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Influenza viruses exploit host factors to successfully replicate in infected cells. Using small interfering RNA (siRNA) technology, we identified six human genes required for influenza A virus (IAV) replication. Here we focused on the role of acid phosphatase 2 (ACP2), as its knockdown showed the greatest inhibition of IAV replication. In IAV-infected cells, depletion of ACP2 resulted in a significant reduction in the expression of viral proteins and mRNA, and led to the attenuation of virus multi-cycle growth. ACP2 knockdown also decreased replication of seasonal influenza A and B viruses and avian IAVs of the H7 subtype. Interestingly, ACP2 depletion had no effect on the replication of Ebola or hepatitis C virus. Because ACP2 is known to be a lysosomal acid phosphatase, we assessed the role of ACP2 in influenza virus entry. While neither binding of the viral particle to the cell surface nor endosomal acidification was affected in ACP2-depleted cells, fusion of the endosomal and viral membranes was impaired. As a result, downstream steps in viral entry were blocked, including nucleocapsid uncoating and nuclear import of viral ribonucleoproteins. Our results established ACP2 as a necessary host factor for regulating the fusion step of influenza virus entry.

Unlabelled: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner., Conclusion: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis., (© 2014 by the American Association for the Study of Liver Diseases.)

During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.

The interplay between pathogen-encoded virulence factors and host cell signaling networks is critical for both the establishment and clearance of microbial infections. Yersinia uptake into host cells serves as an in vitro model for exploring how host cells respond to Yersinia adherence. In this study, we provide insight into the molecular nature and regulation of signaling networks that contribute to the uptake process. Using a reconstitution approach in Fak(-/-) fibroblasts, we have been able to specifically address the interplay between Fak, Cas and Pyk2 in this process. We show that both Fak and Cas play roles in the Yersinia uptake process and that Cas can function in a novel pathway that is independent of Fak. Fak-dependent Yersinia uptake does not appear to involve Cas-Crk signaling. By contrast, Cas-mediated uptake in the absence of Fak requires Crk as well as the protein tyrosine kinases Pyk2 and Src. In spite of these differences, the requirement for Rac1 activity is a common feature of both pathways. Furthermore, blocking the function of either Fak or Cas induces similar morphological defects in Yersinia internalization, which are manifested by incomplete membrane protrusive activity that is consistent with an inhibition of Rac1 activity. Pyk2 also functions in Yersinia uptake by macrophages, which are physiologically important for clearing Yersinia infections. Taken together, these data provide new insight into the host cellular signaling networks that are initiated upon infection with Y. pseudotuberculosis. Importantly, these findings also contribute to a better understanding of other cellular processes that involve actin remodeling, including the host response to other microbial pathogens, cell adhesion and migration.