Cells infected with the murine coronavirus, mouse hepatitis virus (MHV), show decreased host protein synthesis concomitant with an increase in viral protein synthesis. We examined the in vitro translation property of the conserved MHV 5β€²leader RNA sequence by constructing chimeric mRNAs in which the 72-nt 5β€²-leader of M protein mRNA (A59 strain) was positioned upstream of the human Ξ±-globin coding region in a T7 expression vector. Synthetic 5β€²-capped transcripts of these mRNA constructs were translated in cell-free extracts prepared from uninfected and MHV-infected murine DBT cells. Nonviral mRNAs translated readily in both uninfected and infected cell-free extracts. By contrast, replacement of the human Ξ±-globin 5β€²-untranslated region (UR) with the MHV 5β€²-leader increased translation ca. three- to fourfold in cell-free extracts from MHV-infected cells versus translation in extracts from uninfected cells. Chimeric globin mRNA containing the reverse complementary sequence of the viral leader RNA in the 5β€²-UR showed no such increase in translation, indicating sequence specificity for the effect. A 13-nt region (-UCUAAUCCAAACA-) immediately proximal to the start codon was found to be important for the increased translation of the MHV leader-containing mRNAs. These data indicate that the apparent down-regulation of host translation is not primarily due to an inhibition of host translation but also involves a significant stimulation of viral translation in cis by a structural feature of the MHV 5β€²-leader RNA sequence in conjunction with a virus-specified or virus-induced factor. Β© 1994 Academic Press.