent cell growth. Protein stability of LMP1, which colocalizes with LMP2A, was not affected by the LMP2A-associated E3s. Show less

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. LMP2A functions to downregulate B-cell signal transduction and viral reactivation from latency in EBV-immortalized B cells in vitro, and acts to provide B cells with both a survival and developmental signal in vivo. Identification of proteins associated with LMP2A is important for elucidation of the mechanism that LMP2A employs to regulate B-cell signal transduction and EBV latency. LMP2A is constitutively tyrosine phosphorylated and is associated with protein tyrosine kinases such as Lyn and Syk when specific LMP2A tyrosines are phosphorylated. The amino-terminal domain of LMP2A includes multiple proline-rich regions, which may provide binding sites for proteins containing SH3 or WW domains. In this study, we demonstrate that four cellular proteins bind specifically to two PPPPY (PY) motifs present within the LMP2A amino-terminal domain. Protein microsequence analysis determined that three of these proteins were AIP4, WWP2/AIP2, and Nedd4. All of these proteins are members of the Nedd4-like ubiquitin-protein ligases family and have conserved domains including the C2, WW, and ubiquitin-protein ligase domain. The mutation of both PY motifs completely abolished binding activity of these proteins to LMP2A and the interaction of AIP4 and WWP2 with LMP2A was confirmed in cell lines expressing LMP2A, WWP2, and AIP4. Furthermore, a reduction in the level of Lyn and the rapid turnover of LMP2A and Lyn were observed in LMP2A-expressing cells. These findings suggest that LMP2A recruits Nedd4-like ubiquitin-protein ligases and B-cell signal transduction molecules, resulting in the degradation of LMP2A and Lyn by a ubiquitin-dependent mechanism. This provides a new means by which LMP2A may modulate B-cell signal transduction. Show less