***< 0.001. To determine whether this effect is specific for S-phase arrest, we assessed the effect of G0/G1 and G2/M arrest on viral replication. cell cycle arrest in infected cells via non-structural protein 3D, which may provide favorable conditions for virus production. genus of the Picornaviridae family, which has a single-stranded, positive-sense RNA genome of about 7400?bp consisting of 5 and 3 non-translated regions flanking a large open reading frame that encodes a polyprotein of about 2193 amino acids. In host cells, this polyprotein is further cleaved into 4 structural (VP1 to VP4) and 7 non-structural (2A to 3D) proteins via the virus-encoded non-structural 2A and 3C proteases.10 In addition to the 2A and 3C proteins, non-structural 3D protein is an RNA-dependent RNA polymerase that plays an important role in virus replication via the incorporation of nucleotides during RNA elongation.11 Recent studies have demonstrated that 2A,12, 3C,13 and 3D14 exert other roles that affect the life cycle of GW4064 the virus. As part of their pathogenic mechanism, many viruses facilitate their own growth by interacting with genes that regulate the host cell cycle. Examples can be found among DNA viruses, retroviruses, and SLIT1 RNA viruses. Although the DNA viruses, which replicate in the nucleus of host cells, have been the most extensively studied with regard to cell cycle control, some small DNA viruses such as simian virus 40,15 adenovirus 16,17 and human papillomavirus,18 which lack their own polymerases, encode proteins that promote the entrance of cells into the S phase from the G1 phase by using host polymerase. Other large DNA viruses, such as herpesviruses, can induce cell cycle arrest in the G0/G1 phase to avoid competition for cellular DNA replication resources.19 As is true for DNA viruses, cell cycle regulation has been observed for retroviruses, which also replicate in the nucleus. The Vpr protein of human immunodeficiency virus type1 is responsible for eliciting cell cycle arrest in the G2/M phase.20,21 Furthermore, RNA viruses, whose primary site of replication is normally the cytoplasm, have also been demonstrated to interfere with the host cell cycle. In the coronavirus family, infectious bronchitis virus (IBV) induces an S and G2/M-phase arrest to favor viral replication22,23; and mouse hepatitis virus (MHV)24 and some severe acute respiratory syndrome GW4064 coronavirus (SARS-CoV) proteins can induce cell cycle arrest in the G0/G1 phase.25,26 Normally, the cell cycle is controlled by the binding of CDK to the corresponding cyclin regulatory subunits, which are tightly regulated temporally. The G1 phase cyclins, cyclin D and cyclin E, associate predominantly with CDK 4/CDK 6 and CDK 2, respectively, to promote G1 progression and S-phase entry.27 Both cyclin A and cyclin E then combine, mainly with CDK 2, to promote S-phase progression.28 Subsequently, CDK 1 and cyclin B-forming maturation-promoting factor (MPF) regulate mitotic entry.29,30 Some viruses have been reported GW4064 to regulate cell cycle progression by manipulating cyclin and CDK progression,31,32 but the potential effect of EV71 is unknown. In the present study, we examined the potential effects of EV71 infection on the cell cycle. Our data show that EV71 replication induces cell cycle arrest in S phase, and, conversely, that cells arrested in S phase produce favorable conditions for the production of EV71. We further demonstrated that the non-structural 3D protein is responsible for the S-phase arrest. These results further our understanding of the pathogenic mechanisms of EV71 and provide a potential target for the treatment and prevention of HMDF disease. Results EV71-infected cells accumulate in S phase To address whether EV71 affects the cell cycle of host cells, human rhabdomyosarcoma RD cells were infected with EV71 at an MOI of 1 1. The cells were collected at 30?h post-infection, and the cell cycle distribution was analyzed by flow cytometry. An obvious accumulation in the S phase was observed by ModFit analysis, with an increase from 37.37 1.35% to 45.38 0.15% for EV71-infected as compared to mock-infected cells (22.20% increase; Fig. 1A). The S-phase arrest after EV71 infection was most apparent at 24?h to 30?h post-infection (Fig. 1B). These data suggest that EV71 infection induces S-phase accumulation. Open in a separate window Figure 1. S-phase accumulation induced by enterovirus 71 (EV71) infection. (A) Top panel: RD cells were mock-infected (mock) or infected with EV71 (infected) at an MOI of 1 1. At GW4064 30?h post-infection, cells were collected, and the cell cycle profiles were analyzed by flow cytometry..