Annexation of the interchromosomal space during viral infection.

The nucleus is known to be compartmentalized into units of function, but the processes leading to the spatial organization of chromosomes and nuclear compartments are not yet well defined. Here we report direct quantitative analysis of the global structural perturbations of interphase chromosome and interchromosome domain distribution caused by infection with herpes simplex virus-1 (HSV-1). Our results show that the peripheral displacement of host chromosomes that correlates with expansion of the viral replication compartment (VRC) is coupled to a twofold increase in nuclear volume. Live cell dynamic measurements suggest that viral compartment formation is driven by the functional activity of viral components and underscore the significance of spatial regulation of nuclear activities.

Principles of homeostasis in governing virus activation and latency.

The goal of our work is to understand, from the molecular to the organismal level, the principles that drive and sustain lifelong infection by viruses. These infectious agents live in a dynamic equilibrium (homeostasis) with their hosts in which both immune and nonimmune pathways contribute to viral homeostasis. Disruption of these pathways can have dramatic consequences on pathogenesis. Immune responses to infection provide a vital countermeasure by the host but are nonsterilizing. They effect an essential and primary control mechanism for viral growth. Essential nonimmune pathways for effecting control of a viral life cycle relate to the obligate dependency of the virus on its host. For these reasons, we view infections as a highly dynamic interplay that takes place between the pathogen and host. This, in many cases, leads to the establishment of an incurable lifelong infection that remains benign but can become life threatening once key homeostatic pathways are disrupted. We discuss these issues in the context of our studies using cytomegalovirus as a clinically relevant pathogen.

DNA immunization confers protection against murine cytomegalovirus infection.

The murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) encodes an 89-kDa phosphoprotein (pp89) which plays a key role in protecting BALB/c mice against the lethal effects of the MCMV infection. In this report, we have addressed the question of whether "naked DNA" vaccination with a eukaryotic expression vector (pcDNA-89) that contains the MCMV IE1 gene driven by a strong enhancer/promoter can confer protection. BALB/c mice were immunized intradermally with pcDNA-89 or with the plasmid backbone pcDNAI/Amp (pcDNA) and then challenged 2 weeks later with either a lethal or a sublethal intraperitoneal dose of the K181 strain of MCMV. Variable results were obtained for the individual experiments in which mice received a lethal challenge. In four separate trials, an average of 63% of the mice immunized with pcDNA-89 survived, compared with 18% of the mice immunized with pcDNA. However, in two other trials there was no specific protection. The results of experiments in which mice were injected with a sublethal dose of MCMV were more consistent, and significant decreases in viral titer in the spleen and salivary glands of pcDNA-89-immunized mice were observed, relative to controls. At the time of peak viral replication, titers in the spleens of immunized mice were reduced 18- to >63-fold, while those in the salivary gland were reduced approximately 24- to 48-fold. Although DNA immunization elicited only a low level of seroconversion in these mice, by 7 weeks postimmunization the mice had generated a cytotoxic T-lymphocyte response against pp89. These results suggest that DNA vaccination with selected CMV genes may provide a safe and efficient means of immunizing against CMV disease.

In vivo transduction of photoreceptors or ciliary body by intravitreal injection of pseudotyped adenoviral vectors.

Strategies for retargeting adenoviral (Ad) vectors have been developed, but their in vivo efficacy remains to be demonstrated. Gene delivery to specific ocular cell types represents an approach to treating many diseases that cause irreversible blindness. One of these cell types, the photoreceptor (PR), is not infected by standard Ad5-based vectors. We evaluated gene delivery after intraocular injection of Ads pseudotyped with three different fiber proteins and found three distinct patterns of infection. An intravitreally injected Ad5 vector readily infected the iris, corneal endothelium, and ciliary body, while few cells in the retina expressed transgene product. In contrast, an Ad3-pseudotyped virus selectively transduced ciliary body, of interest for treating diseases such as glaucoma. A vector pseudotyped with the fiber protein of Ad37 transduced PRs as well as ciliary body. This finding has potential application to the treatment of retinal degenerative or neovascular diseases. These studies demonstrate cell type-selective gene delivery in vivo with retargeted Ads, provide information about the cellular tropisms of several Ad serotypes, and should lead to improved strategies for preserving vision.