A γ-2 Herpesvirus Nucleocytoplasmic Shuttle Protein Interacts with Importin α1 and α5

Delyth J Goodwin,Adrian Whitehouse

doi:10.1074/jbc.m009513200

Abstract

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus. This is an increasing important subfamily of herpesviruses due to the identification of the first human gamma-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS open reading frame (ORF) 57 protein is a multifunctional trans-regulatory protein homologous to genes identified in all classes of herpesviruses. Recent analysis has demonstrated that ORF 57 has the ability to bind viral RNA and to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts. Here we have investigated the nucleocytoplasmic shuttling mechanism utilized by the ORF 57 protein. The yeast two-hybrid system was employed to identify interacting cellular proteins using ORF 57 as bait. We demonstrate that ORF 57 interacts with importin alpha isoforms 1 and 5. In addition, the binding of ORF 57 to importin alpha was mediated by the importin alpha hydrophobic internal armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as a nuclear localization sequence, was also required for this interaction. Furthermore, the ORF 57 protein is responsible for the redistribution of importin alpha into the nucleoli. These results identify novel cellular interactions essential for the functioning of this important herpesvirus regulatory protein.

Highlights

A number of viruses including herpes, adenovirus, influenza, and retroviruses replicate in the host cell nucleus
Importin ␣1 and ␣5 Interact with Herpesvirus saimiri (HVS) open reading frame (ORF) 57—To identify ORF 57-interacting cellular proteins, 1.5 ϫ 106 independent cDNA clones of a human kidney cDNA library fused to the GAL4 activation domain were screened. 24 clones were identified that activated histidine and ␤-galactosidase reporter gene expression in the presence of the ORF 57-DBD fusion protein
ORF 57-Importin ␣ Interaction Is Required for Viral RNA Nuclear Export—We have previously demonstrated that ORF 57 is a nucleocytoplasmic shuttle protein which mediates the HVS ORF 57 is a member of the virus-encoded nucleocytoplasmic shuttle proteins involved in mediating the nuclear export of virus transcripts

Summary

Introduction

A number of viruses including herpes, adenovirus, influenza, and retroviruses replicate in the host cell nucleus. Recent analysis has demonstrated that ORF 57 has the ability to bind viral RNA and to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts. The results demonstrate that, in both ORF 57-transfected and HVS-infected cells, ORF 57 interacts with both importin ␣1 and ␣5 (Fig. 1).

Results

Conclusion