Studies On The Mechanism Of HCMV Protein PUL49 Modulates The Host Cell Cycle And Proliferation

Human cytomegalovirus(HCMV), a member of the beta herpesvirus family, is widespread among humans, where it establishes a persistent infection that causes disease and mortality in immuno compromised individuals such as newborn, blood transfusion patients, organ transplantation and HIV/AIDS patients.UL49 gene is essential for HCMV replication, it is demonstrated that the virus BAC-Towne-ΔUL49 deficient UL49 open read frame cannot successfully package progeny virus particles after virus infected host cells, downregulation of UL49 expression also seriously influence the virus replication, indicating the importance of UL49 for HCMV. However, the function of UL49 gene in host cells is not very clear. To clarity the functions of p UL49 on host cells, we conducted the following investigations:To study the function of p UL49, we first established and purified lentivirus carring UL49 c DNA. After infecting U373 cells, we screened a cell line that stably expresses p UL49. Firstly, we observed that p UL49 overexpression inhibits cell proliferation but not induce cell apoptosis demonstrated by Annexin V/PI staining and apoptosis-related protein detection. Simultaneously overexpressed p UL49 increased the cell number of G1/S phase, indicating p UL49 overexpression suppressed U373 cell proliferation by arresting cell cycle in G1/S phase.To clarity the detail mechanisms of p UL49 on cell proliferation inhibition, we employed proteomics based immune co-precipitation and total of 33 candidated proteins were found. One of the p UL49 binding partner is FRK, which is a cytoplasmic SRC-like kinase that shares with other SRC-family members a similar structure, consisting of an N-terminal unique domain, a SRC homology 3(SH3) and an SH2 domain, a kinase domain and a short C-terminal regulatory tail. Overexpression of FRK inhibited cell proliferation and migration,arresting cells in G1 / S phase. Co-immunoprecipitation assay was used to further confirm the interaction of UL49 and FRK; Immunofluorescence assay indicated that UL49 and FRK co-localized in nuclear.FRK arrests cell cycle in G1/S phase by inhibiting cyclin D1 nuclear translocation. The interactions and the similar effects in cell proliferation of p UL49 and FRK promote us postulate that p UL49 regulated host cell proliferation via FRK. To demonstrate this hypothesis,we first designed si RNA targetingp UL49 and FRK. Knockdown FRK or p UL49 inhibited U373 cell proliferation, whereas, FRK knockdown can partially reverse cell proliferation inhibition induced by p UL49 overexpression. Indicating p UL49 inhibited host cell proliferation via interacting with FRK.Then we observed whether p UL49 regulated cell cycle via FRK in U373 cells. The results showed that knockdown p UL49 and FRK decreased cell number arresting in G1 phase induced by p UL49 overexpression, and this effect was potential in cells with p UL49 and FRK knockdown simultaneously. Moreover, p UL49 overexpression inhibited cyclin D1 trans-located into nuclear, and knockdown FRK can partially reversed this effects. These observation provided evidences that p UL49 inhibited cycling D1 nuclear translocation via interacting FRK.We also detected several signaling pathway regulating cell growth, including JNK(T183 / Y185), IRS-1(S636/S639), P38MAPK(T180 / Y182), MEK1(S217 / S221), P70S6K(T421/S424) phosphorylated protein levels by high-throughput liquid chip technology. We found overexpression of p UL49 increased IRS-1(S636/S639)、AKT(S473/T308) 、 PDK1(S241) 、 m TOR(S2448) 、 P70S6K(T421/S424) phosphorylated protein levels, these results were further confirmed by western blotting. Whereas, other protein phosphorylation did not change. m TOR(S2448)、P70S6K(T421/S424) inhibitor potential the inhibition of cell proliferation induced by p UL49 overexpression, indicating p UL49 may function through IRS-1 signaling pathway to mantain host cell survival.Previous study found that deletion 143 amino acid of N-terminal affected p UL49 nuclear localization. We constructed several p UL49 truncated fragment including p UL49(ΔN143), p UL49(ΔN100), p UL49(143), p UL49(100). After overexpression of these fragment into U373 cells, we detected IRS-1 signaling pathway and found p UL49(ΔN143), p UL49(ΔN100) retain the ability that increase IRS-1(S636 / S639) and P70S6K(T421/S424) phosphorylation, suggesting that p UL49 without N-terminal 143 amino acid also play a functional role.In this study, we provide evidence that p UL49 arrests cell cycle in G1/S phase and inhibited cell proliferation via interacting with FRK. In addition, p UL49 may function through IRS-1 signaling pathway to mantain host cell survival...