Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

Abstract

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription͞ export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA.mRNA export ͉ nucleolus ͉ virus T he eukaryotic cell nucleus is a highly organized environment containing distinct and often dynamic compartments (1). Of these, the nucleolus is the most prominent, and, for many years, its exclusive role was thought to be the site of ribosomal RNA transcription, processing, and assembly into the ribosome subunits (2). Recent studies, however, suggest that it has additional nonclassical roles in many aspects of cell biology, including cell cycle regulation, viral replication, tumorigenesis, and cellular stress responses (3-5). This plurifunctional nature of the nucleolus has been highlighted by extensive proteomic analysis of human nucleoli (6, 7). To date, the nucleolar proteome database archives 728 nucleolar proteins, and functional classification of these proteins reinforces the multiple roles of the nucleolus (8). Furthermore, there is constant dynamic trafficking of nucleolar proteins, and this concomitant dynamic nature of nucleolar structure may provide regulation of nonclassical nucleolar functions.Interestingly, an increasing number of key proteins from both RNA and DNA viruses have been shown to localize to the nucleolus. These proteins include those encoded by viruses such as coronaviruses, influenza, HIV-1, adenoviruses, and herpesviruses (9). Therefore, virus-nucleolar interactions are likely to have important implications in the life cycle of many viruses. However, at present, the precise functional role of these virus-nucleolar colocalizations has not been determined. One such key viral protein that traffics to the nucleolus is the herpesvirus saimiri (HVS) ORF57 protein. HVS is the prototype ␥-2 herpesvirus, or rhadinovirus (10), which has become an important family of viruses since the identification of the first human ␥-2 herpesvirus, the oncogenic Kaposi's sarcoma-associ...

Rhesus Monkey Rhadinovirus ORF57 Induces gH and gL Glycoprotein Expression through Posttranscriptional Accumulation of Target mRNAs

“…Instead, we observed higher levels of gH and gL total RNA, which seemed to be contributed from both nucleus and cytoplasm proportionally in the presence of ORF57. The preferential accumulation of target RNA in the cytoplasm has been used to support a role for some ORF57s in the egress of RNA from the nucleus (4,6,29,41). In our study, the overall increase of total RNA levels of the target gene was contributed by nuclear and cytoplasmic compartments (Fig.…”

“…Examples include translation and mRNA stability, the nuclear egress of mRNA and mRNA stability, and the transcription and nuclear egress of mRNA. In looking at the literature on ORF57 action, it is often difficult to distinguish what is multifunctional activity from what is a single activity manifest in different, linked, functional processes (6,17,20,26,32). Similar difficulties can be seen in the data reported here.…”

“…However, as the name MTA (mRNA transcript accumulation) in KSHV implies, a posttranscriptional mechanism of activation by ORF57 orthologues has been studied and documented most extensively (4,14,21,29). It has been reported that increased expression appears to be achieved in many cases via the facilitation of the nuclear export of target mRNAs (4,6,8,29,41). Many herpesviral mRNAs, most of which are intronless and therefore do not undergo splicing, seem to be poor substrates for nuclear export by the regular cellular transport machineries.…”

The major tegument structural protein VP22 targets areas of dispersed nucleolin and marginalized chromatin during productive herpes simplex virus 1 infection

“…The nucleolus is necessary for protein translation {reviewed in (Melese and Xue, 1995;Pederson and Politz, 2000)}. It has been proposed that the herpesvirus saimiri ORF57 protein uses nucleoli to export intronless viral messages (Boyne and Whitehouse, 2006;Williams et al, 2005). The HSV-1 ICP27, ICP0, γ 1 34.5, and U S 11 proteins were reported to possess nucleolar targeting sequences (Cheng et al, 2002;MacLean et al, 1987;Mears et al, 1995;Morency et al, 2005) but the implications of these are unknown.…”

General, rapid, and transcription-dependent fragmentation of nucleolar antigens in S. cerevisiae mRNA export mutants

“…Molecular flux from the chromatin-rich area to the nucleolus is known to occur; e.g., snoRNAs are imported into the nucleolus where they function in rRNA biogenesis (Terns and Dahlberg 1994). Although an involvement of the nucleolus in the maturation and nuclear export of mRNA has also been suggested Ideue et al 2004;Boyne and Whitehouse 2006), such a functional connection between the two compartments has not yet been firmly established. In contrast, real-time analysis in mammalian cells of GFP-decorated mRNAs suggests that mRNPs diffuse from their sites of synthesis and dock at the nuclear membrane without a substantial nucleolar phase (Shav-Tal et al 2004).…”