The execution of apoptosis depends on the hierarchical activation of caspases. The initiator procaspases become autoproteolytically activated through a less understood process that is triggered by oligomerization. Procaspase-8, an initiator caspase recruited to death receptors, is activated through two cleavage events that proceed in a de®ned order to generate the large and small subunits of the mature protease. Here we show that dimerization of procaspase-8 produces enzymatically competent precursors through the stable homophilic interaction of the procaspase-8 protease domain. These dimers are also more susceptible to processing than individual procaspase-8 molecules, which leads to their cross-cleavage. The order of the two interdimer cleavage events is maintained by a sequential accessibility mechanism: the separation of the large and small subunits renders the region between the large subunit and prodomain susceptible to further cleavage. In addition, the activation process involves an alteration in the enzymatic properties of caspase-8; while procaspase-8 molecules speci®cally process one another, mature caspases only cleave effector caspases. These results reveal the key steps leading to the activation of procaspase-8 by oligomerization.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

To investigate the infections of severe fever with thrombocytopenia syndrome virus (SFTSV) in domesticated animals, we sampled a total of 3,039 animals in 2 counties in Shandong Province, People’s Republic of China, from April to November 2011. SFTSV-specific antibodies were detected in 328 (69.5%) of 472 sheep, 509 (60.5%) of 842 cattle, 136 (37.9%) of 359 dogs, 26 (3.1%) of 839 pigs, and 250 (47.4%) of 527 chickens. SFTSV RNA was detected in all sampled animal species, but the prevalence was low, ranging from 1.7% to 5.3%. A cohort study in 38 sheep was conducted to determine when seroconversion to SFTSV occured. SFTSVs were isolated from sheep, cattle, and dogs and shared >95% sequence homology with human isolates from the same disease-endemic regions. These findings demonstrate that natural infections of SFTSV occur in several domesticated animal hosts in disease-endemic areas and that the virus has a wide host range.

Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell-specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre-T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.

e Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, multiorgan dysfunction, and a high fatality rate between 12 and 30%. It is caused by SFTS virus (SFTSV), a novel Phlebovirus in family Bunyaviridae. Although the viral pathogenesis remains largely unknown, hemopoietic cells appear to be targeted by the virus. In this study we report that human monocytes were susceptible to SFTSV, which replicated efficiently, as shown by an immunofluorescence assay and real-time reverse transcription-PCR. We examined host responses in the infected cells and found that antiviral interferon (IFN) and IFN-inducible proteins were induced upon infection. However, our data also indicated that downregulation of key molecules such as mitochondrial antiviral signaling protein (MAVS) or weakened activation of interferon regulatory factor (IRF) and NF-B responses may contribute to a restricted innate immunity against the infection. NSs, the nonstructural protein encoded by the S segment, suppressed the beta interferon (IFN-␤) and NF-B promoter activities, although NF-B activation appears to facilitate SFTSV replication in human monocytes. NSs was found to be associated with TBK1 and may inhibit the activation of downstream IRF and NF-B signaling through this interaction. Interestingly, we demonstrated that the nucleoprotein (N), also encoded by the S segment, exhibited a suppressive effect on the activation of IFN-␤ and NF-B signaling as well. Infected monocytes, mainly intact and free of apoptosis, may likely be implicated in persistent viral infection, spreading the virus to the circulation and causing primary viremia. Our findings provide the first evidence in dissecting the host responses in monocytes and understanding viral pathogenesis in humans infected with a novel deadly Bunyavirus.