Detection of specific reticuloendotheliosis virus sequence and protein from REV-integrated fowlpox virus strains.

The detection is described of reticuloendotheliosis virus (REV) protein in tissue culture of chicken embryonated cells (CEFs) infected with field isolates of fowl poxvirus (FPV). By the polymerase chain reaction (PCR), five out of the six field isolates, but two out of the seven vaccine strains of FPV, were found to have had a 291 bp repeat sequence of REV-LTR integrated in their genomic DNA. An immunofluorescence (IF) method was employed using a monoclonal antibody (MAb) known to specify strain common envelope proteins for REV and allowed to detect the presence of a specific REV protein. The IF results indicate the localization of REV proteins in boundaries defined precisely within cells infected with these field strains of FPV carrying REV (FPV-REV). Furthermore, by immunoblotting (IB) using a chemiluminescent detection kit, the REV protein reacted specifically with the MAb and had a relative molecular mass (RMM) of 62 kDa. The data have the potential to advance substantially the current understanding of the integrated REV in FPV strains; and the identification of a unique protein associated with variant forms of FPV will also offer great potential for identification of novel vaccine candidates for use in poultry against variant forms of FPV.

Cross-packaging of human immunodeficiency virus type 1 vector RNA by spleen necrosis virus proteins: construction of a new generation of spleen necrosis virus-derived retroviral vectors.

The ability of the nonlentiviral retrovirus spleen necrosis virus (SNV) to cross-package the genomic RNA of the distantly related human immunodeficiency virus type 1 (HIV-1) and vice versa was analyzed. Such a model may allow us to further study HIV-1 replication and pathogenesis, as well as to develop safe gene therapy vectors. Our results suggest that SNV can cross-package HIV-1 genomic RNA but with lower efficiency than HIV-1 proteins. However, HIV-1-specific proteins were unable to cross-package SNV RNA. We also constructed SNV-based gag-pol chimeric variants by replacing the SNV integrase with the HIV-1 integrase, based on multiple sequence alignments and domain analyses. These analyses revealed that there are conserved domains in all retroviral integrase open reading frames (orf), despite the divergence in the primary sequences. The transcomplementation assays suggested that SNV proteins recognized one of the chimeric variants. This demonstrated that HIV-1 integrase is functional in the SNV gag-pol orf with a lower transduction efficiency, utilizing homologous (SNV) RNA, as well as the heterologous vector RNA of HIV-1. These findings suggest that homology in the conserved sequences of the integrase protein may not be fully competent in the replacement of protein(s) from one retrovirus to another, and there are likely several other factors involved in each of the steps related to replication, integration, and infection. However, further studies to dissect the gag-pol region will be critical for understanding the mechanisms involved in the cleavage of reverse transcriptase, RNase H, and integrase. These studies should provide further insight into the design and development of novel molecular approaches to block HIV-1 replication and to construct a new generation of SNV-based vectors.

Cell-type-specific gene delivery into neuronal cells in vitro and in vivo.

The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain.

Avian I kappa B alpha is transcriptionally induced by c-Rel and v-Rel with different kinetics.

The Rel/NF-kappa B family of transcription factors participates in the regulation of genes involved in defense responses, inflammation, healing and regeneration processes, and embryogenesis. The control of the transcriptional activation potential of the Rel/NF-kappa B proteins is mediated, in part, by their association with inhibitory proteins of the I kappa B family. This association results in the cytoplasmic retention of these factors until the cell receives a proper stimulatory signal. The I kappa B alpha gene is a target for regulation by the Rel/NF-kappa B proteins and is in fact upregulated in response to Rel/NF-kappa B activation. A naturally occurring oncogenic variant of the Rel/NF-kappa B family, v-rel, transforms avian lymphocytes, bone marrow cells, monocytes, and fibroblasts. Avian I kappa B alpha expression is upregulated in cells transformed by v-Rel. Avian I kappa B alpha is also upregulated in fibroblasts overexpressing c-Rel and oncogenic variants of c-Rel. c-Rel, a carboxy-terminally truncated variant of c-Rel, and v-Rel are all able to directly transactivate the expression of the avian I kappa B alpha gene. However, c-Rel was the most potent activator of this gene, and the induction of I kappa B alpha expression showed faster kinetics in cells overexpressing c-Rel than in those overexpressing v-Rel. The regulation of I kappa B alpha induction by the Rel proteins was shown to be dependent on a 362-bp region of the I kappa B alpha promoter that contains two potential NF-kappa B binding sites and one AP-1-like binding site. Results of electrophoretic mobility shift assays using these NF-kappa B binding sites indicate that major changes in the profile of DNA binding complexes in fibroblasts overexpressing v-Rel correlated temporally with the kinetic changes in v-Rel's ability to activate the expression of the I kappa B alpha gene.