SUMMARY Integrated Stress Response is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism resulting in partial but not complete translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits “foamy cell” development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

Background: Protein synthesis control is important for ␤-cell fate during ER stress. Results: Increased protein synthesis during chronic ER stress in ␤-cells involves the transcriptional induction of an amino acid transporter network. Conclusion: Increased amino acid uptake in ␤-cells during ER stress promotes apoptosis. Significance: Induced expression of a network of amino acid transporters in islets can contribute to chronic ER stress-induced diabetes.

cis-acting structures in positive-strand [(ϩ)-strand] RNA virus genomes that function as signals for genome translation, transcription, and replication are potential sites for antiviral drug design. The fact that replication signals from evolutionarily divergent coronaviruses can be exchanged, for example, from the 3Ј untranslated region (UTR) of the (group 2a) bovine coronavirus (BCoV) to the (group 2a) mouse hepatitis coronavirus (MHV) (21)(22) or from the (group 2b) severe acute respiratory syndrome (SARS) coronavirus (SCoV) to MHV (15), would suggest that designed therapeutic agents against common signals might be broadly effective. Several studies have identified cis-replication structures in the 3Ј UTR of MHV, heretofore recognized as the group 2 type species, and in the 3Ј UTR of BCoV that are likely involved in the initial steps of genome translation and initiation of negativestrand [(Ϫ)-strand] synthesis. These elements, identified in either a helper virus-dependent defective interfering (DI) RNA of MHV or BCoV or by reverse genetics in the fulllength MHV genome, are the following: (i) the 3Ј poly(A) tail (26, 34), (ii) the 3Ј-terminal 55 nucleotides (nt) of the 3Ј UTR (14,23,26,42), and (iii) an upstream bulged stem-loop (SL) and associated hairpin pseudoknot (13,(21)(22)36). Curiously, an ϳ140-nt hypervariable region in the 3Ј UTR that comprises part of a 3Ј-proximal bulged stem-loop and harbors a coronavirus-universal octamer sequence (GGAAGAGC) is not required for MHV replication in cell culture but plays a role in MHV pathogenesis (14).cis-replication structures have also been identified in the 5Ј-proximal region of group 2 coronaviruses, but few studies have characterized their common signaling features. In BCoV, the very 5Ј end of the genome exhibits a puzzling hypervariability during virus replication in that 5Ј extensions of up to 9 nt and varying sequences within the first 14 nt are found, but the full significance of this in virus replication is unknown (19). The higher-order RNA structures within the BCoV 5Ј UTR were initially predicted by the Tinoco algorithm (35) and more recently by the mfold algorithm of Zuker (31, 41). They have been characterized as SLs I to IV (Fig. 1B) and were shown to be consistent with enzyme structure probing analyses (5-6, 32-33). More recent characterizations based on comparisons with MHV have shown SLI to be comprised of two smaller stem-loops, named SL1 and SL2 (Fig. 1B) (24)(25)(27)(28). In the context of the DI RNA in BCoV-infected cells, it was not feasible to determine whether SLI or SLII is a bona fide cisreplication element, since SLI resides within the 65-nt leader and SLII resides largely within the leader, and transfected DI RNAs containing an experimentally mutated leader rapidly acquired the leader sequence of the helper virus genome in a process known as "leader switching" (5-6, 29). SLII, furthermore, harbors the leader-associated transcription regulatory core sequence (UCUAAAC) responsible for the RNA-depen-* Corresponding author. Mailing addres...

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-celllike phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

Higher-order RNA structures in the 5= untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ϳ36% nucleotide sequence divergence. In a previous study, each of three 5=-end-proximal cis-acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stemloops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ϳ200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5= UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5= UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5= UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture. In positive-strand RNA viruses that replicate in the cytoplasm, genomic 5=-end-proximal RNA structures carry out several functions required for virus reproduction. In coronaviruses, these are thought to include (i) translation initiation, commonly presumed to occur by a canonical cap-dependent 5=-terminal ribosomal entry mechanism, to synthesize the replicase enzymes from open reading frame 1 (13, 14, 31); (ii) signaling an RNA-dependent RNA polymerase template switch during minus-strand synthesis at a heptameric transcription-regulatory sequence (UCUAAAC in the case of mouse hepatitis coronavirus [MHV] and bovine coronavirus [BCoV]) (Fig. 1A and B) for placement of a common leader on subgenomic mRNAs (sgmRNAs) (43,48,49,55); (iii) encoding signals on the 3= end of minus-strand genomic RNA (the antigenome) for initiating synthesis of plus-strand genomic mRNAs and sgmRNAs (9, 43, 48, 55); (iv) possibly harboring signals that act in trans to initiate synthesis of nascent plus-strand genomes (46); (v) possibly directly influencing initiation of minus-strand synthesis at the 3= end of the genome (33); and (vi) harboring a genome packaging signal (10,18). A mechanistic understanding of these events may aid in the devel...

An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5= untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5=-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5=-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-crosslinking analyses that cellular proteins of ϳ60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5 untranslated region (UTR)-and one 3 UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5 UTR with ϳ2.5 M affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.Coronaviruses (CoVs) (59) cause primarily respiratory and gastroenteric diseases in birds and mammals (35,71). In humans, they most commonly cause mild upper respiratory disease, but the recently discovered human CoVs (HCoVs), HCoV-NL63 (65), HCoV-HKU1 (73), and severe acute respiratory syndrome (SARS)-CoV (40) cause serious diseases in the upper and lower respiratory tracts. The SARS-CoV causes pneumonia with an accompanying high (ϳ10%) mortality rate (69). The ϳ30-kb positive-strand CoV genome, the largest known among RNA viruses, is 5Ј capped and 3Ј polyadenylated and replicates in the cytoplasm (41). As with other characterized cytoplasmically replicating positive-strand RNA viruses (3), translation of the CoV genome is an early step in replication, and terminally located cis-acting RNA signals regulate translation and direct genome replication (41). How these happen mechanistically in CoVs is only beginning to be understood.In the highly studied group 2 mouse hepatitis coronavirus model (MHV A59 strain) and its close relative the bovine CoV (BCoV Mebus strain), five higher-order cis-replication signals have been identified in the 5Ј and 3Ј untranslated regions (UTRs). These include two in the 5Ј UTR required for BCoV defective interfering (DI) RNA replication (Fig. 1A) described as stem-loop III (SLIII) (50) and SLIV (51). Recently, the SLI regi...

The inability of cells to adapt to increased environmental tonicity can lead to inflammatory gene expression and pathogenesis. The Rel family of transcription factors TonEBP and NF-κB p65 play critical roles in the switch from osmoadaptive homeostasis to inflammation, respectively. Here we identified PACT-mediated PKR kinase activation as a marker of the termination of adaptation and initiation of inflammation in Mus musculus embryonic fibroblasts. We found that high stress-induced PACT-PKR activation inhibits the interaction between NF-κB c-Rel and TonEBP essential for the increased expression of TonEBP-dependent osmoprotective genes. This resulted in enhanced formation of TonEBP/NF-κB p65 complexes and enhanced proinflammatory gene expression. These data demonstrate a novel role of c-Rel in the adaptive response to hyperosmotic stress, which is inhibited via a PACT/PKR-dependent dimer redistribution of the Rel family transcription factors. Our results suggest that inhibiting PACT-PKR signaling may prove a novel target for alleviating stress-induced inflammatory diseases.