The upstream end of the 3 untranslated region (UTR) of the mouse hepatitis virus genome contains two essential and overlapping RNA secondary structures, a bulged stem-loop and a pseudoknot, which have been proposed to be elements of a molecular switch that is critical for viral RNA synthesis. It has previously been shown that a particular six-base insertion in loop 1 of the pseudoknot is extremely deleterious to the virus. We have now isolated multiple independent second-site revertants of the loop 1 insertion mutant, and we used reverse-genetics methods to confirm the identities of suppressor mutations that could compensate for the original insertion. The suppressors were localized to two separate regions of the genome. Members of one class of suppressor were mapped to the portions of gene 1 that encode nsp8 and nsp9, thereby providing the first evidence for specific interactions between coronavirus replicase gene products and a cis-acting genomic RNA element. The second class of suppressor was mapped to the extreme 3 end of the genome, a result which pointed to the existence of a direct base-pairing interaction between loop 1 of the pseudoknot and the genomic terminus. The latter finding was strongly supported by phylogenetic evidence and by the construction of a deletion mutant that reduced the 3 UTR to its minimal essential elements. Taken together, the interactions revealed by the two classes of suppressors suggest a model for the initiation of coronavirus negative-strand RNA synthesis.RNA virus genomes contain cis-acting sequences and structures that interact with viral and cellular components to initiate a variety of essential functions. Such interactions can mediate RNA replication and transcription, the selective translation of viral transcripts, and the specific packaging of progeny genomic RNA (gRNA) into assembling virions. Initiation of RNA virus genome replication occurs at the extreme 3Ј end of gRNA in order to produce a strand of complementary polarity. For positive-strand RNA viruses, the initiation of negativestrand RNA synthesis requires the specific recognition of the viral gRNA template and the juxtaposition of replicative proteins and the 3Ј genomic terminus. cis-acting sequences are believed to play pivotal roles in these processes. An intriguing example of how cis-acting sequences can coordinate the assembly and targeting of a replication complex to the initiation site of negative-strand RNA synthesis has been provided by studies of poliovirus replication (4, 23, 57). A crucial initial event in this system is the formation of a ribonucleoprotein complex around a cloverleaf RNA structure at the 5Ј end of the poliovirus genome. This complex consists of the poliovirus proteasepolymerase precursor 3CD and the cellular poly(rC) binding protein, both of which bind to the 5Ј cloverleaf (1,2,7,17). In order to target poliovirus 3CD to the initiation site of negativestrand RNA synthesis, another cellular protein, poly(A) binding protein, becomes involved. Poly(A) binding protein is associated with...

The 3 cis-acting element for mouse hepatitis virus (MHV) RNA synthesis resides entirely within the 301-nucleotide 3 untranslated region (3 UTR) of the viral genome and consists of three regions. Encompassing the upstream end of the 3 UTR are a bulged stem-loop and an overlapping RNA pseudoknot, both of which are essential to MHV and common to all group 2 coronaviruses. At the downstream end of the genome is the minimal signal for initiation of negative-strand RNA synthesis. Between these two ends is a hypervariable region (HVR) that is only poorly conserved between MHV and other group 2 coronaviruses. Paradoxically, buried within the HVR is an octanucleotide motif (oct), 5-GGAAGAGC-3, which is almost universally conserved in coronaviruses and is therefore assumed to have a critical biological function. We conducted an extensive mutational analysis of the HVR. Surprisingly, this region tolerated numerous deletions, rearrangements, and point mutations. Most striking, a mutant deleted of the entire HVR was only minimally impaired in tissue culture relative to the wild type. By contrast, the HVR deletion mutant was highly attenuated in mice, causing no signs of clinical disease and minimal weight loss compared to wild-type virus. Correspondingly, replication of the HVR deletion mutant in the brains of mice was greatly reduced compared to that of the wild type. Our results show that neither the HVR nor oct is essential for the basic mechanism of MHV RNA synthesis in tissue culture. However, the HVR appears to play a significant role in viral pathogenesis.Coronaviruses are large, positive-strand RNA viruses with a reproductive cycle that involves both replication of genomic RNA (gRNA) and transcription of a 3Ј nested set of subgenomic mRNAs (sgRNAs) (34). RNA sequences and structures that participate in these functions are embedded at various loci in the genome and are thought to interact with viral and host cellular components (2). The cis-acting elements that are required for coronavirus replication have been studied principally through the use of defective interfering (DI) RNAs, which are extensively deleted genomic remnants that parasitize the RNA synthetic machinery of a helper virus. Most DI RNA studies have been performed with mouse hepatitis virus (MHV) and bovine coronavirus (BCoV), closely related members of the second of the three phylogenetic groups into which coronaviruses are classified.The 3Ј end of the genome is the site of initiation of synthesis of negative-strand gRNA and sgRNA. Independent deletion analyses of MHV DI RNAs concluded that the minimal amount of the genomic 3Ј terminus that can support RNA synthesis ranges between 378 and 462 nucleotides (nt) (23,28,32). This suggested that the 3Ј genomic cis-acting element of MHV must encompass the entire 301-nt 3Ј untranslated region (3Ј UTR) as well as a portion of the adjacent nucleocapsid (N) gene. However, genetic manipulations of the intact viral genome have demonstrated that the 3Ј UTR and the 3Ј cis-acting

Recent studies have shown that CD4+ memory T cells persist in nonlymphoid organs following infections. However, the development and phenotype of these peripheral memory cells are poorly defined. In this study, multimerized MHC-Ig fusion proteins, with a covalently attached peptide sequence from the Sendai virus hemagglutinin/neuraminidase gene, have been used to identify virus-specific CD4+ T cells during Sendai virus infection and the establishment of peripheral CD4+ memory populations in the lungs. We show declining frequencies of virus-specific CD4+ T cells in the lungs over the course of ∼3 mo after infection. Like peripheral CD8+ T cells, the CD4+ have an acutely activated phenotype, suggesting that a high level of differentiation is required to reach the airways and persist as memory cells. Differences in CD25 and CD11a expression indicate that the CD4+ cells from the lung airways and parenchyma are distinct memory populations.