No abstract

RNA virus genomes contain cis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3 untranslated region (3 UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3 UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.Embedded in the genomes of RNA viruses are cis-acting sequence and structural elements that participate in replication, transcription, translation, and packaging. Some of these signals are thought to facilitate the selective interactions of viral RNAs with the machinery of RNA synthesis, while others enable or modulate events that occur during viral protein synthesis or assembly. The coronaviruses and arteriviruses, members of the order Nidovirales, are positive-strand RNA viruses with very large genomes (13 to 15 kb for arteriviruses, 27 to 31 kb for coronaviruses). In addition to the production of progeny genome copies, nidovirus replication entails the synthesis of a 3Ј nested set of subgenomic (sg) mRNAs (15). Each sgRNA contains a 5Ј leader segment connected to a body segment that is identical to the 3Ј end of the genome, starting at a given point preceding one of the downstream open reading frames. Accumulating evidence, particularly from landmark studies using a full-length infectious cDNA of equine arterivirus, supports a model in which the discontinuous step in sgRNA formation occurs during negative-strand RNA synthesis (1,28,31,35). Thus, for nidoviruses, the earliest steps of both genome replication and sgRNA transcription initiate at the 3Ј end of the genome; therefore, it is reasonable to expect that at least part of the regulation of these processes is implicit in the sequence and structure of the 3Ј untranslated region (3Ј UTR). Indeed, studies of defective interfering...

For positive-strand RNA viruses, genomic RNA (gRNA) functions both as a message for translation and as a template for the viral RNA-dependent RNA polymerase. As part of their infectious cycle, these viruses often extensively remodel host cellular membranous compartments to facilitate replication and transcription of gRNA. These expropriated membranes are thought to provide surfaces for the concentration of enzymatic and substrate components of RNA synthesis, to furnish general protection from cellular ribonucleases, and to allow evasion of host innate immunity through the sequestration of double-stranded viral RNA intermediates. Among all known RNA viruses, coronaviruses have the largest genomes, ranging from 27 to 32 kb, and the most complex machinery for RNA synthesis (21,35,74). The coronavirus replicase-transcriptase complex (here called the replicase) is assembled from 16 nonstructural proteins (nsp), which collectively total some 803 kDa for mouse hepatitis virus (MHV). The nsp are autoproteolytically processed from two overlapping polyprotein precursors, which are produced by translation of gRNA. Almost all of the replicase components are associated with or embedded in convoluted membranes and double-membrane vesicles that are generated during coronavirus infection (29).In addition to the replicase, the nucleocapsid protein (N) is known to play an important role in coronavirus RNA synthesis, although that role is, as yet, poorly defined (1,51,60,76). Multiple investigations have shown intracellular colocalization of N with replicase components at early stages of infection by MHV or by severe acute respiratory syndrome coronavirus (SARS-CoV) (14,55,58,61). The participation of N protein in an early event in RNA synthesis is also implied by the poor adherence of coronaviruses to the nearly universal rule that the gRNA of positive-strand RNA viruses is completely infectious when introduced into susceptible host cells. Some time ago, we noted that the infectivity of purified MHV gRNA was markedly enhanced by cotransfection of synthetic N mRNA (36); this phenomenon was rediscovered when full-length reversegenetic systems were devised for coronaviruses. For systems that are launched by transfection of gRNA, the provision of cotransfected N mRNA or N-encoding plasmid either is greatly stimulatory (10,(69)(70)(71) or is absolutely required (6) for the recovery of infectious virus.In the present study, we analyzed the consequences of replacing the MHV N open reading frame (ORF) with its counterpart from the closely related bovine coronavirus (BCoV). This substitution resulted in the discovery of a previously unsuspected genetic interaction between the serine-and argininerich (SR) region of the N protein and a region of the nsp3 subunit of the viral replicase. We confirmed this interaction through biochemical pulldown experiments. Moreover, we found that there is a correlation between the N-nsp3 interaction and the ability of N protein to stimulate the infectivity of MHV gRNA. Our results offer an explanation for t...