Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus.

Naturally occurring strains of Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. Here, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of reverse genetics. Mice bearing s.c. implanted CT26 tumors were treated with intratumoral (i.t.) injections of a recombinant NDV modified to contain a highly fusogenic F protein. These treated mice exhibited significant reduction in tumor development compared with mice treated with the unmodified virus. Furthermore, mice in a CT26 metastatic tumor model treated with an i.v. injection of the genetically engineered NDV exhibited prolonged survival compared with wild-type control virus. In addition, we examined whether the oncolytic properties of NDV could be improved by expression of immunostimulatory molecules. In this regard, we engineered several NDVs to express granulocyte macrophage colony-stimulating factor, IFN-gamma, interleukin 2 (IL-2), or tumor necrosis factor alpha, and evaluated their therapeutic potential in an immunocompetent colon carcinoma tumor model. Mice bearing s.c. CT26 tumors treated with i.t. injections of recombinant NDV expressing IL-2 showed dramatic reductions in tumor growth, with a majority of the mice undergoing complete and long-lasting remission. Our data show the use of reverse genetics to develop enhanced recombinant NDV vectors as effective therapeutic agents for cancer treatment.

Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses.

Most RNA viruses that lack a DNA phase replicate in the cytoplasm. However, several negative-stranded RNA viruses such as influenza, Thogoto, and Borna disease viruses replicate their RNAs in the nucleus, taking advantage of the host cell's nuclear machinery. A challenge faced by these viruses is the trafficking of viral components into and out of the nucleus through the nuclear membrane. The genomic RNAs of these viruses associate with proteins to form large complexes called viral ribonucleoproteins (vRNPs), which exceed the size limit for passive diffusion through the nuclear pore complex (NPC). To insure efficient transport across the nuclear membrane, these viruses use nuclear import and export signals exposed on the vRNPs. These signals recruit the cellular import and export complexes, which are responsible for the translocation of the vRNPs through the NPC. The ability to control the direction of vRNP trafficking throughout the viral life cycle is critical. Various mechanisms, ranging from simple post-translational modification to complex, sequential masking-and-exposure of localization signals, are used to insure the proper movement of the vRNPs.

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein.

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.

Newcastle disease virus V protein is a determinant of host range restriction.

It has been demonstrated that the V protein of Newcastle disease virus (NDV) functions as an alpha/beta interferon (IFN-alpha/beta) antagonist (M. S. Park, M. L. Shaw, J. Muñoz-Jordan, J. F. Cros, T. Nakaya, N. Bouvier, P. Palese, A. García-Sastre, and C. F. Basler, J. Virol. 77:1501-1511, 2003). We now show that the NDV V protein plays an important role in host range restriction. In order to study V functions in vivo, recombinant NDV (rNDV) mutants, defective in the expression of the V protein, were generated. These rNDV mutants grow poorly in both embryonated chicken eggs and chicken embryo fibroblasts (CEFs) compared to the wild-type (wt) rNDV. However, insertion of the NS1 gene of influenza virus A/PR8/34 into the NDV V(-) genome [rNDV V(-)/NS1] restores impaired growth to wt levels in embryonated chicken eggs and CEFs. These data indicate that for viruses infecting avian cells, the NDV V protein and the influenza NS1 protein are functionally interchangeable, even though there are no sequence similarities between the two proteins. Interestingly, in human cells, the titer of wt rNDV is 10 times lower than that of rNDV V(-)/NS1. Correspondingly, the level of IFN secreted by human cells infected with wt rNDV is much higher than that secreted by cells infected with the NS1-expressing rNDV. This suggests that the IFN antagonist activity of the NDV V protein is species specific. Finally, the NDV V protein plays an important role in preventing apoptosis in a species-specific manner. The rNDV defective in V induces apoptotic cell death more rapidly in CEFs than does wt rNDV. Taken together, these data suggest that the host range of NDV is limited by the ability of its V protein to efficiently prevent innate host defenses, such as the IFN response and apoptosis.

Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins.

We have generated a recombinant Newcastle disease virus (NDV) that expresses the green fluorescence protein (GFP) in infected chicken embryo fibroblasts (CEFs). This virus is interferon (IFN) sensitive, and pretreatment of cells with chicken alpha/beta IFN (IFN-alpha/beta) completely blocks viral GFP expression. Prior transfection of plasmid DNA induces an IFN response in CEFs and blocks NDV-GFP replication. However, transfection of known inhibitors of the IFN-alpha/beta system, including the influenza A virus NS1 protein and the Ebola virus VP35 protein, restores NDV-GFP replication. We therefore conclude that the NDV-GFP virus could be used to screen proteins expressed from plasmids for the ability to counteract the host cell IFN response. Using this system, we show that expression of the NDV V protein or the Nipah virus V, W, or C proteins rescues NDV-GFP replication in the face of the transfection-induced IFN response. The V and W proteins of Nipah virus, a highly lethal pathogen in humans, also block activation of an IFN-inducible promoter in primate cells. Interestingly, the amino-terminal region of the Nipah virus V protein, which is identical to the amino terminus of Nipah virus W, is sufficient to exert the IFN-antagonist activity. In contrast, the anti-IFN activity of the NDV V protein appears to be located in the carboxy-terminal region of the protein, a region implicated in the IFN-antagonist activity exhibited by the V proteins of mumps virus and human parainfluenza virus type 2.