Kaposi`s sarcoma-associated herpesvirus (KSHV) plays a significant role in the development of Kaposi`s sarcoma, primary effusion lymphoma and some forms of multicentric Castleman`s disease. The KSHV open reading frame K9 encodes the viral interferon factor 1 (vIRF1), which down-regulates interferon (IFN)- and IRF-mediated transcriptional activation, and leads to cellular transformation in rodent fibroblasts and induction of tumors in nude mice. Here we show that vIRF1 directly associates with the tumor suppressor p53 and represses its functions. The vIRF1 interaction domains of p53 are DNA binding domain (a.a. 100-300) and tetramerization domain (a.a. 300-393) of p53. p53 interacts with the central region (a.a. 152-360) of vIRF1. vIRF1 suppresses p53-dependent transcription and deregulates its apoptotic activity. These results suggest that vIRF1 may regulate cellular function by inhibiting p53. In addition, using the yeast two-hybrid assay, we identified genes associated with retinoid-IFN-induced mortality-19 (GRIM19), which interacts directly with vIRF1, both in vivo and in vitro. The N-terminal region of vIRF1 is required for binding GRIM19. Colocalization of vIRF1 and GRIM19 was observed in 293T cells and BCBL-1 cells. The vIRF1 protein deregulates GRIM19-induced apoptosis in the presence of IFN/all-trans-retinoic acid (RA) and inhibits IFN/RA-induced cell death. Other DNA tumor viral protein, human papillomavirus type 16 E6, also bind GRIM19, suggesting that this is a general target of viral proteins. Our results collectively indicate that vIRF1 modulates IFN/RA-cell death signals via interactions with GRIM19. vIRF1 also inhibits TGF- β-induced transcription and recovers TGF- β-induced growth inhibition. vIRF1 directly interacts with Smad3 and Smad4. vIRF1 disrupts Smad3-Smad4 complex formation and its DNA biniding. These results suggest that suppression of Smad-induced signaling by vIRF1 may contribute to KSHV-associated carcinogenesis.
Nuclear factor κB (NF-κB) is a transcription factor that plays an important role in the immune system and cell death. Many viral proteins modulate κB to escape host immune surveillance, promote cell survival, and enhance viral replication. In the present study we show that κB activity is down-modulated by viral interferon regulatory factor 3 (vIRF3), which is encoded by Kaposi’s sarcoma-associated herpesvirus open reading frame K10.5. vIRF3 repressed κB -dependent transcription in a dose-dependent manner, and inhibited DNA binding of κB induced by tumor necrosis factor-α. In vivo studies showed vIRF3 inhibited IκB kinase beta activity, but not I κB kinase alpha activity, resulting in reduced I κB phosphorylation. Immunofluorescence assays showed that vIRF3 interfered with nuclear translocation of NF- κB . In addition, consistent with inhibition of NF- κB activity, vIRF3 sensitized cells to TNF-alpha-induced apoptosis and inhibited major histocompatibility complex class I promoter activity. Our results indicate that vIRF3 can regulate the host immune system and apoptosis via inhibition of NF- κB activity.
카포시 육종 허피스 바이러스는 사람에게 감염되는 8번째 허피스 바이러스로서, 카포시 육종, primary effusion lymphoma, Castleman disease을 직접적으로 일으키는 병인으로 알려져 있다. 카포시 육종 허피스 바이러스의 open reading frame K9에서 발현되는 vIRF1 단백질은 인터페론에 의한 신호전달을 저해하고, 세포를 형질전환시켜 종양을 유발한다. 이 연구에서는 vIRF1이 p53단백질과 결합하여 p53의 기능을 저해하는 것을 확인하였다. 또한 이스트를 이용한 two-hybrid screening을 통하여 vIRF1이 GRIM19과 상호작용함을 밝혔다. vIRF1은 GRIM19과 in vivo, in vitro에서 결합할 뿐만 아니라 두 단백질의 세포내 위치도 일치하였다. vIRF1은 GRIM19과의 상호작용을 통해 인터페론과 레티노이드에 의해 일어나는 세포 사멸을 저해하였다. vIRF1은 Smad단백질을 저해하여 TGF-b에 의해 유발되는 유전자 발현 및 성장억제를 저해하였다. vIRF1은 Smad3와 Smad4의 결합을 막고, Smad단백질이 DNA에 결합하는 것을 억제하였다.
NF- κB 는 면역 체계와 세포 사멸의 조절에 중요한 역할을 하는 전사인자이다. 따라서 많은 바이러스 단백질이 NF- κB 의 활성을 조절함으로써 바이러스가 유리한 환경으로 만들어 간다. 카포시 육종 허피스 바이러스의 vIRF3는 NF- κB 의 활성을 저해하였다. 이러한 저해는 vIRF3가 IκB 인산화 효소β 를 저해함으로써 나타난다. 뿐만 아니라 vIRF3는 TNF-α 에 의해 일어나는 세포사멸을 촉진하였다. 이러한 결과는 vIRF3가 NF- κB 를 저해함으로써 숙주세포의 면역체계와 세포사멸을 조절할 수 있다는 사실을 보여준다.
