No abstract

PRV-Bartha ͉ PRV 152 ͉ suprachiasmatic nucleus ͉ retinal ganglion cell T he use of neurotropic alphaherpesviruses has greatly advanced our ability to visualize ensembles of neurons that contribute to multisynaptic circuits in the central nervous system (CNS) (1). In particular, the attenuated vaccine strain of pseudorabies virus (PRV-Bartha) has been used successfully as a self-amplifying neural tracer after peripheral application or direct injection into brain parenchyma (2-4). The usefulness of PRV as a neural tracer relies on its ability to infect chains of hierarchically connected neurons via specific transsynaptic passage of progeny virus rather than infection by lytic release into the extracellular space (4, 5). Typically, PRV infects the CNS by invading neurons in the periphery and then replicating and spreading to the CNS via synaptically linked neurons. However, PRV can also invade neurons through their somata if the viral concentration is sufficient (6), as evidenced by primary infection of retinal ganglion cells (RGCs) after intravitreal injection of PRV (7-9). Infection of RGCs with PRV-Bartha, followed by viral replication, results in the anterograde transsynaptic infection of a restricted set of retinorecipient neurons [i.e., suprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL), pretectum (PT), and lateral terminal nucleus]. Intravitreal injection of the wild-type virus, PRV-Becker, produces transneuronal infection of neurons in all retinorecipient subcortical regions (7). The factors that determine the specificity of PRV-Bartha infection of selective retinorecipient targets are not completely understood, although deletion of specific genes in PRV-Becker results in a restricted neurotropism identical to that demonstrated with PRV-Bartha (10).Although viral transsynaptic tracing represents an important methodological advance for the analysis of CNS circuits, functional analysis of virus-infected neurons has been limited to sensory or sympathetic ganglia in culture (11, 12) because of the inability to identify virus-infected neurons in situ. Analyses of electrophysiological properties of neurons, in the context of known functional connections of the recorded neuron, would represent a further important methodological advance for the analysis of CNS circuits.The development of retrogradely transported fluorescent tracers has allowed investigators to examine the physiology of neurons with known projections (13-15). However, such studies usually require direct and accurate injection of a target region followed by retrograde transport to identify first-order neurons projecting to the target. Other ''prelabeling'' studies have used constructs of green fluorescent protein to label neurons that possess a particular genetic phenotype, such as expression of gonadotropin-releasing hormone (16). Both of these techniques have allowed examination of the physiological properties of neurons in vitro that possess presumed anatomical or functional correlates in the intact animal.We now report a neuron-labeling...

cHerpes simplex virus 2 (HSV-2) is an important human pathogen that is the major cause of genital herpes infections and a significant contributor to the epidemic spread of human immunodeficiency virus infections. The UL21 gene is conserved throughout the Alphaherpesvirinae subfamily and encodes a tegument protein that is dispensable for HSV-1 and pseudorabies virus replication in cultured cells; however, its precise functions have not been determined. To investigate the role of UL21 in the HSV-2 replicative cycle, we constructed a UL21 deletion virus (HSV-2 ⌬UL21) using an HSV-2 bacterial artificial chromosome, pYEbac373. HSV-2 ⌬UL21 was unable to direct the production of infectious virus in noncomplementing cells, whereas the repaired HSV-2 ⌬UL21 strain grew to wild-type (WT) titers, indicating that UL21 is essential for virus propagation. Cells infected with HSV-2 ⌬UL21 demonstrated a 2-h delay in the kinetics of immediate early viral gene expression. However, this delay in gene expression was not responsible for the inability of cells infected with HSV-2 ⌬UL21 to produce virus insofar as late viral gene products accumulated to WT levels by 24 h postinfection (hpi). Electron and fluorescence microscopy studies indicated that DNA-containing capsids formed in the nuclei of ⌬UL21-infected cells, while significantly reduced numbers of capsids were located in the cytoplasm late in infection. Taken together, these data indicate that HSV-2 UL21 has an early function that facilitates viral gene expression as well as a late essential function that promotes the egress of capsids from the nucleus.

The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.