Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.

Transfection of wounds with DNA-encoding growth factors has the potential to improve healing, but current means of nonviral gene delivery are inefficient. Repeated high doses of DNA, necessary to achieve reliable gene expression, are detrimental to healing. We assessed the ability of in vivo electroporation to enhance gene expression. Full-thickness cutaneous excisional wounds were created on the dorsum of female mice. A luciferase- encoding plasmid driven by a CMV promoter was injected at the wound border. Following plasmid administration, electroporative pulses were applied to injection sites. Pulse parameters were varied over a range of voltage, duration, and number. Animals were euthanized at intervals after transfection and the luciferase activity measured. Application of electric pulses consistently increased luciferase expression. The electroporative effect was most marked at a plasmid dose of 50 micro g, where an approximate tenfold increase was seen. Six 100- micro s-duration pulses of 1750 V/cm were found to be the most effective in increasing luciferase activity. High numbers of pulses tended to be less effective than smaller numbers. This optimal electroporation regimen had no detrimental effect on wound healing. We conclude that electroporation increases the efficiency of transgene expression and may have a role in gene therapy to enhance wound healing.