Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-␤-D-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 M and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.

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Induced pluripotent stem (iPS) cells can be generated from somatic cells by transduction with several transcription factors in mouse and human. However, direct reprogramming in other species has not been reported. Here, we generated monkey iPS cells by retrovirus-mediated introduction of monkey transcription factors OCT4, SOX2, KLF4, and c-MYC.

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.Human embryonic stem cells (hESCs) 3 are capable of essentially unlimited self-renewal and retain the developmental potential to differentiate into almost any cell type. These characteristics of hESCs make them attractive for tissue and cellbased therapies (1, 2). Previously, basic fibroblast growth factor and activin A were identified as self-renewal factors (3-6). However, for reasons that are not clear, hESCs often display high rates of spontaneous apoptosis and differentiation in culture, thus making the process of expanding these cells highly inefficient (3, 7-10). For example, Dravid et al. (8) reported that, under routine culture conditions, Ͼ30% of hESCs undergo spontaneous apoptosis. Furthermore, Ezashi et al. (12) showed that nearly 40% of hESCs undergo spontaneous differentiation after 12 days of culture in normoxic conditions. Finally, Maitra et al. (13) reported that multiple passages of hESCs can cause genomic alterations, which may limit the therapeutic application of hESCs. In contrast to hESCs, mouse embryonic stem cells (mESCs) undergo lower rates of spontaneous apoptosis and differentiation (14). Moreover, they maintain their pluripotency and genomic stability longer than hESCs (15). The reason for these different species-specific phenotypes in embryonic stem cells is currently unknown.The p53 tumor suppressor gene is a strong candidate for playing a role in the observed phenotypes of hESCs, because it regulates various cellular processes, including apoptosis, differentiation, and genomic integrity (16). In many cell types p53 plays a crucial role in controlling apoptosis and cell cycle arrest when these cells are exposed to stress-inducing conditions (17). In response to stress, p53 accumulates and transactivates downstream target genes such as mdm2 (responsible for the feedback degradation circuitry of p53), p21 (responsible for cell cycle control), bax, noxa, and puma (responsible for DNA damage-induced ...

Cholesterol present in the plasma membrane of target cells has been shown to be important for the infection by SARS-CoV. We show that cholesterol depletion by treatment with methyl-beta-cyclodextrin (m beta CD) affects infection by SARS-CoV to the same extent as infection by vesicular stomatitis virus-based pseudotypes containing the surface glycoprotein S of SARS-CoV (VSV-Delta G-S). Therefore, the role of cholesterol for SARS-CoV infection can be assigned to the S protein and is unaffected by other coronavirus proteins. There have been contradictory reports whether or not angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV, is present in detergent-resistant membrane domains. We found that ACE2 of both Vero E6 and Caco-2 cells co-purifies with marker proteins of detergent-resistant membranes supporting the notion that cholesterol-rich microdomains provide a platform facilitating the efficient interaction of the S protein with the cellular receptor ACE2. To understand the involvement of cholesterol in the initial steps of the viral life cycle, we applied a cell-based binding assay with cells expressing the S protein and cells containing angiotensin-converting enzyme 2 (ACE2). Alternatively, we used a soluble S protein as interaction partner. Depletion of cholesterol from the ACE2-expressing cells reduced the binding of S-expressing cells by 50% whereas the binding of soluble S protein was not affected. This result suggests that optimal infection requires a multivalent interaction between viral attachment protein and cellular receptors.