What's in the name? That which we call a rose by any other name would smell as sweet.

de Breyne, Sylvain, Nathalie Chamond, Didier Décimo, Mary-Anne Trabaud, Patrice Andre, Bruno Sargueil, and Théophile Ohlmann. 2012. “In Vitro Studies Reveal That Different Modes of Initiation on HIV-1 mRNA Have Different Levels of Requirement for eIF4F.” The FEBS Journal (July 3). doi:10.1111/j.1742-4658.2012.08689.x.

What I would like to say about this manuscript is that it has nothing to do with HIV-1 mRNA translation. It's just irrelevant and reproduces old data. Bold statement indeed, but I'll try to convince you. What do the authors think they study? HIV-1 translation. As far as I know, there exists a set of HIV-1 mRNAs, all of them possessing 5'UTRs (1). However, the authors continue to use an artificial mRNA without any 5'UTR, thereby strating directly with gag AUG-codon. Somehow they do not bother if this is physiologically relevant or no. So shall we.

Prior to reading this manuscript I highly recommend reading two undeservedly forgotten researches, which were remotely mentioned by the authors. Here they are: (2) and (3). In the former translation of an artificial mRNA with two separated AUG-codons was studied in RRL. And the latter research deals with a behavior of leaderless mRNA in RRL.

Testing different concentrations of an mRNA with 5'UTR and two AUG codons.

de Breyne et al., Figure 1B: increasing mRNA (HIV1 5'UTR-AUG1-AUG2) concentration in RRL results in a continuous increase of initiation frequency at the second AUG codon and, starting from a certain concentration, decrease in initiation frequency at the first AUG codon.

Dasso et al., Figure 4A: increasing mRNA concentration (irrelevant 5'UTR-AUG1-AUG2) in RRL results in a continuous increase of initiation frequency at the second AUG codon and, starting from a certain concentration, decrease in initiation frequency at the first AUG codon.

Addition of a competitor to an mRNA with 5'UTR and two AUG codons.

de Breyne et al., Figure 1C: addition of a competitor (HIV-1 5'UTR) results in an increase of initiation frequency at the second AUG.
Dasso et al., Figure 5: addition of a competitor (E.coli 16S rRNA) results in an increase of initiation frequency at the second AUG.

Effect of 5'-terminal m7G-cap on AUG1 and AUG2

de Breyne et al., Figure 2 and S2: capping simulates translation at AUG1 and does not affect initiation at AUG2.
Dasso et al., Figure 7: initiation frequency at AUG2 is not affected by mRNA capping

Requirements for eIF4A

de Breyne et al., Figure 4: eIF4A R362Q mutant does not inhibit translation initiation at AUG1 of leaderless mRNA
Andreev et al., Figure 3: 48S complex is efficiently formed on the leaderless mRNA in the absence of eIF4F or eIF4A.

In the absence of proper control (any other mRNA with two AUG codons or any other leaderless mRNA which both would behave essentially similar to the corresponding mRNAs used in this study) all the authors say is totally irrelevant to HIV-1. And, sadly, there is nothing new in these data. Is a corpus of published papers so huge that those referees have forgotten these papers (2,3) or are they just unfamiliar with them?

1. Yilmaz, Alper, Cheryl Bolinger, and Kathleen Boris-Lawrie. 2006. “Retrovirus Translation Initiation: Issues and Hypotheses Derived From Study of HIV-1..” Current HIV Research 4 (2): 131–139.

2. Dasso, M C, S C Milburn, J W Hershey, and R J Jackson. 1990. “Selection of the 5'-Proximal Translation Initiation Site Is Influenced by mRNA and eIF-2 Concentrations.” European Journal of Biochemistry / FEBS 187 (2): 361–371.

3. Andreev, Dmitri E, Ilya M Terenin, Yan E Dunaevsky, Sergei E Dmitriev, and Ivan N Shatsky. 2006. “A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors.” Molecular and Cellular Biology 26 (8): 3164–3169.

Less is more

Pan, Meng, Xiaorong Yang, Lei Zhou, Xinna Ge, Xin Guo, Jinhua Liu, Dabing Zhang, and Hanchun Yang. 2012. “Duck Hepatitis a Virus Possesses a Distinct Type IV Internal Ribosome Entry Site Element of Picornavirus..” Journal of Virology 86 (2): 1129–1144.

In this study the authors have characterized IRES found in 5'UTR of DAHV. Almost all the data presented here are in line with earlier suggestion that this IRES belongs to wide group of hepacivirus/pestivirus (HP) IRESs (1). Not in line with this suggestion is the finding that translation of a bicistronic mRNA with DAHV IRES in the intercistronic position is highly sensitive to eIF4G cleavage by SVDV 2A protease.

However, this test was performed in vivo by means of DNA transfection. This approach suffers from a plenty of possible artifacts, for example cryptic promotor activity, e.g. present in the HCV IRES cDNA (2). One might argue that using T7 RNA polymerase vaccinia virus system overcomes these shortcomings, but the transfected DNA goes to the nucleus anyway and can be transcribed there, generating short capped monocistronic Fluc containing mRNAs, which translation would be perfectly inhibited by 2A protease. Therefore, RNA controls should have been performed: Nothern, RT-PCR, or RNAi against the first cistron (3). Also, a simple way to show that the intact eIF4G is required is translation in vitro with the addition of recombinant protease.

Funny is the authors' statement about "~0.5-fold stimulation" (and it was stimulated indeed) of the EMCV IRES which normally means 2-fold inhibition. :) Finally, the authors draw their conclusion:

These data indicate that the intact eIF4G is required for DHAV internal initiation of translation and that the HAV IRES element is no longer the only one abolished by cleavage of eIF4G.

Well, not exactly. Translation of the DAHV IRES was stimulated ~0.4-fold inhibited ~2.5 fold upon eIF4G cleavage. (Where is a control with HCV IRES, by the way?) This is not what one would call abolished. If a factor is required, there's no translation in it's absence: EMCV IRES translation is abolished by eIF4A R362Q mutant. Therefore, you can't contend that translation is eIF4G-dependent until you perform a set of in vitro translations with addition of hippuristanol (4) or eIF4A R36Q mutant or anything of this kind.

That's surprising, because this fact is the only one in the whole manuscript that makes a researcher blink at. But there is a single experiment lacking controls. And you can be sure that in a forthcoming review this IRES will be cited as a novel. Which is not proved yet. 

1. Hellen, Christopher U T, and Sylvain de Breyne. 2007. “A Distinct Group of Hepacivirus/Pestivirus-Like Internal Ribosomal Entry Sites in Members of Diverse Picornavirus Genera: Evidence for Modular Exchange of Functional Noncoding RNA Elements by Recombination..” Journal of Virology 8 (11): 5850–5863.

2. Dumas, Estelle, Cathy Staedel, Marie Colombat, Sandrine Reigadas, Sandrine Chabas, Thérèse Astier-Gin, Annie Cahour, Simon Litvak, and Michel Ventura. 2003. “A Promoter Activity Is Present in the DNA Sequence Corresponding to the Hepatitis C Virus 5' UTR..” Nucleic Acids Research 3 (4): 1275–1281.

3. van Eden, Marc E, Marshall P Byrd, Kyle W Sherrill, and Richard E Lloyd. 2004. “Demonstrating Internal Ribosome Entry Sites in Eukaryotic mRNAs Using Stringent RNA Test Procedures..” RNA 10 (4): 720–730.

4. Bordeleau, Marie-Eve, Ayaka Mori, Monika Oberer, Lisa Lindqvist, Louisa S Chard, Tatsuo Higa, Graham J Belsham, Gerhard Wagner, Junichi Tanaka, and Jerry Pelletier. 2006. “Functional Characterization of IRESes by an Inhibitor of the RNA Helicase eIF4A.” Nature Chemical Biology 2 (4): 213–220.