Modification of the second translation initiation site restricts the replication of foot-and-mouth disease virus in PK-15 cells

Hong Yuan,Yimei Cao,Zaixin Liu,Na Li,Zengjun Lu,Pinghua Li,Xueqing Ma,Pu Sun,Kun Li,Jie Zhang,Jing Zhang,Yingli Chen,Huifang Bao,Yuanfang Fu,Xiaohua Gong,Dong Li,Xingwen Bai

doi:10.1007/s00253-020-10810-w

Abstract

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.

Highlights

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly infectious disease of domestic and wild cloven-hoofed animals including cattle, swine, sheep, and goats (Grubman and Baxt 2004)
Two alternative translation initiation sites are invariably noted in L coding sequence, and they are found to be responsible for the initiation of viral protein synthesis
We demonstrated that for FMDV O/HN/CHN/93 used in the present study, the individual mutation of Lab AUG was tolerated

Summary

Introduction

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly infectious disease of domestic and wild cloven-hoofed animals including cattle, swine, sheep, and goats (Grubman and Baxt 2004). The production of inactivated vaccine requires expensive high-containment facilities where live FMDVs are grown in large volumes, which poses a risk of virus escape from the manufacturing facilities (Cottam et al 2008; King et al 1981). There does have been an association of disease outbreaks with incomplete chemical inactivation and escape of virus from vaccine manufacturing facilities (Cottam et al 2008). To overcome this serious production risk, one of the approaches is to develop attenuated FMDV strains. It is crucial to obtain FMDV with reduced replication

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Conclusion