Báo cáo khoa học: The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue

The Potyviral Virus Genome-Linked Protein VPg Forms a Ternary Complex with the Eukaryotic Initiation Factors eIF4E and eIF4G and Reduces eIF4E Affinity for a mRNA Cap Analogue

Tác giả: Thierry Michon, Yannick Estevez, Jocelyne Walter, Sylvie German-Retana and Olivier Le Gall

Lĩnh vực: Tương tác giữa thực vật và virus

Nội dung tài liệu: Nghiên cứu này khám phá vai trò của protein liên kết với bộ gen virus (VPg) của potyvirus trong việc tương tác với các yếu tố khởi đầu dịch mã của sinh vật nhân thực, cụ thể là eIF4E và eIF4G. Thông qua các thí nghiệm phổ huỳnh quang và kéo xuống GST, các nhà nghiên cứu đã chứng minh rằng VPg có thể tạo thành phức hợp ba ngôi với eIF4E và eIF4G. Ngoài ra, VPg làm giảm ái lực của eIF4E đối với một chất tương tự mũ mRNA, cho thấy VPg có thể điều chỉnh chức năng sinh hóa của eIF4E. Dữ liệu cũng cho thấy VPg có thể liên kết với eIF4G một cách gián tiếp thông qua sự liên kết với eIF4E, gợi ý một cơ chế mới trong tương tác virus-vật chủ.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 10 October 2005, revised 23 January 2006, accepted 26 January 2006)
• doi:10.1111/j.1742-4658.2006.05156.x
• The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5′ end.
• We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV).
• GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation.
• Lettuce mosaic virus (LMV), is a member of the genus Potyvirus [1].
• Eukaryotic mRNAs are capped by the addition of a 7-methylated guanine (m7G).
• Abbreviations
• VPg abolished its interaction with eIF4E, and this correlated with a defect in infectivity in Brassica perviridis [11].
• The simplest hypothesis is that VPg recruits eIF4E to initiate the translation of the potyvirus polyprotein [12].
• Results and discussion
• Interaction between VPg and eIF4E
• We initially evaluated the perturbation of the intrinsic fluorescence of eIF4E upon its interaction with VPg.
• Binding-site characterization
• To obtain a better fluorimetric response of eIF4E upon its interaction with VPg, we attempted to follow indirectly the complex formation using the cap analogue m’GDP.
• Fig. 1. Fluorescence emission spectra of eIF4E upon VPg addition.
• Fig. 2. Effect of VPg on m’GDP binding to eIF4E at 25 °C.
• Fig. 3. (A) Binding isotherms of m’GDP with eIF4E in the presence of high VPg concentrations.
• Scheme 1. Two plausible models for the interactions between eIF4E, VPg and the cap analogue m’GDP.
• GST pull-down assay of plant proteins forming complexes with VPg
• Several studies have reported specific interactions between the VPg from potyvirus and eIF4E or one of its isoforms [9].
• Fig. 4. Analysis of the plant soluble proteins complexed with VPg.
• We demonstrate here that the VPg-eIF4E interaction previously described in other pathosystems such as TuMV/B. perviridis is also found in the LMV/lettuce system.
• The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, is enhanced by VPg
• The eukaryotic eIF4F initiation complex is a heterotrimer consisting of eIF4E, eIF4A (an RNA helicase) and eIF4G [26].
• Fig. 6. Effect of VPg on the association between eIF4E and pep4G.
• Scheme 2. Hypothetical pathways of eIF4E and eIF4G recruitment.
• In fact, pep4G has a small effect on cap binding, whereas whole eIF4G is a strong enhancer of eIF4E binding to the mRNA cap structure [29].
• Experimental Procedures
• Materials
• Protein expression and isolation
• Specific isolation procedures
• Pull-down assays
• Fluorescence measurements
• Data analysis
• Acknowledgements
• References