C., M. activation by targeting the host cell DNA and probably interfering with normal DNA replication. Human immunodeficiency computer virus type 1 (HIV-1) is the causative agent of AIDS, which is usually characterized by continual loss of CD4+ T lymphocytes and enhanced susceptibility to opportunistic infections and malignancies. To achieve optimal replicative efficiency, HIV-1 manipulates host cell processes such as gene regulation, chromatin remodeling, signal transduction, and regulations of major histocompatibility complex class 1 surface expression, cell cycle, and apoptosis, as well as overcoming host antiviral mechanisms and targeting bystander cells (50, 54). These multiple activities of HIV are mediated by the specific interactions of viral proteins with various cellular components. As a complex retrovirus, HIV-1 encodes not only the essential structural proteins, Gag, Pol, and Env, but also several regulatory (Tat and Rev) and accessory MIM1 (Vpr, Vif, Vpu, and Nef) proteins. These accessory proteins, while in the MIM1 beginning thought to be dispensable for contamination, have now been shown to be important for HIV infectivity and pathogenesis in vivo (8, 16, 19, 20, 50). Among them, Vpr (viral protein R) (51) is unique in that it is incorporated in the HIV-1 virion at a high copy number (10), suggesting that it may play a significant role in the early stage of contamination. Vpr is a small (96-amino-acid) basic protein conserved in HIV-1, HIV-2, and simian immunodeficiency computer virus (55). Even though molecular mechanisms of Vpr function during viral replication remain elusive, it has some interesting biological activities. Vpr localizes to the nucleus of the infected cell and, together with other virion components, promotes nuclear transport of HIV-1 preintegration complex (7, 18, 25, 33, 39). This function is critical for HIV-1 replication in macrophages and other nondividing cells (4, 12, 25). Vpr can also modestly activate transcription of the HIV-1 long terminal repeat and other cellular promoters (2, 11, 56). Notably, Vpr has the capacity to arrest cell cycle at the G2 phase (26, 37, 38, 44). Several studies have related this function of Vpr to HIV-1 replication and pathogenicity. For example, transcription from your viral long terminal repeat has been shown to be enhanced in G2 regardless of whether the arrest was induced by Vpr or by other means (21), and the ability of Vpr to increase viral replication correlates with G2 arrest (22). This suggests that the G2 arrest induced by Vpr provides a favorable environment for computer virus production. Accumulating evidence indicates that Vpr-induced G2 arrest depends on signaling events analogous to the DNA damage response (24, 26, TNC 41). Specifically, it requires activation of the ATR (ataxia-telangiectasia and Rad3-related)-mediated checkpoint transmission pathway (45, 68). ATR, the kinase related to ATM (Ataxia-Telangiectasia-Mutated) and Rad3, belongs to a conserved family of phosphatidylinositol 3-kinase-like protein kinases. ATR plays an essential role in maintaining genome integrity. In response to a variety of DNA-damaging brokers, ATR is activated and initiates signaling MIM1 cascade by phosphorylating a broad range of downstream substrates, which in turn implement transcriptional regulation, MIM1 checkpoint control, and DNA repair functions (1, 47, 53, 58, 67). In G2/M checkpoint control, ATR-dependent activation of Chk1 kinase (23, 28, 64) prospects to Cdc25A degradation (65) and Cdc25C cytosolic sequestration (36, 48). This prevents the dephosphorylation and activation of the cyclin-dependent kinase 1-cyclin B complex, resulting in arrest of cell cycle in G2 phase. Although previous studies have clearly exhibited the utilization of the ATR pathway by Vpr, the molecular mechanism by which Vpr activates ATR is not known. It is not obvious whether Vpr causes DNA lesions and thus indirectly activates the ATR pathway or whether Vpr directly binds ATR and/or its regulatory proteins, so altering the activity of ATR. Our studies uncover that Vpr binds to chromatin and suggest that Vpr interferes with ongoing DNA replication and in doing so activates the ATR-dependent replication checkpoint pathway following viral infection. MATERIALS AND METHODS Cell culture and genotoxic brokers. HeLa and 293T cells were produced in Dulbecco’s altered Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum. In experiments with drug treatment, cells were incubated in medium made up of 2 mM hydroxyurea (HU) (Sigma) for 2 h prior to analysis. Ionizing radiation was performed using a 137Cs source. UV light was delivered in a single.
