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Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro.

ABSTRACT: Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.

SUBMITTER: Gradi A

PROVIDER: S-EPMC371048 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro.

Gradi Alessandra A Foeger Nicole N Strong Rebecca R Svitkin Yuri V YV Sonenberg Nahum N Skern Tim T Belsham Graham J GJ

Journal of virology 20040401 7

Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage ...[more]

PMID: 15016848

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Project description:The translation initiation of foot-and-mouth disease virus (FMDV) occurs at two alternative initiation sites (Lab AUG and Lb AUG). Usually, the Lb AUG is more favorably used to initiate protein synthesis than the Lab AUG. To explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, this initiation codon was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA). Fortunately, the modifications did not prevent viral viability but influenced replication characteristics of some FMDV mutants in a cell-specific manner, as was shown by the similar replication in BHK-21 cells and delayed growth kinetics in PK-15 cells. This attenuated phenotype of FMDV mutants in PK-15 cells was found to be correlated with reduced abilities to cleave eIF4GI and suppress interference (IFN) expression. As leader (L) protein was reported to be responsible for eIF4GI cleavage and inhibition of IFN expression, the in vivo L protein synthesis was examined during the infection of FMDV mutants. Our results showed that not only the total yield of L proteins was severely influenced but also the individual yield of L protein was seen to be affected, which implied that both the relative usage of the two initiation sites and overall translation efficiency were changed by Lb AUG modifications. In addition, the in vitro translation activity was also negatively regulated by Lb AUG mutations. Collectively, these findings suggested that the restricted replications of Lb AUG-modified FMDVs were related to the delayed eIF4GI cleavage and decreased ability to block IFN expression but were mainly determined by the inefficient translation initiation. FMDVs precisely with modifications of Lb AUG initiation codon may represent safer seed viruses for vaccine production. KEY POINTS: • The polyprotein translation of FMDV initiates at two alternative initiation sites (Lab AUG and Lb AUG). In order to explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, the Lb initiation AUG was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA), and four FMDV mutants with Lb AUG modification were generated. • We found that partial FMDV mutants grew almost as well as WT virus in BHK-21 cells, a typical cell line used for FMD vaccine production, but displayed impaired replication in IFN-competent PK-15 cells. • The attenuation of mutant FMDVs in PK-15 cells was found to be correlated with delayed eIF4GI cleavage and decreased ability to block IFN expression. • We proved that the attenuated phenotype of Lb AUG-modified FMDVs was mainly determined by the inefficient translation initiation, as demonstrated by the decrease of total yield of L proteins and individual production of L protein. • We successfully generated genetically engineered FMDV with attenuated phenotype. The approach of precise engineering of FMDV with the modification of initiation codon provides a safe platform to produce inactivated antigen vaccines.

Project description:Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (type III-like IRES) and classical swine fever virus (type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions.

Dataset Information

Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro.

Publications

Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro.

Similar Datasets

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