Nuclear Translocation and Regulation of Intranuclear Distribution of Cytoplasmic Poly(A)-Binding Protein Are Distinct Processes Mediated by Two Epstein Barr Virus Proteins

Richard Park,Sumit Borah,Lee Heston,George Miller,Mate Nagy,Su-Fang Lin,Ayman El-Guindy,Joan Steitz,Henri-Jacques Delecluse,Kuan-Ping Yu,Alison J Sinclair

doi:10.1371/journal.pone.0092593

Richard Park, Sumit Borah + Show 9 more

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https://doi.org/10.1371/journal.pone.0092593

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Abstract

Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

Highlights

Viruses promote a widespread reduction of host cell gene expression to reduce competition for cellular resources, to decrease expression of cellular factors that elicit an immune response to viral infection, and to facilitate the establishment of viral latency
PABPC was present in the nucleus in cells that were positive for diffuse early antigen (EA-D) a viral protein that functions as a DNA polymerase processivity factor during lytic replication (Fig. S1: v, vi)
When the Epstein Barr Virus (EBV) lytic cycle was induced by transfection of a plasmid expressing ZEBRA, PABPC localized to the nucleus (Fig. 1B: x, xi, xii, xiv, xvi, xvii; blue arrows)

Summary

Introduction

Viruses promote a widespread reduction of host cell gene expression to reduce competition for cellular resources, to decrease expression of cellular factors that elicit an immune response to viral infection, and to facilitate the establishment of viral latency. This process, termed viral host shutoff (vhs), is mediated by modulation of transcription, mRNA splicing, nuclear export of mRNA, mRNA decay, translation, and proteolysis [1]. NSP3 (non-structural protein 3) evicts PABPC from eukaryotic mRNA poly(A) tails and disrupts the interaction between PABPC and eIF4G [6,7]. PABPC accumulates in the nucleus as the result of an interaction of NSP3 with a cellular protein, RoXaN [8,9]

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