Understanding the development of cortical interneuron phenotypic diversity is critical because interneuron dysfunction has been implicated in several neurodevelopmental disorders. Here, tyrosine hydroxylase (TH)-immunoreactive neurons in the developing and adult rat cortex were characterized in light of findings regarding interneuron neurochemistry and development. Cortical THimmunoreactive neurons were first observed two weeks postnatally and peaked in number three weeks after birth. At subsequent ages, the number of these cell profiles was gradually reduced, and they were seen less frequently in adults. No DNA fragmentation or active caspase 3 was observed in cortical TH cells at any age examined, eliminating cell death as an explanation for the decrease in cell number. Although cortical TH cells reportedly fail to produce subsequent catecholaminergic enzymes, we found that the majority of these cells at all ages contained phosphorylated TH, suggesting that the enzyme may be active and producing L-DOPA as an end-product. Morphological criteria and colocalization of some TH cells with glutamic acid decarboxylase suggests that these cells are interneurons. Previously, parvalbumin, somatostatin, and calretinin were demonstrated in nonoverlapping subsets of interneurons. Cortical TH neurons colocalized with calretinin but not with parvalbumin or somatostatin. These findings suggest that the transitory increase in TH cell number is not due to cell death but possibly due to alterations in the amount of detectable TH present in these cells, and that at least some cortical TH-producing interneurons belong to the calretinin-containing subset of interneurons that originate developmentally in the caudal ganglionic eminence.

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH 2 -terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH 2 -terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5=-3= polymerase activity. We identified three pre-NH 2 -terminal Pol mutants that exhibited 5=-3= polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants Pol⌬N43 and Pol⌬N52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA 6 , in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the pol⌬N43 mutant displayed replication kinetics similar to those of wild-type virus, while pol⌬N52 and polA 6 mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both pol⌬N52 and polA 6 viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH 2 -terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.

Murine gammaherpesvirus 68 (MHV68) infection of laboratory strains of mice has provided a tractable small animal model for dissecting gammaherpesvirus pathogenesis. The MHV68 latency associated antigen M2 promotes viral latency establishment in germinal center (GC) B cells and plays an important role in virus infection of plasma cells (PCs), which is linked to virus reactivation. More recently, M2 has been highlighted as a potent immunomodulatory molecule capable of hindering both cell-mediated and humoral immunity to MHV68 infection and subsequent challenges. M2 expression in B cells results in activation of B cell receptor signaling pathways that promote proliferation, differentiation, and cytokine production—a hallmark of gammaherpesviruses. In this study, we utilized an adoptive transfer model to explore the biological consequence of M2 expression in activated B cells in vivo. Secondly, we engineered and validated two independent MHV68 M2 reporter viruses that track M2 protein expression in latently infected B cells during infection. Here we demonstrate that upon adoptive transfer into naive mice, M2 expression promotes activated primary B cells to competitively establish residency in the spleen as either a GC B cell or a PC, most notably in the absence of an ongoing GC reaction. Moreover, M2 antigen drives robust PC differentiation and IL10 production in vivo in the absence of other viral factors. Lastly, we confirm that M2 expression during MHV68 infection is localized to the GC compartment, which is a long term latency reservoir for gammaherpesviruses. Overall, these observations are consistent with, and extend upon previous reports of M2 function in B cells and within the context of MHV68 infection. Moreover, this work provides support for a model by which M2-driven dysregulation of B cell function compromises multiple aspects of antiviral immunity to achieve persistence within the infected host.

The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) is essential for viral DNA synthesis and production of infectious virus in cell culture. While mutations that affect 59-39 polymerase activity have been evaluated in animal models of HSV-1 infection, mutations that affect other functions of HSV-1 Pol have not. In a previous report, we utilized bacterial artificial chromosome technology to generate defined HSV-1 pol mutants with lesions in the previously uncharacterized pre-NH 2 -terminal domain. We found that the extreme N-terminal 42 residues (deletion mutant polDN43) were dispensable for replication in cell culture, while residues 44-49 (alanine-substitution mutant polA 6 ) were required for efficient viral DNA synthesis and production of infectious virus. In this study, we sought to address the importance of these conserved elements in viral replication in a mouse corneal infection model. Mutant virus polDN43 exhibited no meaningful defect in acute or latent infection despite strong conservation of residues 1-42 with HSV-2 Pol. The polA 6 mutation caused a modest defect in replication at the site of inoculation, and was severely impaired for ganglionic replication, even at high inocula that permitted efficient corneal replication. Additionally, the polA 6 mutation resulted in reduced latency establishment and subsequent reactivation. Moreover, we found that the polA 6 replication defect in cultured cells was exacerbated in resting cells as compared to dividing cells. These results reveal an important role for the conserved motif at residues 44-49 of HSV-1 Pol for ganglionic viral replication.