ΔAflII-SpeI(+) 5'GACCCGGGGTACCAAGCTTGAGTTTAAACGCTAGCCAGC'3  Further Characterisation of the Translational Termination-Reinitiation Signal of the Influenza B Virus Segment 7 RNA Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAA UG and ∼10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAA UG, known as the ‘termination upstream ribosome binding site’ (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAA UG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning. Eukaryotic viruses have evolved a variety of translational control strategies to facilitate expression of downstream open reading frames (ORFs) on polycistronic mRNAs and examples have been described at all three steps of protein synthesisinitiation, elongation and termination [1] . These include leaky scanning of 40S ribosomal subunits past the most 59 AUG [2] , the de novo recruitment of ribosomes to intercistronic internal ribosomal entry sites (IRESs; [3] ); programmed ribosomal frameshifting [4, 5] and the circumvention of normal termination by programmed stop codon readthrough [6, 7] . Generally, these processes allow the expression of two (or more) proteins from a single mRNA and may also permit a level of control over their relative quantities. Another way of accessing a downstream ORF in viral mRNAs is by termination-dependent reinitiation of translation (termination-reinitiation), a phenomenon first described in the expression of the influenza B virus BM2 protein [8] . The dicistronic mRNA that is derived from genomic segment 7 of this virus has two ORFs encoding matrix protein 1 (M1) and BM2, with the termination codon of M1 in close proximity to the start codon of the BM2 ORF (UAAUG; stop codon of M1 in bold, start codon of BM2 underlined) [8] [9] [10] . Following translation of M1, some 10-20% of ribosomes terminating at the M1 stop codon go on to reinitiate translation at the immediately adjacent BM2 start codon [8, 11] . This capacity to reinitiate protein synthesis following translation of a long upstream ORF was unexpected. During the elongation phase, initiation factors are likely to be rapidly lost, thus reinitiation of translation following termination was believed to be restricted to cases where the upstream ORF (uORF) is very short [12] [13] [14] . Our knowledge of the mRNA signals that allow efficient reinitiation following translation of a long upstream ORF has largely been obtained from studies of caliciviruses, namely in the expression of the VP2 protein of feline calicivirus (FCV; [15] [16] [17] ) and the VP10 protein of rabbit haemorrhagic disease virus (RHDV; [18, 19] ). Here, expression of the downstream ORF by termination-reinitiation requires a stretch of mRNA (between 69 and 87 nt in length) upstream of the stop codon of the first ORF (termed the termination upstream ribosome binding site or TURBS) and the close proximity of the stop and start codons of the two ORFs [15, 17, 19] . Within the TURBS, two essential sequence motifs (motifs 1 and 2) have been described. Motif 1 is highly conserved amongst caliciviruses and is composed of a short stretch of 6-9 nt (with a conserved core of UGGGA) with complementarity to the apical loop of helix 26 of 18S rRNA. This motif, which lies towards the 59 boundary of the TURBS, likely acts to tether the 40S subunit to the mRNA posttermination, facilitating the recruitment of the initiation factors necessary for reinitiation [15, 19] . Motif 2, which lies some 15-20 nt upstream of the termination-reinitiation window (AUGA in FCV), is a second essential stretch of 4-8 nt but shows little sequence conservation amongst caliciviruses. Recent work has revealed that this motif in fact forms the 39 arm of a stem-loop structure whose formation is necessary for efficient terminationreinitiation [16] .