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...

Coronavirus assembly results from an accumulation of interactions among four structural proteins, the positive-sense RNA genome, and a host membrane envelope obtained from the site of budding, which is the endoplasmic reticulum-Golgi intermediate compartment. Three of the four structural proteins are embedded in the virion envelope: the spike protein (S), the membrane protein (M), and the small envelope protein (E). The fourth, the nucleocapsid protein (N), resides in the virion interior, wrapping the positive-strand RNA genome into a helical nucleocapsid.The most abundant viral constituent is M, a 25-kDa protein containing three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M protein is the central organizer of assembly, in that it self-associates (8, 10, 21), captures S for virion incorporation (36, 37), and selectively packages the fraction of N protein that is bound to genomic RNA (32-34). The S protein is responsible for viral attachment to host cell receptors and for the membrane fusion event that initiates infection. This type I membrane protein, shortly after its synthesis, folding, and oligomerization, forms complexes with M protein in the endoplasmic reticulum (36, 37). The M-interacting domain of the S protein was localized to the transmembrane-endodomain region of the molecule, based on the assembly of chimeric S proteins into virus-like particles (VLPs) (16) or virions (18, 23). More recently, this determinant was further localized to the endodomain of S, which could confer virion assembly competence if transferred to a heterologous transmembrane protein (48).The general (1, 3, 4, 31, 45), but not universal (20), consensus from studies of VLPs is that formation of coronaviruses is mediated by just the M and the E proteins, and that neither S protein nor the nucleocapsid plays an obligate role in virion morphogenesis. The precise role of E protein in this process is enigmatic, with some evidence pointing to a direct interaction between E and M (1, 5) and other observations suggesting that E acts independently of M in the budding compartment (4,26,41). A very recent finding that may shed light on the workings of the E protein is the demonstration that the E protein of severe acute respiratory syndrome coronavirus has the properties of a cation-selective ion channel (47). The construction of E protein mutants of mouse hepatitis virus (MHV) has confirmed the critical role of E in viral assembly (14). Surprisingly, however, MHV remains viable, although severely impaired, following deletion of the E gene (25), whereas disruption of the E gene of porcine transmissible gastroenteritis virus (TGEV) is lethal (6,38).The interaction between M protein and N protein has been previously explored by biochemical and molecular biological methods for both MHV (33,34, 44) and TGEV (12). In a genetic approach, we constructed and analyzed a highly defective MHV mutant with a carboxy-terminal truncated M protein, M⌬2, and we identified suppressors of this...