This study reports that recombinant adenovirus-mediated human bone morphogenetic protein-2 gene transfer can induce mesenchymal progenitor cell differentiation and bone formation. The recombinant adenovirus with the human bone morphogenetic protein-2 gene was constructed, and mature human bone morphogenetic protein-2 expression mediated by adenovirus gene transfer was detected by specific antibody. Under adenovirus-mediated bone morphogenetic-protein gene transfer, mesenchymal progenitor cell line C3H/10T 1/2 showed cell proliferation dependent on adenovirus bone morphogenetic-protein dose. The C3H/10T 1/2 cells transduced by adenovirus bone morphogenetic protein also exhibited differentiation to osteoblast phenotype, which indicates alkaline phosphatase activity. Injection of the C3H/10T 1/2 cells into the thigh muscles of nude mice led to ossicle development detectable on radiographs. Histological analysis indicated that the new ossicles that developed in the thigh muscles of the mice had different osseous components including bone trabeculae, bone marrow, and chondrified tissue. The results of this study demonstrate the potential for gene therapy by adenovirus-mediated bone morphogenetic-protein gene transfer.
The nucleus represents a cellular compartment where the discrimination of self from non-self nucleic acids is vital. While emerging evidence establishes a nuclear non-self DNA sensing paradigm, the nuclear sensing of non-self RNA, such as that from nuclear-replicating RNA viruses, remains unexplored. Here, we report the identification of nuclear-resident RIG-I actively involved in nuclear viral RNA sensing. The nuclear RIG-I, along with its cytoplasmic counterpart, senses influenza A virus (IAV) nuclear replication leading to a cooperative induction of type I interferon response. Its activation signals through the canonical signaling axis and establishes an effective antiviral state restricting IAV replication. The exclusive signaling specificity conferred by nuclear RIG-I is reinforced by its inability to sense cytoplasmic-replicating Sendai virus and appreciable sensing of hepatitis B virus pregenomic RNA in the nucleus. These results refine the RNA sensing paradigm for nuclear-replicating viruses and reveal a previously unrecognized subcellular milieu for RIG-I-like receptor sensing.
DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus-induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation. IMPORTANCEDDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in the influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza virus infection.
Elimination of infected cells by programmed cell death is a well-recognized host defense mechanism to control the spread of infection. In addition to apoptosis, necroptosis is also one of the mechanisms of cell death that can be activated by viral infection. Activation of necroptosis leads to the phosphorylation of mixed-lineage kinase domain-like protein (MLKL) by receptor-interacting protein kinase 3 (RIPK3) and results in MLKL oligomerization and membrane translocation, leading to membrane disruption and a loss of cellular ion homeostasis. It has recently been reported that influenza A virus (IAV) infection induces necroptosis. However, the underlying mechanism of the IAV-mediated necroptosis process, particularly the roles of IAV proteins in necroptosis, remains unexplored. Here, we report that IAV infection induces necroptosis in macrophages and epithelial cells. We demonstrate that the NS1 protein of IAV interacts with MLKL. Coiled-coil domain 2 of MLKL has a predominant role in mediating the MLKL interaction with NS1. The interaction of NS1 with MLKL increases MLKL oligomerization and membrane translocation. Moreover, the MLKL-NS1 interaction enhances MLKLmediated NLRP3 inflammasome activation, leading to increased interleukin-1 (IL-1) processing and secretion. IMPORTANCE Necroptosis is a programmed cell death that is inflammatory in nature owing to the release of danger-associated molecular patterns from the ruptured cell membrane. However, necroptosis also constitutes an important arm of host immune responses. Thus, a balanced inflammatory response determines the disease outcome. We report that the NS1 protein of IAV participates in necroptosis by interacting with MLKL, resulting in increased MLKL oligomerization and membrane translocation. These results reveal a novel function of the NS1 protein and the mechanism by which IAV induces necroptosis. Moreover, we show that this interaction enhances NLRP3 inflammasome activation and IL-1 processing and secretion. This information may contribute to a better understanding of the role of necroptosis in IAV-induced inflammation.
Limitations of the current vaccines and antivirals against influenza A virus (IAV) pandemic underscore the urgent need for developing novel anti-influenza strategies. RNA interference (RNAi) induced by small interfering RNA (siRNA) has become a powerful new means to inhibit viral infection in a gene-specific manner. However, the efficacy of the siRNA delivery platform and the relatively high cost of administration have hindered widespread application of siRNA. In this study, we developed a microRNA (miRNA)-30-based lentivirus delivery system by embedding a synthetic short hairpin RNA (shRNA) stem into the context of endogenous precursor of miRNA-30 (shRNAmir) to express a silencer of the influenza gene. We showed that the miRNA-based lentivirus vector was able to express and process a single nucleoprotein (NP)-targeting shRNAmir, which could potently inhibit IAV replication. We further showed that miRNA-based lentivirus vector carrying tandemly linked NP and polymerase PB1 shRNAmirs could express and process double shRNAmirs. Despite the relatively low levels of NP and PB1 miRNAs produced in the stably transduced cells, the combination of two miRNAs exerted a great degree of inhibition on influenza infection. Given the advantage of combinatorial RNAi in preventing emergence of mutant virus, miRNA-based lentiviral vectors are valuable tools for anitiviral activities. To the best of our knowledge, this is the first study demonstrating that a miRNA-based RNAi strategy can be applied for better control of influenza virus infection.
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