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The open reading frame 57 gene product of herpesvirus saimiri shuttles between the nucleus and cytoplasm and is involved in viral RNA nuclear export - PubMed

The open reading frame 57 gene product of herpesvirus saimiri shuttles between the nucleus and cytoplasm and is involved in viral RNA nuclear export

D J Goodwin et al. J Virol. 1999 Dec.

Abstract

The herpesvirus saimiri open reading frame (ORF) 57 is homologous to genes identified in all classes of herpesviruses. It has previously been shown to regulate gene expression through a posttranscriptional mechanism. We demonstrate in this report that the expression of the ORF 57 protein leads to the cytoplasmic accumulation of glycoprotein B and capsid mRNAs. We also demonstrate that ORF 57 has the ability to specifically bind viral RNA transcripts. Utilizing an interspecies heterokaryon assay, we show that ORF 57 has the ability to shuttle between the nucleus and the cytoplasm. Furthermore, we show that ORF 57 contains a relatively leucine-rich sequence which shares some homology with nuclear export signals (NES) found in a number of proteins with the ability to shuttle between the nucleus and the cytoplasm. Moreover, we demonstrate that the ORF 57 NES enables the nuclear export of a heterologous protein and that mutation of the conserved leucine residues contained within the ORF 57 NES signal abrogates the ability of the ORF 57 protein to shuttle between the nucleus and cytoplasm. These results suggest that ORF 57 is involved in mediating the nuclear export of viral transcripts.

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Figures

**FIG. 1**
ORF 57 increases cytoplasmic transport of viral mRNA. Cos-7 cells transfected with pUCgB (a) and pEcoB (b) in the absence or presence of pRSVORF57. Total (lanes 1 and 2), nuclear (lanes 3 and 4), and cytoplasmic (lanes 5 and 6) RNA was then isolated and separated by elecrophoresis on a 1% denaturing formaldehyde agarose gel. The RNA was transferred to Hybond-N membranes and hybridized with ³²P-radiolabelled randomly primed probes specific for the HVS gB and the capsid coding sequence.

**FIG. 2**
The ORF 57 protein binds viral mRNA. (a) Coomassie blue-stained gel of GST (lane 1) and GST-ORF 57 (lane 2) proteins expressed in *E. coli* DH5α and purified from the crude lysate by incubation with glutathione-Sepharose 4B beads according to manufacturer’s specifications. (b) RNA-binding assay of the ORF 57 protein. GST and GST-ORF 57 proteins were separated on an SDS–12% polyacrylamide gel and transferred onto nitrocellulose (Amersham). The nitrocellulose bound proteins were denatured by using 6 M guanidine-HCl and then renatured by using six 50% stepwise dilutions of the guanidine-HCl in binding buffer. The filter-bound ORF 57 protein was then hybridized with ³²P-radiolabelled RNA probes specific for HVS gB (i) and actin (ii).

**FIG. 3**
(a) Hoechst dye staining of monkey and mouse cell nuclei. Hoechst dye allowed differentiation between monkey (i) and mouse (ii) nuclei. Monkey cells stained diffusely throughout the nuclei, whereas mouse nuclei stained with a distinctive speckled pattern. (b) The ORF 57 protein shuttles between the nucleus and cytoplasm. Cos-7 cells seeded at 2 × 10⁵ cells per 35-mm-diameter petri dish were transiently transfected with 2 μg of pRSVORF57. After 18 h, mouse 3T3 cells (5 × 10⁵ cells/well) were plated onto the Cos-7 cells in medium containing 50 μg of cycloheximide per ml. Four hours later the cells were washed in PBS and fused by the addition of 2 ml of 50% polyethylene glycol (wt/wt) in PBS. After being washed, the cells were returned to medium containing 50 μg of cycloheximide per ml for 60 min. Cells were incubated with a 1:100 dilution of ORF 57 antibody and then costained with 0.5 μg of Hoechst dye (i) and fluorescein-conjugated anti-mouse immunoglobulin (ii) per ml.

**FIG. 4**
The ORF 57 gene product expresses a nuclear export signal. Cos-7 cell monolayers were transfected with 2 μg of either pEGFP-C1 (i) or pEGFP-57NES (ii). After 24 h, the subcellular localization of GFP was observed by using fluorescence microscopy.

**FIG. 5**
Mutation of the ORF 57 NES abrogated the ability of the ORF 57 protein to shuttle between the nucleus and cytoplasm. Cos-7 cells seeded at 2 × 10⁵ cells per 35-mm-diameter petri dish were transiently transfected with 2 μg of p57-NES. After 18 h, mouse 3T3 cells (5 × 10⁵ cells/well) were plated onto the Cos-7 cells in medium containing 50 μg of cycloheximide per ml. Four hours later the cells were washed in PBS and fused by the addition of 2 ml of 50% polyethylene glycol (wt/wt) in PBS. After being washed, the cells were returned to medium containing 50 μg of cycloheximide per ml for 60 min. Cells were then incubated with a 1:100 dilution of ORF 57 antibody and costained with 0.5 μg of Hoechst dye (i) and fluorescein-conjugated anti-mouse immunoglobulin (ii) per ml.

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The open reading frame 57 gene product of herpesvirus saimiri shuttles between the nucleus and cytoplasm and is involved in viral RNA nuclear export - PubMed