Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA - PubMed (original) (raw)

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

James R Boyne et al. Proc Natl Acad Sci U S A. 2006.

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 (transcription/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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Redistribution of TREX components by ORF57 into the nucleolus. (a) ORF57 localizes to the nucleolus during HVS infection. OMK cells were cultured on glass coverslips until fully confluent and then infected with HVS A11-S4 strain and incubated at 37°C for 18 h. Cells were fixed, and coimmunofluorescence was carried out by using an ORF57-specific monoclonal antibody (i) and a nucleolin-specific polyclonal antibody (ii). A merged image is shown in iii. (b) Components of the hTREX complex are redistributed to the nucleolus and colocalize with ORF57 in HVS-infected cells. OMK cells were cultured on glass coverslips until fully confluent and then infected with the HVS A11-S4 strain and incubated at 37°C for 18 h. Cells were fixed, and coimmunofluorescence was carried out by using an ORF57-specific monoclonal antibody (ii, vi, and x) and Aly-, UAP56-, and hTho2-specific polyclonal antibodies (iii, vii, and xi). (i, v, and ix) Uninfected OMK cells served as negative controls.

Fig. 2.

Characterization of a previously unidentified NoLS within ORF57. (a) Schematic representation of the ORF57 deletion and point mutants. Deletion analysis identified two distinct nuclear localization sequences within ORF57. These sequences were deleted from ORF57–GFP constructs and subsequently rendered nonfunctional by the alanine substitution mediated by site-directed mutagenesis. (b) HeLa cells were cultured on glass coverslips to 80% confluency and transfected with the respective ORF57–GFP constructs. Twenty-four hours after transfection, cells were fixed, and immunofluorescence was carried out by using a nucleolin-specific polyclonal antibody as a marker of the nucleolus.

Fig. 3.

ORF57 mutants that lack a NoLS can no longer export viral mRNA. (a) 293T cells were cultured in 35-mm plates to 80% confluency and transfected with the respective ORF57–GFP deletion constructs plus pBK-gB. Cells were incubated at 37°C for 24 h, and total RNA was extracted from nuclear and cytoplasmic fractions by using a PARIS kit. RNA was separated on a denaturing agarose gel, and Northern blotting was carried out by using a gB-specific radiolabeled probe. A probe to the 18S subunit of ribosomal RNA was used as a loading control. (b) The experiment outlined in a was repeated by using the ORF57–GFP point mutants to confirm that these mutants possessed the mRNA export defect.

Fig. 4.

The ORF57 NLS1 mutant retains the ability to bind viral RNA and interact with mRNA export proteins with similar affinity to WT ORF57. (a) 293T cells were transfected with the labeled expression vectors and cultured for 24 h. After UV cross-linking, RNA-IPs were performed by using an antibody to ORF57, and nested RT-PCR was carried out on the extracted RNA for ORF47 mRNA. Total RNA extracted from mock-transfected and ORF47/ORF57-transfected 293T cells was used as a negative and positive control for the RT-PCR (Input). (b) RNA-IPs were repeated to compare the binding affinities of WT ORF57 and ORF57NLS1M. Nested RT-PCRs were performed by using 10-fold dilutions of extracted RNA (10, 1, 0.1, and 0.01 μl). (c) BL-21 cells were transformed with either pGEX-4T or pGEX-Aly, and expression of the fusion protein was induced. GST-Aly and control GST protein was bound to glutathione-agarose beads and purified by washing before incubating bound beads with total cell lysate from 293T cells transfected with the labeled constructs. Beads were washed, and bound proteins were analyzed by Western blot using a GFP-specific antibody. (d) GST pull-down assays were repeated to compare the binding affinity of GST-Aly with WT ORF57 and ORF57NLS1M. GST-Aly bound to glutathione-agarose beads was incubated with 10-fold dilutions (1,000, 100, 10, and 1 μl) of total cell lysate from 293T cells transfected with WT ORF57 or ORF57NLS1M constructs.

Fig. 5.

Insertion of the HIV-1 Rev NoLS at the C terminus of pORF57NLS1M reconstitutes nucleolar localization and restores viral mRNA export. (a) HeLa cells were cultured on glass coverslips to 80% confluency and transfected with the labeled constructs. Twenty-four hours after transfection, cells were fixed, and immunofluorescence was carried out by using a nucleolin-specific polyclonal antibody. (b) 293T cells were cultured in 35-mm plates to 80% confluency and transfected with the respective ORF57–GFP constructs plus pBK-gB. Cells were incubated at 37°C for 24 h, and total RNA was extracted from nuclear and cytoplasmic fractions by using a PARIS kit. RNA was separated on a denaturing agarose gel, and Northern blotting was carried out by using a gB-specific radiolabeled probe. A probe to the 18S subunit of ribosomal RNA was used as a loading control.

Fig. 6.

Proposed model for viral mRNA export in HVS. These data led to the findings that ORF57 trafficking through the nucleolus is essential for viral mRNA nuclear export. WT ORF57 is able to traffic through the nucleolus, bind intronless viral mRNAs, and associate with cellular mRNA export proteins to achieve viral mRNA nuclear export (i). However, ORF57 mutants incapable of nucleolar trafficking are unable to export bound viral mRNA, even though assembly of the export complex appears to be unperturbed (ii).

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Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA - PubMed (original) (raw)