Previous studies on the termination-reinitiation process have suggested the importance of the close proximity of the stop codon of the upstream ORF and the start codon of the downstream ORF [8, 11, 15, 17, 18, 32] . This is believed to reflect a restricted mobility of the tethered ribosome; that is, it may be able to undergo only limited movement following termination. However, we show here that the distance between the terminating ribosome and the TURBS is more critical to reinitiation efficiency ( Figure 5 ) and that when the start codon of the BM2 ORF is placed downstream of the stop codon, reinitiation is detectable even when the start codon is moved up to 63nt downstream of its original position ( Figure 6 ). This suggests that it is not solely the distance between the start and stop codons per se that affects reinitiation efficiency but rather how well the ribosome can be tethered (by virtue of the distance between where the ribosome terminates and the TURBS). However, whilst efficient reinitiation requires an AUG at, or very near, the wild-type site ( Figure 6A and 6B) , other reinitiation sites can be used when the native reinitiation codon is absent, albeit at lower efficiency. Under the latter circumstance, ribosomes can reinitiate at a downstream AUG codon ( Figure 6B ), or at a near-cognate initiation codon (such as the -1 AUA codon, Figure 6 ) close to the native reinitiation site. However, if given a choice of a wild-type AUG and one located downstream, the ribosome always selects the wild-type AUG, even if the ribosome artificially terminates downstream of the reinitiation window ( Figure 6B) , suggesting that the TURBS may cause the tethered 40S subunit to 'snap back' to the proper site of reinitiation. Importantly, we show that there is no requirement for eIF4G in location of either wild-type or downstream reinitiation sites. As such, location of downstream AUGs is likely due to the 40S subunit being transferred directly to the AUG whilst tethered to the TURBS (perhaps in complex with eIF3 and other factors) by an RNA-looping mechanism. Figure S1 Recombinant 4EBP1 has no significant effect on reinitiation on the BM2 ORF. Motif 1 and TURBS deletion mutants were translated in the absence or presence of 250nM 4EBP1 (details as in Figure 7) . The fold-stimulation over the reinitiation levels observed in the absence of 4EBP1 are shown below the autoradiographs. (EPS) RNA and DNA Bacteriophages as Molecular Diagnosis Controls in Clinical Virology: A Comprehensive Study of More than 45,000 Routine PCR Tests Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids. Real-time (rt) PCR and reverse transcription (RT) PCR techniques are rapid and versatile diagnostic procedures broadly used in clinical virology where there are mostly considered as diagnostic ''gold standards'' [1] . Monitoring rt-PCR and rt-RT-PCR assays and validation of the results rely on the use of relevant external or internal controls (ECs or ICs) [1, 2] and commercial kits including such control systems are being increasingly improved for the molecular diagnosis of a number of pathogens such as HIV, hepatitis viruses, influenza viruses etc.. However, one of the main strengths of rt-PCR is versatility, which provides the opportunity to set-up ''in-house'' protocols for specific pathogens. The scientific literature now includes an impressive number of 'home made'' assays for various viral agents. Whilst most commercial kits include both ICs and ECs allowing accurate validation of the results [3] , ''home made tests'' are frequently performed in the absence of ICs and therefore without any possible individual monitoring of each diagnostic reaction. For example, the detection of technical errors or PCR amplification inhibitors is intrinsically impossible if only ECs are used. In addition, ECs are usually undistinguishable from the native genome.