Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin

Abstract

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.The UL24 protein is conserved throughout the Herpesviridae family, and to the best of our knowledge, a UL24 homolog has been identified in all Herpesvirales genomes sequenced to date with the exception of the channel catfish virus (9, 10, 19). UL24 of herpes simplex virus 1 (HSV-1) is required for efficient virus replication both in vitro and in vivo and for reactivation from latency in a mouse model of ocular infection (18). UL24 is one of the few HSV-1 genes, along with gB, gK, and UL20, in which mutations have been identified that cause the formation of syncytial plaques (2,7,34,36,39). The UL24-associated syncytial phenotype is only partially penetrant at 37°C but is fully penetrant at 39°C. Indications are that gK and UL20 have an inhibitory effect on the formation of syncytia (1), while certain mutations in gB entrain an uncontrolled fusogenic activity (11,13,15).UL24 is a highly basic protein of 269 amino acids that is expressed with leaky-late kinetics (31). Five homology domains (HDs), which consist of stretches of amino acids with a high percentage of identity between homologs, are present in the UL24 open reading frame (ORF) (19). In addition, a PD-(D/ E)XK endonuclease motif has been identified that falls within the HDs (20); however, a role for this motif has yet to be demonstrated. In infected cells, UL24 is detected in the nucleus and the cytoplasm a...

Genome-wide analysis of G-quadruplexes in herpesvirus genomes

“…HHV-1 and HHV-8 DNA were available to us (b) The presence of at least one PQS in the regulatory region of the gene (c) Conserved functions across all human herpesviruses or genes with a well-established role in viral pathogenesis. The UL2 gene codes for uracil DNA-glycosidase [45], A DNA repair protein and the UL24 gene codes for a nucleolin transporter protein [46] ;essential for efficient viral replication. These two proteins are present in all human herpesviruses.…”

Efficient Induction of Nuclear Aggresomes by Specific Single Missense Mutations in the DNA-binding Domain of a Viral AP-1 Homolog

“…Lytic induction also caused redistribution of nucleolin; rather than being confined to a discrete dot, nucleolin within cells expressing ZEBRA was visibly dispersed. Dispersal of nucleolin also occurs during lytic infection of HSV-1 and is mediated by the UL24 protein (51,52). In cells transfected with Z(R183E), nucleolin was not recruited to aggresomes (supplemental Fig.…”

Virus manipulation of cell cycle

“…This result indicates that this alteration of the nucleoli in HSV-1-infected cells is partially dependent on UL24 (Lymberopoulos et al 2011). The fact that deletion of UL24 conserved homology domains, including the putative endonuclease motifs, resulted in loss of nucleolin and B23 dispersal activity suggests that this function may be shared among all herpesviruses and must be relevant for the viral life cycle (Bertrand et al 2010;Bertrand and Pearson 2008;Lymberopoulos et al 2011). Indeed, a study with UL24 homologues from human herpesviruses representative of each subfamily (HSV-1 UL24, HCMV UL76, and KSHV ORF20) showed that all these genes are able to induce cell cycle arrest followed by apoptosis.…”

“…During infection with HSV-1, UL24 family gene homologues are detected predominantly in the nucleus and transiently localize in the nucleoli (Hong-Yan et al 2001;Nascimento and Parkhouse 2007;Pearson and Coen 2002;Wang et al 2000Wang et al , 2004. Expression of the N-terminal domain of UL24 is sufficient to induce the redistribution of nucleolin in the nucleus (Bertrand et al 2010;Bertrand and Pearson 2008). However, cells infected with two independent UL24-deficient viruses, UL24XB and UL24XG, retain the prominent foci of nucleolin staining, although the pattern was not identical to the uninfected cells.…”