Translation of Sindbis Subgenomic mRNA is Independent of eIF2, eIF2A and eIF2D

Area of Science:

• Molecular Biology
• Virology
• Cell Biology

Background:

• Translation of viral subgenomic mRNA (sgmRNA) in mammalian cells can proceed even after eIF2 phosphorylation.
• Alternative translation initiation factors, such as eIF2A and eIF2D, have been proposed to substitute for eIF2 under these conditions.

Purpose of the Study:

• To investigate the role of eukaryotic initiation factor 2A (eIF2A) and eukaryotic initiation factor 2D (eIF2D) in Sindbis virus sgmRNA translation.
• To determine if eIF2A or eIF2D can compensate for the phosphorylation-induced inactivation of eIF2 during viral infection.

Main Methods:

• Utilized CRISPR/Cas9 genome engineering to create HAP1 human cell lines deficient in eIF2A, eIF2D, or both.
• Compared Sindbis virus infection and viral protein synthesis in knockout cell lines versus wild-type cells.
• Assessed the impact of siRNA-mediated silencing of eIF2A and eIF2D on viral protein production.
• Modified the initiation codon (AUGi) in sgmRNA to other codons and evaluated translation efficiency.

Main Results:

• Sindbis virus infection kinetics and viral protein synthesis were comparable across wild-type and eIF2A/eIF2D knockout cell lines, even when eIF2α was phosphorylated.
• Silencing eIF2A or eIF2D using siRNAs had minimal impact on late-stage viral protein synthesis.
• sgmRNA variants with modified initiation codons could still direct viral protein synthesis, indicating translation was not abrogated.
• No significant differences in translation were observed between cell lines, suggesting eIF2A and eIF2D are not involved in this sgmRNA translation.

Conclusions:

• Eukaryotic initiation factor 2A (eIF2A) and eukaryotic initiation factor 2D (eIF2D) are not required for the translation of Sindbis virus sgmRNA when eIF2α is phosphorylated.
• The translation of this specific viral sgmRNA is independent of eIF2A and eIF2D, even when alternative initiation codons are used.