Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are ubiquitous human pathogens that cause a variety of serious diseases. Primary HSV infections typically initiate at mucosal surfaces, where lytic replication in epithelial cells generates mucosal lesions. Peripheral replication amplifies the virus load and increases uptake of virus into sensory nerve termini. Establishment of latent infections in sensory neurons secures shelter for the virus throughout the life of the host. Periodic reactivations result in disease recurrence and provide an opportunity for transmission to new hosts.The virion host shutoff (vhs) protein of HSV plays a significant role in promoting pathogenesis at the cell and organismal levels. In the infected cell, vhs possesses both endo-and exonuclease activity (8, 9, 57) and mediates the rapid shutoff of protein synthesis via degradation of both cellular and viral mRNAs (22,23,39,43). As a component of the virion tegument, vhs is released directly into the cytosol and can immediately exert its effects on the newly infected cell. Cellular mRNAs are almost completely degraded within 6 h of infection with HSV-1, and within just 2 h by HSV-2 (19). The activity of HSV-2 vhs is also approximately 40-fold stronger than that of HSV-1 (10, 11). In vivo, vhs null mutants of HSV-1 and HSV-2 are profoundly attenuated, implicating vhs as a virulence factor that helps HSV establish robust infection. HSV-1 lacking vhs activity replicates to 1,000-fold-lower titers in the cornea, trigeminal ganglia, and brain of mice than wildtype virus and has impaired capacity to enter the central nervous system, establish latency, and undergo reactivation (48-50). vhs-deficient HSV-2 strains (333-vhsB or 333d41) also replicate much less efficiently than wild-type virus in the genital mucosa and nervous tissue of mice and cause less disease (47). vhs has been implicated in down-regulating major histocompatibility complex (MHC) class I (18, 54) and class II (55) molecules. This activity has functional implications because it is associated with reduced cytotoxic T-lymphocyte recognition

The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8؊ virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. ؊ mutant virus. The data indicate that inactivation of vhs in a replicationincompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.Herpes simplex virus type 1 (HSV-1) is a common human pathogen, infecting approximately 80% of individuals by adulthood (49). The virus typically enters the body at epithelial and mucosal surfaces, where lytic infection of epithelial cells and fibroblasts leads to infection of sensory neurons innervating the mucosa and to the rapid establishment of latent infection in the neuronal cell bodies. In this latent reservoir, HSV infection is maintained for the life of the host. Either initial infection or reactivation can result in serious human disease, including rare but devastating encephalitis and keratitis, which is the second most common cause of nontraumatic corneal blindness (49). A vaccine to obviate or therapeutically alleviate these HSV-1-mediated diseases is a desirable goal.Development of an antiviral vaccine requires consideration of both safety and immunogenicity. An effective balance between these can be difficult to achieve, especially when faced with HSV that has a complex and persistent lifestyle. Immunization with live attenuated virus has the potential advantages of generating immune responses to a broad spectrum of viral proteins and induction of type 1 T-cell as well as humoral responses. In the development of prototypic live virus vaccines, several viral proteins that regulate host cell and viral synthetic processes have been manipulated to advantage. During infection, one of the earliest viral activities is that mediated by the virion host shutoff (vhs) protein, a product of the UL41 gene. This viral tegument component exerts its effects immediately upon entry into the cell, prior to viral gene expression (13,39). The vhs protein is associated with degradation of both cellular and viral mRNAs (24-26, 36, 39, 43) and endoribonucleolytic activity (9, 52), and the destabilization of viral messages mediated by vhs has been theorized to promote the switch from transcription of one kinetic class of viral genes to the next (43). We have previously shown that mice immunized with an HSV-1 strain that is deficient in vhs activity, UL41NHB, are significantly pro...

During lytic infection, the virion host shutoff (vhs) protein mediates the rapid degradation of mRNA and the shutoff of host protein synthesis. In vivo, herpes simplex virus type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. Homologs of vhs exist in all of the neurotropic herpesviruses, and the goal of this study was to determine the virulence of HSV-2 mutants lacking vhs. Two HSV-2 recombinants were used in this study: 333-vhsB, which has a lacZ cassette inserted into the N terminus of vhs, and 333d41, which has a 939-bp deletion in vhs. As expected, both 333-vhsB and 333d41 failed to induce the cellular RNA degradation characteristic of HSV. Corneal, vaginal, and intracerebral routes of infection were used to study pathogenesis. Both viruses grew to significantly lower titers in the corneas, trigeminal ganglia, vaginas, dorsal root ganglia, spinal cords, and brains of mice than wild-type and rescue viruses, with a correspondingly reduced induction of disease. Both viruses, however, reactivated efficiently from explanted trigeminal ganglia, showing that vhs is dispensable for reactivation. The lethality of 333d41 following peripheral infection of mice, however, was significantly higher than that of 333-vhsB, suggesting that some of the attenuation of 333-vhsB may be due to the presence of a lacZ cassette in the vhs locus. Taken together, these data show that vhs represents an important determinant of HSV-2 pathogenesis and have implications for the design of HSV-2 recombinants and vaccines.Herpes simplex virus (HSV) induces a rapid destabilization of mRNA due to the product of the viral UL41 gene, known as the virion host shutoff (vhs) protein. This 58-kDa phosphoprotein, packaged within the tegument of the virus, is released directly into the cytoplasm upon infection, where it immediately begins to degrade mRNA prior to any viral gene expression (7,23). Recent studies have demonstrated that vhs has endoribonucleolytic activity near the 5Ј end of target mRNA (3, 4, 15), which requires a cellular factor (18) but no other viral proteins (14,20). Viruses containing mutations within any of the four conserved domains in the UL41 gene do not cause RNA destabilization upon infection (8,22,23,25). Such mutants are viable in cell culture, and the effect of vhs deletion on viral replication in tissue culture is minimal (22).The genomes of HSV type 1 (HSV-1) and HSV-2, varicellazoster virus, equine herpesvirus, and pseudorabies virus all have homologs of vhs (1). The conservation of vhs in these and other neurotropic alphaherpesviruses and its apparent absence in beta-and gammaherpesviruses suggest that vhs plays a role in neuropathogenesis, although the role of vhs in this context has only been studied in HSV-1. The goal of this study was therefore to examine the contribution of vhs to the pathogenesis of HSV-2.The pathogenesis of HSV-1 and HSV-2 infections in humans and animal models has some general similarities and some important differences. HSV-1 is primarily associated with orolabial lesions, st...

CRSBP-1, a membrane glycoprotein, can mediate cell-surface retention of secreted growth factors containing CRS motifs such as PDGF-BB. CRSBP-1 has recently been found to be identical to LYVE-1, a specific marker for lymphatic capillary endothelial cells. The in vivo role of CRSBP-1/LYVE-1 is unknown. CRSBP-1-null mice are overtly normal and fertile but exhibit identifiable morphological and functional alterations of lymphatic capillary vessels in certain tissues, marked by the constitutively increased interstitial-lymphatic flow and lack of typical irregularly-shaped lumens. The CRSBP-1 ligands PDGF-BB and HA enhance interstitial-lymphatic flow in wildtype mice but not in CRSBP-1-null animals.

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.Herpes simplex virus (HSV) causes the rapid shutoff of macromolecular synthesis in infected cells. The factor responsible for this shutoff is the product of the UL41 gene, known as the virion host shutoff (vhs) protein (21,33,38). Vhs is a 58-kDa phosphoprotein, and approximately 200 copies are packaged within the tegument of the virus, allowing it to exert its effects immediately upon infection prior to de novo viral gene expression (11,36). vhs-induced shutoff leads to polysomal disaggregation and nonspecific cytoplasmic degradation of cellular mRNAs (23)(24)(25)35) and viral mRNAs belonging to all three kinetic classes (32). Although the exact mechanism remains to be determined, recent studies demonstrate that vhsdependent endoribonucleolytic activity can be selectively targeted by specific cis-acting elements in the RNA (7,8,47). Two advantages to the virus may result from vhs-induced shutoff. First, it depletes the pool of translatable host mRNA such that viral mRNA can be preferentially translated; second, it prevents the overexpression of immediate-early and early viral genes, allowing efficient transition between the sequential kinetic classes. Despite these putative advantages, the effect of vhs deletion on the efficiency of viral replication in cell culture is minimal (35,36,42).Primary sequence analysis of five of the...