In vivo dynamics of AAV-mediated gene delivery to sensory neurons of the trigeminal ganglia.

The ability to genetically manipulate trigeminal ganglion (TG) neurons would be useful in the study of the craniofacial nervous system and latent alphaherpesvirus infections. We investigated adeno-associated virus (AAV) vectors for gene delivery to the TG after intradermal whiskerpad delivery in mice. We demonstrated that AAV vectors of serotypes 1, 7, 8, and 9 trafficked from the whiskerpad into TG neurons and expressed transgenes within cell bodies and axons of sensory neurons in all three branches of the TG. Gene expression was highest with AAV1, and steadily increased over time up to day 28. Both constitutive and neuronal-specific promoters were able to drive transgene expression in TG neurons. Levels of vector genomes in the TG increased with input dose, and multiple transgenes could be co-delivered to TG neurons by separate AAV vectors. In conclusion, AAV1 vectors are suitable for gene delivery to TG sensory neurons following intradermal whiskerpad injection.

DNA cleavage enzymes for treatment of persistent viral infections: recent advances and the pathway forward.

Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application.

Assessment of mitochondrial toxicity by analysis of mitochondrial protein expression in mononuclear cells.

BACKGROUND: Real-time PCR has quantified decreased mitochondrial DNA levels in association with nucleoside reverse transcriptase inhibitor (NRTI) therapy of HIV-infected populations. However, real-time PCR is best suited to distinguish log differences in an analyte. In an effort to monitor individuals in more detail, we developed a flow cytometric assay to gauge mitochondrial function. METHODS: Flow cytometric quantification of a mitochondrial DNA-encoded mitochondrial protein (cytochrome c oxidase subunit I (COX-I)) and a nuclear DNA-encoded mitochondrial protein [ATP synthase subunit D (Sub-D)] was optimized and validated. RESULTS: Intra-assay and interassay variability was low using peripheral blood mononuclear cells (PBMCs) (CV of 6.15% for COX-I and 7.11% Sub-D, and 9.38% and 9.83% for COX-I and Sub-D, respectively). Mitochondrial protein depletion was evident with in vitro treatment of cells with ethidium bromide (EtBr) and zalcitabine (ddC). Mitochondrial protein expression in 40 healthy adults clustered tightly. Depletion of mitochondrial protein, however, was neither detected in cryopreserved PBMC from NRTI-treated children (n = 9) nor in adults with a history of symptoms consistent with mitochondrial toxicity or ongoing treatment with didanosine (ddI) or stavudine (d4T) (n = 51). CONCLUSIONS: A validated flow cytometric assay allows simultaneous detection of mitochondrial DNA and nuclear DNA encoded proteins at the single cell level, offering a method to monitor for mitochondrial function. Prospective studies are required to evaluate whether mitochondrial protein loss is observed in at-risk patients prior to the onset of symptoms from mitochondrial dysfunction.

The antiapoptotic herpes simplex virus glycoprotein J localizes to multiple cellular organelles and induces reactive oxygen species formation.

The Us5 gene of herpes simplex virus (HSV) encodes glycoprotein J (gJ). The only previously reported function of gJ was its ability to inhibit apoptosis. However, the mechanism by which gJ prevents apoptosis is not understood, and it is not known whether gJ mediates additional cellular effects. In this study, we evaluated the expression, localization, and cellular effects of Us5/gJ. Us5 was first expressed 4 h after infection. gJ was detectable at 6 h and was expressed in glycosylated and unglycosylated forms. Us5 was regulated as a late gene, with partial dependency on DNA replication for expression. Us5 expression was delayed in the absence of ICP22; furthermore, expression of Us5 in trans protected cells from apoptosis induced by an HSV mutant with deletion of ICP27, suggesting that the antiapoptotic effects of ICP22 and ICP27 are mediated in part through effects on gJ expression. Within HSV-infected or Us5-transfected cells, gJ was distributed widely, especially to the endoplasmic reticulum, trans-Golgi network, and early endosomes. gJ interacted with F(o)F(1) ATP synthase subunit 6 by a yeast two-hybrid screen and had strong antiapoptotic effects, which were mediated by protein rather than mRNA. Antiapoptotic activity required the extracellular and transmembrane domains of gJ, but not the intracellular domain. Consistent with inhibition of F(o)F(1) ATP synthase function, Us5 was required for HSV-induced reactive oxygen species (ROS) formation, and gJ was sufficient to induce ROS in Us5-transfected cells. Thus, HSV gJ is a multifunctional protein, modulating other cellular processes in addition to inhibition of apoptosis.

Apoptosis and antigen receptor function in T and B cells following exposure to herpes simplex virus.

T cells are an essential component of the immune response against herpes simplex virus (HSV) infection. We previously reported that incubation of T cells with HSV-infected fibroblasts inhibits subsequent T cell antigen receptor signal transduction. In the current study, we found that incubation of T cells with HSV-infected fibroblasts also leads to apoptosis in exposed T cells. Apoptosis was observed in Jurkat cells, a T cell leukemia line, and also in CD4(+) cells isolated from human peripheral blood mononuclear cells. Direct infection of these cells with HSV also resulted in apoptosis. Clinical isolates of both HSV type 1 and 2 induced apoptosis in infected T cells at comparable levels to cells infected with laboratory strains of HSV, suggesting an immune evasion mechanism that may be clinically relevant. Further understanding of these viral immune evasion mechanisms could be exploited for better management of HSV infection.