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.

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.

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.

Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin.Proline-rich tyrosine kinase 2 (Pyk2 1 ; also known as CAK␤, RAFTK, and CADTK) is a recently described cytoplasmic tyrosine kinase that is related to the focal adhesion kinase (FAK) (1-4). Recent studies have shown that Pyk2 is involved in calcium-induced regulation of ion channel and mitogen-activated protein kinase activation (1), stress-induced c-Jun Nterminal kinase activation (5), and Src-mediated activation of the mitogen-activated protein kinase signaling pathway in PC12 cells (6). Although these studies indicate that Pyk2 is involved in several signal transduction pathways, many questions concerning the regulation and function of Pyk2, especially the role of Pyk2 in cell adhesion, remain unanswered.Pyk2 and FAK share a similar structural organization with a tyrosine kinase domain flanked by non-catalytic domains at both the N and C termini. These two kinases are approximately 60% identical in the central catalytic domain and share approximately 40% identity in both the N-and C-terminal domains (1-3). Because of the high sequence homology and similar overall organization between Pyk2 and FAK, it is especially interesting to compare the regulation of Pyk2 with that of FAK, in particular their responses to integrin-mediated cell adhesion. Several recent reports have presented somewhat conflicting data regarding regulation of Pyk2 by integrin-mediated cell adhesion in different cell types. It has been reported that Pyk2 displays an integrin-dependent phosphorylation and is localized in focal contacts in B lymphocytes, megakaryocytes, and transfected COS cells (7,8); however, in transfected 3Y1 cells, Pyk2 phosphorylation is not increased upon plating on fibronectin (FN) an...

Viruses have adopted various mechanisms to evade or subvert host antiviral responses initiated by viral RNA or DNA through Toll-like receptor (TLR) (1, 2) and RIG-I-like receptor (RLR) (3-5) signaling pathways. Viral double-stranded RNA (dsRNA) can be sequestered by VP35 of Ebola virus (6) to avoid the activation of TLRs or RLRs. Viral proteins that contain the TIR domain can be interferon (IFN) antagonists such as A46R of vaccinia virus, which blocks upstream IFN signaling by directly targeting MyD88, MAL, TRIF, and TRAM, the TIR domain-containing adaptors (2). RLR sensors such as RIG-I and MDA5 and adaptor IPS1 are targeted by NS1 of influenza virus (7), V protein of paramyxoviruses (3), and the protease precursor protein 3ABC of hepatitis A virus (5). The adaptor protein TRIF of the TLR signaling pathway can be targeted for degradation by viral proteins containing protease activities, such as NS3/4A of flavivirus hepatitis C virus (HCV) (8) and 3C of picornavirus EV71 (9), which can also cleave transcription factor (TF) IRF7, compromising IFN induction. In addition, the N-terminal fragment of cleaved IRF7 interacts with and inhibits IRF3, another TF essential for IFN induction, thereby enhancing viral infection (10). Downstream TFs are also targeted by viral proteins, such as NS1 of West Nile virus, which inhibits TLR3 signaling by preventing IRF3 translocation into the nucleus (11). Between TLR/RLR sensors and downstream TFs, IKK complexes are also targeted by proteins encoded by HCV, vaccinia virus, and rabies virus (12-14), among others.Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus, SFTS bunyavirus (SFTSV), in the family Bunyaviridae (15). SFTSV is a vector-borne zoonotic arbovirus transmitted to humans by tick bites, causing high fever, loss of white blood cells and platelets, and, in severe cases, multiorgan failure. It is a single-stranded negativesense RNA virus with three genomic segments, L, M, and S (15). The L segment encodes viral RNA polymerase, while the M segment encodes the two viral envelope glycoproteins, Gc and Gn. The S segment is an ambisense RNA of 1,744 bases encoding a nucleoprotein (NP) and a nonstructural protein (NSs). Our recent study demonstrated that NSs inhibits host IFN-␤ and NF-B responses, although the mechanism remains unclear (16). Here we report that NSs forms unique cytoplasmic inclusion bodies (IBs) in infected or transfected cells that appear to play a role in immune evasion through the interaction of NSs and the IKK complex. Our data indicate that SFTSV has adopted a novel host immune evasion strategy involving the sequestration of IKK complex components TBK1, IKKε, and IRF, which are critical for the activation of the IFN signaling pathway, into IBs. MATERIALS AND METHODSCells, viruses, and reagents. African green monkey kidney Vero, HeLa, human embryonic kidney 293T, and MDCK cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovin...

H7N9 avian influenza is an emerging viral disease in China caused by avian influenza A (H7N9) virus. We investigated host cytokine and chemokine profiles in serum samples of H7N9 patients by multiplex-microbead immunoassays. Statistical analysis showed that IP-10, IL-6, IL-17, and IL-2 were increased in H7N9 infected patients. Furthermore, IL-6 and the chemokine IP-10 were significantly higher in severe H7N9 patients compared to nonsevere H7N9 cases. We suggest that proinflammatory cytokine responses, characterized by a combined Th1/Th17 cytokine induction, are partially responsible for the disease progression of patients with H7N9 infection.