Crosses between the two North American rodent species Peromyscus polionotus (PO) and Peromyscus maniculatus (BW) yield parent-of-origin effects on both embryonic and placental growth. The two species are approximately the same size, but a female BW crossed with a male PO produces offspring that are smaller than either parent. In the reciprocal cross, the offspring are oversized and typically die before birth. Rare survivors are exclusively female, consistent with Haldane's rule, which states that in instances of hybrid sterility or inviability, the heterogametic sex tends to be more severely affected. To understand these sex- and parent-of-origin-specific patterns of overgrowth, we analysed reciprocal backcrosses. Our studies reveal that hybrid inviability is partially due to a maternally expressed X-linked PO locus and an imprinted paternally expressed autosomal BW locus. In addition, the hybrids display skewing of X-chromosome inactivation in favour of the expression of the BW X chromosome. The most severe overgrowth is accompanied by widespread relaxation of imprinting of mostly paternally expressed genes. Both genetic and epigenetic mechanisms underlie hybrid inviability in Peromyscus and hence have a role in the establishment and maintenance of reproductive isolation barriers in mammals.

The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.A remarkable property of the alphaherpesvirus life cycle in the natural host is invasion and controlled spread within the peripheral nervous system (PNS) with exceedingly rare incursions into the central nervous system. The basic unit of a herpesvirus infection is the extracellular virion, a complex particle comprising several thousand protein molecules (31). Herpes virions, in general, are approximately 200 nm in diameter with a membrane envelope containing more than 12 virusborne membrane proteins. This host-derived membrane surrounds a tegument layer of at least 12 soluble proteins, which, in turn, surrounds an icosahedral capsid containing the 142-kb genome (36, 59). The assembly and movement of these distinct virion structures must be understood at the cellular level as these properties directly influence herpesvirus pathogenesis and transmission. Pseudorabies virus (PRV), an animal pathogen, has served as a model for study of directional spread of the neurotropic herpesviruses, which include the human pathogens herpes simplex virus (HSV) and varicella-zoster virus (VZV). After replication of PRV at exposed mucosal surfaces, virion components enter the axon terminals of PNS neurons, and unenveloped capsids move toward the c...

SUMMARY The sense of balance depends on the intricate architecture of the inner ear, which contains three semicircular canals used to detect motion of the head in space. Changes in the shape of even one canal cause drastic behavioral deficits, highlighting the need to understand the cellular and molecular events that ensure perfect formation of this precise structure. During development, the canals are sculpted from pouches that grow out of a simple ball of epithelium, the otic vesicle. A key event is the fusion of two opposing epithelial walls in the center of each pouch, thereby creating a hollow canal. During the course of a gene trap mutagenesis screen to find new genes required for canal morphogenesis, we discovered that the Ig superfamily protein Lrig3 is necessary for lateral canal development. We show that this phenotype is due to ectopic expression of the axon guidance molecule Netrin1 (Ntn1), which regulates basal lamina integrity in the fusion plate. Through a series of genetic experiments, we show that mutually antagonistic interactions between Lrig3 and Ntn1 create complementary expression domains that define the future shape of the lateral canal. Remarkably, removal of one copy of Ntn1 from Lrig3 mutants rescues both the circling behavior and the canal malformation. Thus, the Lrig3/Ntn1 feedback loop dictates when and where basement membrane breakdown occurs during canal development, revealing a new mechanism of complex tissue morphogenesis.

The tegument of herpesvirus virions is a distinctive structure whose assembly and function are not well understood. The herpes simplex virus type 1 VP22 tegument protein encoded by the UL49 gene is conserved among the alphaherpesviruses. Using cell biology and viral genetics, we provide an initial characterization of the pseudorabies virus (PRV) VP22 homologue. We identified three isoforms of VP22 present in PRV-infected cells that can be resolved by polyacrylamide gel electrophoresis. The predominant form is not phosphorylated and is present in virions, while the other two species are phosphorylated and excluded from virions. VP22 localized to the nucleus by 6 h postinfection, as determined by immunofluorescence and cell fractionation. VP22 immunofluorescence in the nucleus was both diffuse and in punctate structures. The punctate nuclear localization was the most pronounced form of staining and did not localize exclusively to sites of viral DNA replication. Unexpectedly, a VP22 null mutant had no obvious phenotypes during tissue culture infections and was similar to the wild type in all respects. Moreover, the VP22 null mutant was as virulent and neuroinvasive as the wild-type virus after infection of the rodent eye and spread to the brain using both anterograde and retrograde neuronal circuits.Herpesvirus virions are characterized in part by the presence of tegument, the distinctive proteinaceous layer between the nucleocapsid and envelope. Despite the fact that tegument is an important structural component of virions, there exists rudimentary and often conflicting information on the site(s) of tegument assembly, and the organization, stoichiometry and function of tegument proteins. Recent reports of mRNAs in the virions of human cytomegalovirus (4) and herpes simplex virus (HSV) (45), a subset of which may be acquired along with tegument during final envelopment (22), underscore the complex biology inherent in the herpesvirus tegument. Tegument components are delivered to the cell immediately after fusion of the virus envelope with the plasma membrane and therefore can have an immediate effect on the cell. Some tegument proteins have been characterized as transcription activators, kinases, or RNases (42) affecting viral and host gene expression.The alphaherpesviruses encode a conserved cluster of four tegument genes named UL46, UL47, UL48, and UL49 (1,25,34,44,54). These genes encode four tegument proteins, known in HSV-1 as VP11/12 (UL46), VP13/14 (UL47), VP16 (UL48), and VP22 (UL49) (34). Of these proteins, the transcription activator VP16 has been studied extensively, whereas considerably less is known about the function of the other three proteins in this cluster. Interestingly, VP16 interacts with each of the other three tegument proteins encoded in the cluster (7,14,24,30,55), but the structure and function of these complexes are not well characterized.The HSV-1 VP22 protein has attracted recent attention because it has been shown to have the property of intercellular spread (11,16,38). VP22 also binds...