A furoviral replicase recruits host HSP70 to membranes for viral RNA replication

Jian Yang,Fen Zhang,Tong-Quan Zhu,Nian-Jun Cai,Jian-Ping Chen,Xuan Chen,Ne Wu,Xiang-Feng Meng,Heng-Mu Zhang,Jing Li

doi:10.1038/srep45590

Abstract

Many host factors have been identified to be involved in viral infection. However, although furoviruses cause important diseases of cereals worldwide, no host factors have yet been identified that interact with furoviral genes or participate in the viral infection cycle. In this study, both TaHSP70 and NbHSP70 were up-regulated in Chinese wheat mosaic furovirus (CWMV)-infected plants. Their overexpression and inhibition were correlated with the accumulation of viral genomic RNAs, suggesting that the HSP70 genes could be necessary for CWMV infection. The subcellular distributions of TaHSP70 and NbHSP70 were significantly affected by CWMV infection or by infiltration of RNA1 alone. Further assays showed that the viral replicase encoded by CWMV RNA1 interacts with both TaHSP70 and NbHSP70 in vivo and vitro and that its region aa167–333 was responsible for the interaction. Subcellular assays showed that the viral replicase could recruit both TaHSP70 and NbHSP70 from the cytoplasm or nucleus to the granular aggregations or inclusion-like structures on the intracellular membrane system, suggesting that both HSP70s may be recruited into the viral replication complex (VRC) to promote furoviral replication. This is the first host factor identified to be involved in furoviral infection, which extends the list and functional scope of HSP70 chaperones.

Highlights

Heat shock protein 70 s (HSP70s) are central to the cellular chaperone network, and have long been recognized as among the most abundant conserved molecular chaperones in all living organisms[2,3], assisting nascent protein folding in normal physiological conditions as well as under biotic and abiotic stress
In cells of Chinese wheat mosaic furovirus (CWMV)-infected plants either expressing the non-fused GFP or co-expressing NbHSP70:GFP or TaHSP70-GFP with the CWMV RNA2 clone, the fluorescence patterns were similar to those of the cells expressing non-fused GFP, NbHSP70:GFP, or TaHSP70-GFP alone. These results suggest that CWMV infection modifies the subcellular distribution pattern of TaHSP70 and NbHSP70 and that the proteins encoded by CWMV RNA1 are responsible for this subcellular modification
The tobacco rattle virus (TRV)-based silencing of NbHSP70 resulted in distinct crinkles and abnormal veins on leaves of N. benthamiana plants at 10 dpi (Fig. 3A,B) and even the death of young leaves and shoot apical meristem at 14 dpi, which were effects similar to those seen in N. benthamiana plants with PVX-based silencing of NbHSP70c-1, a cytoplasmic member of the HSP70 family[27,28]

Summary

Introduction

HSP70s are central to the cellular chaperone network, and have long been recognized as among the most abundant conserved molecular chaperones in all living organisms[2,3], assisting nascent protein folding in normal physiological conditions as well as under biotic and abiotic stress. A major staple crop plant, quantitative PCR and proteomic analysis showed that its HSP70 genes are expressed throughout the early grain development stages[10,11,12]; and are up-regulated by the addition of CaCl213, by heat stress[10,14] and by infection with the stripe rust fungus. RNA1 (7,147 nt) encodes three proteins: (1) a 153 KDa replicase protein (p153); (2) a 212 KDa protein (p212) produced by occasional read-through of the UGA termination codon of p153 to incorporate an RdRP domain; (3) a cell-to-cell movement protein (MP). The expression of a host HSP70 gene was found to be regulated by CWMV infection and required for replication of CWMV. Its functions during viral infection were further dissected in host plants

Methods

Results

Conclusion