The generation of infectious rabies virus (RV), a non‐segmented negative‐stranded RNA virus of the Rhabdoviridae family, entirely from cloned cDNA is described. Simultaneous intracellular expression of genetically marked full‐length RV antigenome‐like T7 RNA polymerase transcripts and RV N, P and L proteins from transfected plasmids resulted in formation of transcriptionally active nucleocapsids and subsequent assembly and budding of infectious rabies virions. In addition to authentic RV, two novel infectious RVs characterized by predicted transcription patterns were recovered from modified cDNA. Deletion of the entire non‐translated pseudogene region, which is conserved in all naturally occurring RVs, did not impair propagation of the resulting virus in cell culture. This indicates that non‐essential genetic material might be present in the genomes of non‐segmented RNA viruses. The introduction of a functional extra cistron border into the genome of another virus resulted in the transcription of an additional polyadenylated mRNA containing pseudogene sequences. The possibility of manipulating the RV genome by recombinant DNA techniques using the described procedure‐‐potentially applicable also for other negative‐stranded viruses‐‐greatly facilitates the investigation of RV genetics, virus‐host interactions and rabies pathogenesis and provides a tool for the design of new generations of live vaccines.

Summary Virally-based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19ΔG transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion-mutant strain, CVS-N2cΔG, and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19ΔG, the CVS-N2cΔG strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2cΔG strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian central nervous system.

Prostratin is a unique phorbol ester that stimulates protein kinase C activity but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo human immunodeficiency virus type 1 (HIV-1) infection yet up-regulate viral expression from latent proviruses. Prostratin's lack of tumor promotion, coupled with its ability to block viral spread yet induce latent proviral expression, prompted studies to determine whether this compound could serve as an inductive adjuvant therapy for patients treated with highly active antiretroviral therapy (HAART).