Mutagenesis of the Active-Site Cysteine in the Ubiquitin-Specific Protease Contained in Large Tegument Protein pUL36 of Pseudorabies Virus Impairs Viral Replication In Vitro and Neuroinvasion In Vivo

Abstract

Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein.Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C 26 S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C 26 S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.Herpesviruses possess a large double-stranded DNA genome, comprising more than 70 open reading frames (ORFs) encoding viral proteins, of which at least 30 are incorporated into the mature virus particle. The most complex virion component is the tegument, which, in the alphaherpesviruses, contains more than 15 different viral proteins. Operationally, the tegument has been divided into an "inner," capsid-associated part and an "outer," envelope-juxtaposed portion (reviewed in references 42 and 43). The innermost part contains the largest herpesvirus tegument protein, which, in the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1), is the product of the UL36 gene. Homologs of this protein, for clarity henceforth named pUL36, are present in all members of the Herpesviridae which have been analyzed in this respect to date.Previous studies revealed two conserved domains within the N termini of pUL36 homologs. One is required for interaction with pUL37, another conserved tegument protein (4,22,33,44,50), whereas the second specifies a ubiquitin-specific cysteine protease activity (20,24,27,47,48,51). This activity is not required for herpesvirus replication, as has been shown for human cytomegalovirus (HCMV) (51) and Marek's disease virus (MDV) (24). Nevertheless, abrogation of deubiquitinating activity resulted in impairment of replication in vitro (51) and in vivo (24).In pseudorabies virus (PrV), pUL36 is the only truly essential tegument protein (14), and its deletion completely blocks viral replication. Interestingly, recent data demonstrated that about one-third of PrV pUL36 located in the C-terminal half can be deleted without drastic impairment of viral replication. In contrast, the extreme C terminus of pUL36 is essential (6, 35), probably due to its association with the capsid-associated pUL25 (10). Moreover, deletion of an N-terminal domain of about 200 amino acids comprising the deubi...

Dynamic ubiquitination drives herpesvirus neuroinvasion

“…4C) (35). The ΔDUB virus was incapable of transmission to the SCG despite productive replication in the iris, which is consistent with the prior observation that DUB activity is required for PRV spread from the AC to the brain, and demonstrates that this established CNS neuroinvasive defect is due to an upstream loss of PNS neuroinvasion (7,8). The K442R virus phenocopied the DUB mutant demonstrating that ubiquitination, in addition to deubiquitination, at K442 is critical for invasion of the PNS.…”

“…Recent findings have provided insights into this complex phenotype, but the molecular mechanisms directing nervous system invasion are poorly understood (1)(2)(3)(4)(5). A critical effector of this process is a conserved herpesvirus deubiquitinase (DUB) (a cysteine protease that removes ubiquitin from target posttranslationally modified proteins) (6)(7)(8). The DUB is housed in the amino terminus of the pUL36 protein (protein encoded by the 36th open reading frame in the unique-long segment of the viral genome), amounting to less than 10% of the total mass of this large (>250 kDa) structural component of the virion (9).…”

Autocatalytic Activity of the Ubiquitin-Specific Protease Domain of Herpes Simplex Virus 1 VP1-2

“…These studies indicated that an active USP was not essential for virus replication but that inactivity abrogated the infection process at some stage. Consistent with this, studies in pseudorabies virus (PRV) demonstrated that a virus with a substitution in the proposed catalytic cysteine of the PRV VP1-2 USP (C26S) resulted in a 20-to 30-fold reduction in virus yields in single-step growth curves, a reduction in plaque size (30%), and a delayed onset of pathogenic features after intranasal infection of mice (6). Moreover, in ultrastructural analysis of infection in culture, accumulations in nonenveloped cytoplasmic capsids were observed for the mutant virus, consistent with a defect in the known role of VP1-2 in virus assembly (6).…”

“…Consistent with this, studies in pseudorabies virus (PRV) demonstrated that a virus with a substitution in the proposed catalytic cysteine of the PRV VP1-2 USP (C26S) resulted in a 20-to 30-fold reduction in virus yields in single-step growth curves, a reduction in plaque size (30%), and a delayed onset of pathogenic features after intranasal infection of mice (6). Moreover, in ultrastructural analysis of infection in culture, accumulations in nonenveloped cytoplasmic capsids were observed for the mutant virus, consistent with a defect in the known role of VP1-2 in virus assembly (6). In studies of a similar PRV mutant containing a C26A mutation, virus yields in culture were delayed and lower (10-to 20-fold), and a very significant defect was observed in animal models of neuroinvasion from peripheral tissues (27).…”

Blocking ESCRT-Mediated Envelopment Inhibits Microtubule-Dependent Trafficking of Alphaherpesviruses In Vitro

“…These green fluorescent protein (GFP)-tagged HSV capsids (39) demonstrated kinesin-and dynein-mediated motility with a velocity and processivity similar to those seen in vivo (6). Furthermore, the in vitro motility was dependent (40) upon the large virally encoded tegument protein UL36p (VP1/2) that is known to recruit motors and to be required for motility and invasiveness in vivo (24)(25)(26)(41)(42)(43)(44)(45)(46). In this study, we modified our in vitro trafficking system to permit the study of HSV and PRV particle motility in bulk cytosol containing both naked and enveloped viral capsids.…”