The influenza viral membrane protein hemagglutinin (HA) is required at high concentrations on virion and host-cell membranes for infectivity. Because the role of actin in membrane organization is not completely understood, we quantified the relationship between HA and host-cell actin at the nanoscale. Results obtained using superresolution fluorescence photoactivation localization microscopy (FPALM) in nonpolarized cells show that HA clusters colocalize with actin-rich membrane regions (ARMRs). Individual molecular trajectories in live cells indicate restricted HA mobility in ARMRs, and actin disruption caused specific changes to HA clustering. Surprisingly, the actin-binding protein cofilin was excluded from some regions within several hundred nanometers of HA clusters, suggesting that HA clusters or adjacent proteins within the same clusters influence local actin structure. Thus, with the use of imaging, we demonstrate a dynamic relationship between glycoprotein membrane organization and the actin cytoskeleton at the nanoscale.

Seasonal influenza virus infections cause annual epidemics and sporadic pandemics. These present a global health concern, resulting in substantial morbidity, mortality and economic burdens. Prevention and treatment of influenza illness is difficult due to the high mutation rate of the virus, the emergence of new virus strains and increasing antiviral resistance. Animal models of influenza infection are crucial to our gaining a better understanding of the pathogenesis of and host response to influenza infection, and for screening antiviral compounds. However, the current animal models used for influenza research are not amenable to visualization of host-pathogen interactions or high-throughput drug screening. The zebrafish is widely recognized as a valuable model system for infectious disease research and therapeutic drug testing. Here, we describe a zebrafish model for human influenza A virus (IAV) infection and show that zebrafish embryos are susceptible to challenge with both influenza A strains APR8 and X-31 (Aichi). Influenza-infected zebrafish show an increase in viral burden and mortality over time. The expression of innate antiviral genes, the gross pathology and the histopathology in infected zebrafish recapitulate clinical symptoms of influenza infections in humans. This is the first time that zebrafish embryos have been infected with a fluorescent IAV in order to visualize infection in a live vertebrate host, revealing a pattern of vascular endothelial infection. Treatment of infected zebrafish with a known anti-influenza compound, Zanamivir, reduced mortality and the expression of a fluorescent viral gene product, demonstrating the validity of this model to screen for potential antiviral drugs. The zebrafish model system has provided invaluable insights into host-pathogen interactions for a range of infectious diseases. Here, we demonstrate a novel use of this species for IAV research. This model has great potential to advance our understanding of influenza infection and the associated host innate immune response.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a stress-response gene that has been associated with cancer, but no studies have differentiated among or defined the regulation or function of any of its several recently described expression variants. We found that TNFAIP8 variant 2 (v2) is overexpressed in multiple human cancers, whereas other variants are commonly downregulated in cancer (v1) or minimally expressed in cancer or normal tissue (v3-v6). Silencing v2 in cancer cells induces p53-independent inhibition of DNA synthesis, widespread binding of p53, and induction of target genes and p53-dependent cell cycle arrest and DNA damage sensitization. Cell cycle arrest induced by v2 silencing requires p53-dependent induction of p21. In response to the chemotherapeutic agent doxorubicin, p53 regulates v2 through binding to an intragenic enhancer, together indicating that p53 and v2 engage in complex reciprocal regulation. We propose that TNFAIP8 v2 promotes human cancer by broadly repressing p53 function, in essence offsetting p53-dependent tumor suppression. Cell Death and Differentiation (2017) 24, 181-191; doi:10.1038/cdd.2016 published online 11 November 2016 Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is the founding member of a recently described family of environmental stress response genes that are induced by TNFα. TNFAIP8 has been shown to promote or inhibit apoptosis, depending on cell type and context. 1,2 Although little is known about TNFAIP8, it has recently been found to be overexpressed in a wide range of human cancers. Some studies have suggested protumor functions for TNFAIP8, including enhancement of cell survival, proliferation, and metastasis [3][4][5][6][7][8][9] and resistance to cancer chemotherapeutics in mice. 4,10Nonetheless, nothing is known about how TNFAIP8 influences responses to DNA damage in cancer cells. Moreover, several transcript variants from the TNFAIP8 gene were recently registered in the NCBI reference sequence database, but no study to date has differentiated among them or defined the factors that govern their expression.The tumor suppressor p53 is a transcription factor that regulates many biological processes through its target gene network. Some of the well-characterized p53 target genes including p21/CDKN1A, growth arrest and DNA damageinducible 45a (GADD45A) and Bcl-2-like protein 4 (BAX) together promote senescence, cell cycle arrest, and apoptosis, all of which may contribute to the tumor suppressing functions of p53. 11,12Additional noncanonical tumorsuppressive pathways from p53 have recently been identified. [13][14][15] Improved characterization of the p53 DNAbinding sequence with modern sequencing techniques has led to the identification of a rapidly expanding list of p53 target genes. [16][17][18] Continued identification of these genes and characterization of their mechanisms of regulation and function will be critical to a full understanding of p53 and for full realization of opportunities to intervene upon p53 in cancer.Here, we identify ...

MicroRNAs (miRNAs) 3 are small (ϳ22-nucleotide) noncoding RNAs that regulate gene expression by binding to partially complementary sites in the 3Ј-untranslated regions (UTRs) of specific mRNAs, thereby promoting degradation and/or repressing translation of target mRNAs. The human genome contains Ͼ2500 unique mature miRNAs (1). Because individual miRNAs typically have multiple targets, it is thought that Ͼ60% of all human genes may be subject to regulation by miRNAs (2). The promiscuity of miRNAs for target RNAs is expected to represent a fundamental mechanism of cross-talk and coordination among signaling networks in development, health, and disease.Among the signaling networks that have been shown in recent years to be regulated by miRNAs are the Toll-like receptors (TLRs) of the innate immune system. Multiple TLRs up-regulate miRNAs, including miR-155, miR-146, miR-21, miR-147, and miR-9, whereas activation of TLR4 by lipopolysaccharide (LPS) down-regulates a distinct set of miRNAs (3, 4). In turn, miRNAs "fine tune" the proinflammatory signaling output of TLR cascades by controlling the expression of TLR pathway members (3, 4). Thus, miR-146 suppresses signaling by multiple TLRs via targeting the common signaling hubs IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 (5), whereas miR-155 has complex effects, repressing the TLR adaptor myeloid differentiation primary response 88 (MyD88) (6, 7) and TAK1-binding protein 2 (8), an upstream activator of the mitogen-activated protein kinases, but also promoting cytokine expression through actions on other targets (3). Although virtually all known examples of TLR regulation by miRNAs operate through direct targeting of TLR pathway components, it is expected that miRNAs may also indirectly impact the innate immune response by regulating other networks that cross-talk with TLRs.miR-33a and miR-33b (present in primates but absent in rodents and lower species in which miR-33a is simply referred to as "miR-33") are now known to be master regulators of cho-