Genetic Recoding in HIV-1 and IBV Is Modulated by Alternate RNA Structures

Abstract

RNA has been shown to play significant regulatory roles in many cellular processes, due to its ability to fold into complex three-dimensional structures. Many viral systems use the regulatory ability of RNA to control key steps of the viral life cycle. -1 Ribosomal frameshifting is a form of noncanonical translation used by many viruses, where during the process of translation the ribosome shifts one nucleotide backwards into an overlapping reading frame so that it can continue to translate the downstream gene without encountering a stop codon; this establishes the proper translation ratios of proteins encoded by different reading frames, and is necessary for viral propagation. This work investigates frameshifting in two different viral systems: human immunodeficiency virus-1 (HIV-1), a retrovirus that infects humans; and avian infectious bronchitis virus (IBV), a coronavirus that infects chickens. In both viruses, alternate RNA structures were found to play important roles in establishing frameshifting ratios. In HIV-1, we used in vivo bicistronic dual luciferase reporter assays alongside biophysical structure determination techniques to establish that the HIV-1 frameshifting RNA exists in a monomer-dimer equilibrium, with the dimeric and monomeric structures being the frameshifting active and inactive structures, respectively; the formation of a dimer with complex tertiary interactions is the active driver of frameshifting. This would be the first known characterization of the formation of an asymmetric RNA dimer and would establish the existence of an HIV-1 frameshifting active minor conformation. We likewise used in vivo bicistronic dual luciferase reporter assays alongside biophysical structure determination techniques to show that IBV also uses alternate RNA structures to establish frameshifting rates, through a protonation-driven equilibrium that partitions its population of RNA into frameshifting active and partially inactive dimeric structures. The IBV frameshifting RNA forms a three-stem pseudoknot dimer in a manner similar to that of other coronaviruses, which suggests that the three-stem pseudoknot dimer may be a common mechanism to coronaviruses. The role of dimerization to the mechanisms of these disparate viral systems implicates dimerization as a potentially common theme to all frameshifting systems.