Further Characterization Of The Nodamura Virus Rna2 3\u27-Terminal Stem Loop Structure And Its Role In Viral Rna Replication

Nodamura virus (NoV) is an excellent model system for studying many aspects of the life cycle of positive-strand RNA viruses. NoV\u27s simple genome organization and ability to replicate its genome to high levels in a wide variety of host cells, including plants, yeast, insects and mammals (Ball et al., 1992; Garzon et al., 1978, 1990; Murphy et al., 1970; Price et al., 2005; Scherer & Hurlbut, 1967; Selling et al., 1990; reviewed by Ball & Johnson, 1998), make it a prime model system for studying the basic mechanisms of viral RNA replication. Other positive-strand RNA viruses, including those that are pathogenic for humans, may share these mechanisms. Additionally, these studies together suggest that the host factors involved in NoV RNA replication must be widely conserved across many eukaryotic organisms. NoV is unique in its lethality for both invertebrates and mammals, as other nodaviruses are nonpathogenic for higher vertebrates (Bailey et al., 1975; Longworth & Carey, 1976; Ball & Johnson, 1998) and cannot replicate at 37oC (Ball et al., 1992). These characteristics support the idea of NoV serving as a simplified model system for more complex positive-strand RNA human pathogens, particularly arboviruses and picornaviruses. In defining the minimum essential cis-acting elements required for nodavirus RNA replication, previous work in our laboratory predicted the presence of a conserved stem-loop structure in the 3\u27 untranslated region (UTR) of NoV RNA2, verified its existence biochemically, and showed that it that acts as a cis-acting replication element in chimeric NoV RNA2-based replicons in transformed yeast cells (Rosskopf et al., 2010; Taufer et al., 2008). RNA1structures in the 3\u27 UTRs of viral RNAs represent ubiquitous motifs common to many RNA viruses. These structures function in numerous aspects of the life cycles of these viruses, including RNA replication and translation. This project contains two major foci within the area of nodaviral RNA replication: (i) to elucidate the structural characteristics of a cis-acting element, 3\u27SL, that is required for RNA2 replication, including its potential inclusion in a predicted long-range tertiary structure element; and (ii) to determine the functional implications of another predicted stem-loop structure, 3\u2710SL, at the extreme 3\u27-end of the RNA2 genome segment. Herein, we examine the mechanistic role of the individual components of the 3\u27SL that are responsible for RNA2 replication, specifically the double-stranded stem and the single-stranded loop. We demonstrate that each of these regions retains separate requirements for successfully directing RNA replication. We also illustrate that the NoV RNA2 3\u27SL functions in both strands of RNA2: the positive strand genomic RNA and the negative strand antigenomic RNA replication intermediate. This work examines the specific structural requirements of the NoV 3\u27SL in the context of full- length RNA2 in a mammalian system and its potential functions in RNA2 replication. It will also consider the role in RNA2 replication of a complex RNA tertiary structure in which the loop of 3\u27SL is predicted to base pair with a sequence over 600 nucleotides upstream. Additionally, our work herein demonstrates that the predicted 3\u2710SL at the extreme 3\u27 end of RNA2 is at least partially responsible for protecting this genome segment from enzymatic modification and that it is potentially involved with RNA stability