Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

Uzoma, Ijeoma; Zhu, Heng

Another variation of protein microarray used for investigating pathogen-host interaction involves the human TF array to profile the interactions between KSHV latency proteins and host proteins. In KSHV-associated malignancies, majority of the tumor cells are latently-infected and express viral latency proteins including LANA [45] . LANA functions to maintain KSHV latency by driving viral replication [46, 47] , promoting dysregulated cell growth [48] and dynamically regulating both viral and cell gene transcription [49] [50] [51] . Identification of LA-NA's interacting partners would provide new insights into the mechanisms LANA uses to maintain latent infection. LANA has been an attractive target and previous efforts to identify LANA binding proteins have attempted yeast two-hybrid screens [52] , glutathione S-transferase (GST) affinity immunoprecipitation [53] and MS, resulting in apparent approachdependent binding partners [54] . In a recent study, Shamay et al. purified FLAG-tagged LANA and probed it against the human TF array, which recovered 61 candidate binding partners [36] . Eight candidates validated by co-immunoprecipitation assays included TIP60, protein phosphatase 2A (PP2A), replication protein A (RPA) and XPA. LANA-associated TIP60 retained its acetyltransferase activity and showed enhanced stability, which is consistent with Li et al.'s finding that TIP60 in critical for KSHV lytic replication (see above). The binding interactions between LANA, RPA and XPA seem to echo LANA's role in DNA damage, but further characterization of the LANA's ability to bind to additional RPA complex members, RPA1 and RPA2, spawned a new hypothesis that LANA may also regulate host telomere length. To test this hypothesis, the authors performed ChIP assays with anti-RPA1 and -RPA2 antibodies using primers specific to the telomere regions and found that the presence of LANA drastically reduced the recruitment of both RPA1 and RPA2 to the host telomeres, while it had no impact on the protein level of the RPA complex. This observation raised the possibility that LANA might affect telomere length. Using Southern blot analysis of terminal restriction fragments, the standard method for quantifying telomere length, the authors demonstrated that the average length of telomeres was shortened by at least 50% in both LANA-expressing endothelial cells and KSHV-infected primary effusion lymphoma cells [55] .