Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1.

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hardto-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

Epstein-Barr virus (EBV) has been causally associated with at least five human malignancies. The exact contributions made by EBV to these cancers remain unknown. We demonstrate that one viral protein found in all EBV-associated malignancies, Epstein-Barr nuclear antigen 1 (EBNA-1), is required for survival of one of these cancers, EBV-positive Burkitt's lymphoma. Inhibition of EBNA-1 decreases survival of these tumor cells by inducing apoptosis. Expression of EBNA-1 in uninfected cells also can inhibit apoptosis induced by expression of p53 in the absence of the EBV genome. Our findings demonstrate that EBNA-1 is critical for the continued survival of EBV-associated Burkitt's lymphoma, and, by extension, for the other B cell tumors with which EBV is associated. Efficient inhibitors of EBNA-1's functions would likely prove useful in the therapy of EBV-associated malignancies.

We have demonstrated that Epstein-Barr virus (EBV) confers enhanced growth capability in soft agarose, tumorigenesis in the SCID mouse, and resistance to apoptosis in the Burkitt's lymphoma cell line Akata. Subsequently, we have shown that EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. We constantly observed the upregulation of bcl-2 oncoprotein expression upon EBV infection and expression of EBERs. To test whether these phenotypes were due to the upregulation of bcl-2 expression, we introduced bcl-2 into EBV-negative Akata cells at various levels encompassing the range at which EBV-positive cells expressed it. As cells expressed bcl-2 at higher levels, they became more capable of growing in soft agarose and became resistant to apoptosis. However, clones expressing bcl-2 at a higher level than EBV-positive Akata cells were negative in the tumorigenesis assay in the SCID mouse. On the other hand, introduction of bax into EBV-positive Akata cells reduced the resistance to apoptosis; however, it failed to reduce the growth capability in soft agarose. These data indicate that EBV targets not only bcl-2, but also an unknown pathway(s) to enhance the oncogenic potential of Akata cells.

Characterizing cellular factors involved in the life cycle of human immunodeficiency virus type 1 (HIV-1) is an initial step toward controlling replication of HIV-1. Actin polymerization mediated by the Arp2/3 complex has been found to play a critical role in some pathogens' intracellular motility. We have asked whether this complex also contributes to the viral life cycles including that of HIV-1. We have used both the acidic domains from actin-related protein (Arp) 2/3 complex-binding proteins such as the Wiscott-Aldrich syndrome protein (N-WASP) or cortactin, and siRNA directing toward Arp2 to inhibit viral infection. HIV-1, simian immunodeficiency virus (SIV), and intracellular mature vaccinia virus (IMV) were sensitive to inhibition of the Arp2/3 complex, whereas MLV, HSV-1, and adenovirus were not. Interestingly, pseudotyping HIV-1 with vesicular stomatitis virus G protein (VSV-G) overcame this inhibition. Constitutive inhibition of the Arp2/3 complex in the T-cell line H9 also blocked replication of HIV-1. These data suggested the existence of an Arp2/3 complex-dependent event during the early phase of the life cycles of both primate lentiviruses and IMV. Inhibiting the HIV-1's ability to activate Arp2/3 complex could be a potential chemotherapeutic intervention for acquired immunodeficiency syndrome (AIDS)