All arthropod-borne flaviviruses produce the small noncoding RNA (sfRNA) derived from the highly conserved regions of the viral genome's 3'-untranslated region (UTR). Deleting the stem-loop II (SLII) at the 3'-UTR abolished the formation of the sfRNA in infected cells. The sfRNA-deficient mutant (MT) showed a slower growth rate, smaller plaques, and fewer viral titers than wild-type (WT) viruses. In this study, the small plaques formed by MT viruses could enlarge with prolonged incubation, suggesting that the sfRNA affects cell growth. To investigate host proteins that may involve the slow growth rate of the MT virus, proteomic profiling of uninfected, WT, and MT virus-infected cells was compared. Mass spectrometry screened 2515 proteins with differential expression. One of the most differentially expressed proteins, growth arrest and DNA damage-inducible protein 34 (GADD34), was further examined. GADD34 is upregulated in WT-infected cells but less abundantly expressed in the MT virus-infected cells, as evidenced by qRT-PCR and western blot analyses. Inhibition of GADD34 did not affect uninfected cells but impaired cell viability during both virus infections. Inhibition of GADD34 decreased the NS5 translation of the MT virus but not the WT virus. The function of GADD34 is to mediate the dephosphorylation of the eukaryotic initiation factor alpha subunit (eIF2α), which prevents the inhibition of cap-dependent translation. The eIF2α in the MT virus-infected cells was highly phosphorylated but much less in WT virus-infected cells, leading to less abundant protein expression in the MT virus-infected cells. Taken together, these results provide preliminary evidence that sfRNA may involve in translation and cell growth by association with the host GADD34 protein through dephosphorylation of eIF2α.

INTRODUCTION 1
MATERIALS AND METHODS 7
RESULTS 17
DISCUSSION 23
REFERENCES 27
TABLES AND FIGURES 35

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