UL24 of herpes simplex virus 1 (HSV-1) is widely conserved within the Herpesviridae family. Herein, we tested the hypothesis that UL24, which we have previously shown to induce the redistribution of nucleolin, also affects the localization of the nucleolar protein B23. We found that HSV-1-induced dispersal of B23 was dependent on UL24. The conserved N-terminal portion of UL24 was sufficient to induce the redistribution of B23 in transient transfection assays. Mutational analysis revealed that the endonuclease motif of UL24 was important for B23 dispersal in both transfected and infected cells. Nucleolar protein relocalization during HSV-1 infection was also observed in non-immortalized cells. Analysis of infected cells by electron microscopy revealed a decrease in the ratio of cytoplasmic versus nuclear viral particles in cells infected with a UL24-deficient strain compared to KOS-infected cells. Our results suggest that UL24 promotes nuclear egress of nucleocapsids during HSV-1 infection, possibly though effects on nucleoli.

Herpes simplex virus 1 (human herpesvirus 1) initially infects epithelial cells of the mucosa and then goes on to infect sensory neurons leading ultimately to a latent infection in trigeminal ganglia (TG). UL24 is a core herpesvirus gene that has been identified as a determinant of pathogenesis in several Alphaherpesvirinae, although the underlying mechanisms are unknown. In a mouse model of ocular infection, a UL24-deficient virus exhibited a reduction in viral titres in tear films of 1 log 10 , whilst titres in TG are often below the level of detection. Moreover, the efficiency of reactivation from latency was also severely reduced. Herein, we investigated how UL24 contributed to acute infection of TG. Our results comparing the impact of UL24 on viral titres in eye tissue versus in tear films did not reveal a general defect in virus release from the cornea. We also found that the impairment of replication seen in mouse primary embryonic neurons with a UL24-deficient virus was not more severe than that observed in an epithelial cell line. Rather, in situ histological analyses revealed that infection with a UL24-deficient virus led to a significant reduction in the number of acutely infected neurons at 3 days post-infection (p.i.). Moreover, there was a significant reduction in the number of neurons positive for viral DNA at 2 days p.i. for the UL24-deficient virus as compared with that observed for WT or a rescue virus. Our results supported a model whereby UL24 functions in the dissemination of acute infection from the cornea to neurons in TG.

Herpes simplex virus 1 (HSV-1) infection induces multiple modifications to nucleoli. We and others have observed the redistribution of nucleolar proteins such as nucleolin, fibrillarin, B23 (nucleophosmin), upstream binding factor (UBF) and RNA polymerase I (RNAPI) during HSV-1 infection. UL24 is one of a limited number of HSV-1 proteins that localizes to nucleoli. In a murine model of ocular HSV-1 infection, viruses that do not express the UL24 protein exhibit reduced viral titers in the eye and in trigeminal ganglia, and a reduction in clinical signs of disease. UL24 is conserved among Herpesviridae. Specifically, the N-terminal region of the protein contains several stretches of highly conserved residues. Bioinformatics studies have identified an endonuclease motif in the N-terminal portion of UL24, although as yet, no nuclease activity has been demonstrated for the protein. Previously, we discovered that UL24 transiently localizes to nucleoli during infection, and is both necessary and sufficient to induce the dispersal of nucleolin from nucleoli throughout the nucleus. In contrast, the redistribution of fibrillarin is a UL24-independent event. Moreover, the largest subunit of RNAPI and the transcription factor UBF are redistributed to viral replication compartments and these effects are also UL24-independent. We recently investigated whether UL24 was implicated in other HSV-1-induced nucleolar modifications. We discovered that UL24 was involved in the redistribution of B23 during infection. Furthermore, expression of UL24 in the absence of other viral proteins was sufficient to induce the relocalisation of B23. Similar to what we found for nucleolin, the conserved N-terminal portion of UL24 was important for its effect on B23 redistribution. The impact of the mutant vUL24-E99A/K101A, in which the endonuclease motif has been altered, and of vUL24-G121A, which harbours an amino acid substitution outside of this motif, were tested. While the G121A mutation had only a modest effect on the ability the virus to induce the relocalisation of B23, the E99A/K101A mutation appeared to abolish this function. Interestingly, the E99A/K101A mutation was previously shown to have a large impact on viral pathogenesis in mice. We found that the effect of HSV-1 on the redistribution of B23 was similar in non-immortalized cells such as human foreskin fibroblasts, as in immortalized cell lines such as vero cells. The similarities with regard to the impact of UL24 on the spatial distribution of nucleolin and B23 suggest that these effects may be related to the same function of UL24 during HSV-1 infection.