The polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation. IMPORTANCE The key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

Phage-coded lysin is an enzyme that destroys the cell walls of bacteria. Phage lysin could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics. In this study, a novel staphylococcal phage, GH15, was isolated, and the endogenous lytic enzyme (LysGH15) was expressed and purified. The lysin LysGH15 displayed a broad lytic spectrum; in vitro treatment killed a number of Staphylococcus aureus strains rapidly and completely, including methicillinresistant S. aureus (MRSA). In animal experiments, a single intraperitoneal injection of LysGH15 (50 g) administered 1 h after MRSA injections at double the minimum lethal dose was sufficient to protect mice (P < 0.01). Bacteremia in unprotected mice reached colony counts of about 10 7 CFU/ml within 3.5 h after challenge, whereas the mean colony count in lysin-protected mice was less than 10 4 CFU/ml (and ultimately became undetectable). These results indicate that LysGH15 can kill S. aureus in vitro and can protect mice efficiently from bacteremia in vivo. The phage lysin LysGH15 might be an alternative treatment strategy for infections caused by MRSA.Staphylococcus aureus is a common and dangerous pathogen that causes various infectious diseases, including skin abscesses, wound infections, endocarditis, osteomyelitis, pneumonia, and toxic shock syndrome (2, 23). Treatment of these infections has become ever more difficult due to the emergence of multidrug-resistant strains, especially methicillin-resistant S. aureus (MRSA) (15,25,26,36,37). Vancomycin was effective against MRSA, but certain MRSA strains have already acquired resistance to vancomycin as well (vancomycin-resistant S. aureus [VRSA]), raising serious concerns within the medical community (17,18,37). Therefore, there is an urgent need for novel therapeutic agents directed against this formidable pathogen (2, 9).The phage lysin is encoded by the bacteriophage genome and is synthesized at the end of the phage lytic life cycle to lyse the host cell (30). Lysins belong to the family of mureolytic enzymes that directly destroy peptidoglycans in the bacterial cell wall. Previous studies have suggested that lysins from certain phages were highly efficient in lysing bacteria, especially when applied exogenously (11,14,21,22,29,35). As a potential antibacterial agent, lysins possess several promising features, namely, a distinct mode of action, species or type specificity, and bactericidal activity independent of the antibiotic susceptibility pattern (1). Indeed, there is a low probability that bacteria will develop resistance against lysin (12, 21).Some Staphylococcus phage lysins have been isolated and studied, including LysK, ClyS, MV-L, LysWMY, and ⌽H5; however, only MV-L and ClyS have been studied in in vivo assays (6, 33). In this study, a novel myovirus phage infecting S. aureus was isolated. The lysin derived from this phage, LysGH15, was expressed and refined. The lysin LysGH15 demonstrated a very broad host range and strong lytic activity. We evaluate...

The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.

The transcriptional coactivator with PDZ binding motif (TAZ) has been reported to be one of the nuclear effectors of Hippo-related pathways. TAZ is expressed in many primary tumors and could regulate many biological processes. However, little is known about the role of TAZ in hepatocellular carcinoma (HCC). In the current study, we show that TAZ regulates cellular proliferation and epithelial–mesenchymal transition (EMT) of HCC. TAZ is overexpressed in HCC tissues and cell lines and upregulation of TAZ correlates with a lower overall survival rate of HCC patients after hepatic resection. TAZ knockdown results in inhibition of cancer cell proliferation through decreases in expression of stem cell markers (OCT4, Nanog, and SOX2). Reduction in HCC cell migration and invasion is also evident through reversal of EMT by increases E-cadherin expression, decreases in N-cadherin, vimentin, Snail, and Slug expression, and suppression of MMP-2 and MMP-9 expression. In a xenograft tumorigenicity model, TAZ knockdown could effectively inhibit tumor growth and metastasis through reversal of the EMT pathway. In conclusion, TAZ is associated with the proliferation and invasiveness of HCC cells, and the TAZ gene may contribute to a novel therapeutic approach against HCC.