: Coronaviruses contain a 30Kb single-stranded, positive polarity RNA genome. Using a novel strategy and six adjoining cDNA subclones, we have developed an approach to systematically assemble a full-length infectious construct of the coronavirus, transmissible gastroenteritis virus (TGE), an economically important pathogen in swine. T7 transcripts derived from the full-length TGE construct were infectious and progeny virions were serially passage in permissive host cells. The availability of this cDNA construct will allow us to address fundamental questions in the biology of coronaviruses, which were previously untenable. In this application, we will use reverse genetics and the full length TGE construct to systematically address the role of gene order, cis and trans acting regulatory sequences, and N gene function in coronavirus transcription and replication.
Aim 1. We hypothesize that the highly ordered coronavirus genome structure is selectively maintained to safeguard coordinated levels of virus gene expression. We will study the phenotypic consequences of gene deletion, duplication and rearrangement on TGE transcription and replication in vitro and determine the minimal genome requirements for coronavirus transcription. These studies will also determine if RNA recombination functions to maintain the precise gene order of Nidoviruses.
Aim2. We will use site specific mutagenesis to distinguish between the hypotheses that discontinuous transcription is guided by base pairing during negative or positive strand RNA synthesis. These studies will also define the minimal promoter elements in the TGE leader RNA and intergenic sequence required for expression of heterologous genes like green fluorescent protein and the Norwalk capsid protein in vitro.
Aim3. We will directly test the hypothesis that the N protein functions in virus transcription. It is not clear whether N is necessary for transcription or has some other ancillary role in mRNA, genome or negative strand synthesis. We will assemble TGE replicons either deficient in or encoding the TGE N genes and measure the effects on the replication and transcription of TGE replicon RNAs encoding green fluorescent protein (GFP) in vitro.The assembly of a full-length TGE construct is an important breakthrough for coronavirus research and will benefit all aspects of coronavirus pathogenesis, molecular biology, epidemiology and biochemistry.
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