Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Sm segments in activated splenic B cells. Herein we examine whether mutations are also introduced into the downstream acceptor S regions prior to switch recombination, but ®nd very few mutations in GL Sg3 and Sg1 regions in activated B cells. These data suggest that switch recombination initiates in the Sm segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and speci®city of mutations in GL and recombined Sm segments differ, suggesting different repair mechanisms. Mutations in recombined Sm regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Sm do not. Additionally, induction conditions affect mutation speci®city within the GL Sm segment. Mutations are most frequent near the S±S junctions and decrease rapidly with distance from the junction. Finally, we ®nd that mice expressing a transgene for terminal deoxynucleotidyl transferase (TdT) have nucleotide insertions at S±S junctions, indicating that the recombining DNA ends are accessible to end-processing enzyme activities.

Gene expression among the nonsegmented negative-strand RNA viruses is controlled by distance from the single transcriptional promoter, so the phenotypes of these viruses can be systematically manipulated by gene rearrangement. We examined the potential of gene rearrangement as a means to develop live attenuated vaccine candidates against Vesicular stomatitis virus (VSV) in domestic swine, a natural host for this virus. The results showed that moving the nucleocapsid protein gene away from the single transcriptional promoter attenuated and ultimately eliminated the potential of the virus to cause disease. Combining this change with relocation of the surface glycoprotein gene yielded a vaccine that protected against challenge with wild-type VSV. By incremental manipulation of viral properties, gene rearrangement provides a new approach to generating live attenuated vaccines against this class of virus.

Vesicular stomatitis virus (VSV) is the prototype of the Rhabdoviridae and contains nonsegmented negativesense RNA as its genome. The 11-kb genome encodes five genes in the order 3-N-P-M-G-L-5, and transcription is obligatorily sequential from the single 3 promoter. As a result, genes at promoter-proximal positions are transcribed at higher levels than those at promoter-distal positions. Previous work demonstrated that moving the gene encoding the nucleocapsid protein N to successively more promoter-distal positions resulted in stepwise attenuation of replication and lethality for mice. In the present study we investigated whether moving the gene for the attachment glycoprotein G, which encodes the major neutralizing epitopes, from its fourth position up to first in the gene order would increase G protein expression in cells and alter the immune response in inoculated animals. In addition to moving the G gene alone, we also constructed viruses having both the G and N genes rearranged. This produced three variant viruses having the orders 3-G-N-P-M-L-5 (G1N2), 3-P-M-G-N-L-5 (G3N4), and 3-G-P-M-N-L-5 (G1N4), respectively. These viruses differed from one another and from wild-type virus in their levels of gene expression and replication in cell culture. The viruses also differed in their pathogenesis, immunogenicity, and level of protection of mice against challenge with wild-type VSV. Translocation of the G gene altered the kinetics and level of the antibody response in mice, and simultaneous reduction of N protein expression reduced replication and lethality for animals. These studies demonstrate that gene rearrangement can be exploited to design nonsegmented negative-sense RNA viruses that have characteristics desirable in candidates for live attenuated vaccines.The order Mononegavirales is composed of four families, Rhabdoviridae, Paramyxoviridae, Filoviridae, and Bornaviridae. The viruses in these families contain a single strand of nonsegmented negative-sense RNA and are responsible for a wide range of significant diseases in fish, plants, and animals (31). Viral gene expression is controlled at the level of transcription by the order of the genes on the genome relative to the single 3Ј promoter. Gene order throughout the Mononegavirales is highly conserved; genes encoding products required in stoichiometric amounts for replication are always at or near the 3Ј end of the genome, while those whose products are needed in catalytic amounts are more promoter distal.Vesicular stomatitis virus (VSV) is the prototypic virus of the Rhabdoviridae. Its 11-kb genome has five genes which encode the five structural proteins of the virus: the nucleocapsid protein, N, which is required in stoichiometric amounts for encapsidation of the replicated RNA; the phosphoprotein, P, which is a cofactor of the RNA-dependent RNA polymerase, L; the matrix protein, M; and the attachment glycoprotein, G. The order of genes in the genome is 3Ј-N-P-M-G-L-5Ј, and previous work has shown that expression is obligatorily sequential from a sing...

Transcription of vesicular stomatitis virus is controlled by the position of a gene relative to the single 3 genomic promoter: promoter-proximal genes are transcribed at higher levels than those in more 5 distal positions. In previous work, we generated viruses having rearranged gene orders. These viruses had the promoter-proximal gene that encodes the nucleocapsid protein, N, moved to the second or fourth position in the genome in combination with the glycoprotein gene, G, moved from its usual promoter-distal fourth position to the first or third position. This resulted in three new viruses identified by the positions of the N and G genes in the gene order: G3N4, G1N4, and G1N2. The viruses G3N4 and G1N4 were attenuated for lethality in mice. In the present study, we addressed the basis of this attenuation by measuring the ability of each of the rearranged viruses to travel to and replicate in the olfactory bulb and brain following intranasal inoculation. In addition, the neuropathogenicity, serum cytokine levels, and immunoglobulin G isotype profiles in infected mice were determined. All the viruses reached the olfactory bulb and brain, but the outcomes of these infections were dramatically different. Viruses N1G4(wt) and G1N2 caused lethal encephalitis in 100% of animals within 7 days postinoculation; however, viruses G3N4 and G1N4 were cleared from the brain by 7 days postinoculation and all animals survived without apparent distress. The viruses differed in the distribution and intensity of lesions produced and the type and levels of cytokines induced. Animals inoculated with N1G4(wt) or G1N2 displayed extensive encephalitis and meningitis and had elevated levels of serum gamma interferon compared to what was seen with G3N4-or G1N4-infected mice. In contrast to what occurred with intranasal inoculation, all four viruses caused lethal encephalitis when administered by direct inoculation to the brain, a route that circumvents the majority of the host immune response, demonstrating that G3N4 and G1N4 were not deficient in their abilities to cause disease in the brain. These findings indicate that gene rearrangement and its consequent alteration of gene expression can, without any other changes, alter the viral spread and cytokine response following intranasal infection. Vesicular stomatitis virus (VSV) is the prototypic virus of the orderMononegavirales, which is composed of four families, Rhabdoviridae, Paramyxoviridae, Filoviridae, and Bornaviridae. The viruses in these families are responsible for a wide range of significant diseases in animals, fish, and plants. Viruses in all four families have a single strand of nonsegmented negativesense RNA as their genome (38). VSV is a member of the Rhabdoviridae, and its 11-kb genome has five genes encoding the five structural proteins of the virus: the nucleocapsid protein, N; the phosphoprotein, P; the matrix, M; the glycoprotein, G; and the RNA-dependent RNA polymerase, L. The order of the genes in the genome is 3Ј-N-P-M-G-L-5Ј, and the relative order of these ba...

The ZFY protein is a member of one of the most interesting classes of polydactyl zinc finger proteins. It has a domain of 13 tandem zinc fingers that is organized with an internal repeat of odd-even finger pairs. It has been proposed that each finger pair interacts with six base pairs within a turn of the double helix, the downstream linker crossing the minor groove to place the next finger pair on the following turn of the DNA. Yet putative binding sites for the full-length ZFY protein appear to consist of a six-base AGGCCY consensus sequence that is present in one or two copies. In this study the equilibrium binding of two ZFY-derived zinc finger peptides to 4R DNA with tandem copies of the consensus sequence was investigated. The ZFY5 peptide contains fingers 5-13, including four odd-even finger pairs, and the ZFY11 peptide contains fingers 11-13 and has one odd-even finger pair. Both peptides bound to 4R DNA with equal affinities, forming a bimolecular complex that is mediated by the downstream AGGCCY motif. The additional odd-even finger pairs in ZFY5 made no measurable difference in the mechanism of DNA binding compared to ZFY11. The effects on the DNA-protein interaction of mutations in the 4R DNA and in the key alpha-helical residues of fingers 11-13 indicate that the binding of ZFY to DNA is mediated by the interaction of the GGCC core base pairs with fingers 12 and 13. These results demonstrate that the even-odd repeats in the ZFY zinc finger domain do not make significant contributions to DNA binding.