Moloney murine leukemia virus (MoMLV) expresses Gag and Gag-Pol proteins from the same unspliced mRNA. Both genes lie in the same reading frame and are separated by a single UAG stop codon. Translation of this mRNA primarily yields the Gag polyprotein, but a small percentage (∼2-10%) of ribosomes read through the gag stop codon to produce a Gag-Pol fusion. Gag-Pol is the sole source of the virus-specific enzymes encoded in the pol gene and is essential for viral replication. An RNA pseudoknot located immediately downstream of the gag translation terminator is required for stimulating readthrough. Little is known about the mechanism by which the pseudoknot functions and no high-resolution structural data are currently available.

To further our understanding of pseudoknot-induced readthrough, we used multidimensional nuclear magnetic resonance techniques to determine the structure of the MoMLV pseudoknot. The RNA displays an overall H-type pseudoknot fold, consisting of a stem-loop where bases in the loop hydrogen bond with downstream nucleotides to form a second stem. The two stems of the pseudoknot coaxially stack upon each other to form a collinear quasi-continuous helix. The base of stem 1 contains an unpredicted 1 x 2 internal loop motif and a single nucleotide (A₁₇) packs deeply within the major groove of the helical junction.

We found that the structure of the MoMLV pseudoknot is highly sensitive to pH. The N1 nitrogen of A₁₇ displays an apparent pKa of ∼6.3 and becomes protonated in low pH environments. A₁₇ protonation causes a conformational change at the helical junction that causes stem 2 to bend towards the minor groove. This bend is stabilized by multiple triple base interactions in the minor groove of stem 1.

Using a bicistronic luciferase reporter system, we showed that the readthrough-stimulating activity of the pseudoknot is also pH-sensitive. At higher pH values, the pseudoknot stimulates readthrough at low levels. When the pH is lowered, the pseudoknot induces a significant enhancement of readthrough. We propose that readthrough, and thus retroviral gene expression, may be regulated by a dynamic, proton-driven equilibrium between active and inactive pseudoknot conformations.

In addition, we discovered a host protein, ribosomal protein L4 (Rp14) that augments readthrough. Expression of Rp14 stimulates readthrough in a pseudoknot- and cell line-dependent manner. Rp14 also enhances the frameshifting efficiencies of both HIV-1 and MMTV. These data suggest that Rp14 may function in a biological pathway shared by readthrough and frameshifting to modulate these mechanistically distinct processes.