Examination of determinants for intranuclear localization and transactivation within the RING finger of herpes simplex virus type 1 IE110k protein.
The herpesvirus regulatory protein IE110k possesses a cysteine-rich, RING finger motif required for its role in transactivation and virus replication. IE110k also localizes to subnuclear compartments termed PODs (PML oncogenic domains). Localization to PODs induces redistribution of the proteins associated with this nuclear compartment, including the cellular RING finger protein, PML. Here we construct a series of deletions, RING domain swaps and point mutations to analyse specific requirements within the IE110k RING finger for subnuclear localization, redistribution of PML and transactivation and we examine the relationship between these activities. We find that IE110k localizes to distinct nuclear subdomains that are more numerous than the cellular PODs and that mutation of two residues within a predicted loop of the RING finger, or replacing the IE110k RING finger with a RING finger from a cellular gene abrogates the ability of IE110k to localize to these extra compartments and traps IE110k in the original PODs. We further demonstrate that RING fingers from the cellular genes mdm-2 and Bmi I, when placed within IE110k, alter the nuclear distribution of IE110k, do not transactivate, and do not redistribute PML. We also demonstrate that the majority of wild-type IE110k, like PML, is associated with the nuclear matrix. Although substitutions and deletions within the RING finger abolish transactivation, these mutant proteins remain tightly associated with the matrix. These results further dissect the determinants involved in different aspects of nuclear compartmentalization of IE110k and are discussed in relation to PML, PODs and the IE110k RING finger.
O'Rourke
D
D
Marie Curie Research Institute, The Chart, Oxted, Surrey, UK.
Elliott
G
G
Papworth
M
M
Everett
R
R
O'Hare
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D005816:Genetic Complementation Test; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism*; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D015530:Nuclear Matrix / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014709:Vero Cells; D016335:Zinc Fingers / Q000502:physiology
D017874:Immediate-Early Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1