Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatmentresistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.A ll positive-strand RNA viruses, including picornaviruses, such as poliovirus, rhinovirus, and hepatitis A virus, and flaviviruses, such as dengue virus and hepatitis C virus (HCV), rely heavily on cellular membranes at numerous stages of their infectious cycles. For example, RNA replication complexes must assemble on the topologically cytoplasmic surfaces of intracellular membranes. In some cases, such as poliovirus and hepatitis A virus, these RNA replication complexes are on the convex outer surfaces of discrete vesicles (1). In others, such as dengue virus, RNA replication complexes are assembled on invaginated membrane surfaces that are connected to the cytosol only via narrow openings (2, 3). For dengue virus, newly synthesized viral RNA exits the invaginated cytoplasm and interacts with core protein, which encapsidates the viral RNA and decorates the surfaces of nearby lipid droplets via the high-affinity binding of its N-terminal domain (4, 5). For HCV, a similar interaction of the core protein with lipid droplets has been described and seems to play a critical role in the assembly of viral particles (6-9). During dengue virus infection, formation of the nucleocapsid, subsequent interaction with envelope proteins, and budding into the ER lumen are likely to occur in close proximity (2). In the cis-Golgi, the virion undergoes a conformational change, and the viral prM (prematrix) protein is cleaved by the cellular furin protease into the mature M (matrix) protein and a peptide (pr) (10, 11). Upon cleavage, the pr peptide dissociates from the virion, resulting in the formation of mature progeny viruses that are highly infectious. This finely tuned interplay between cellular membrane remodeling, cellular lipid storage, and viral assembly is not only a fascinating cell biological puzzle, but also provides exciting...

RNA viruses have 5 and 3 untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5 end and has a 3 UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus ( Coronaviruses are members of the newly described order Nidovirales, a group of positive-sense, single-stranded RNA viruses that synthesize a nested set of subgenomic mRNAs during infection (reviewed in reference 9). Coronaviruses possess the largest known RNA virus genome, which is 26 to 30 kb long and contains 9 or 10 open reading frames (ORFs) as well as short untranslated regions (UTRs) at the 5Ј and 3Ј ends (reviewed in reference 27). The genome is capped at the 5Ј end and polyadenylated at the 3Ј end. Virus replication occurs entirely in the cytoplasm, with RNA synthesis being carried out by viral ribonucleoprotein (RNP) complexes that presumably also include cellular proteins.Following coronavirus entry into cells, the plus-strand RNA genome serves as the initial template for both translation of the viral replicase proteins, including the RNA-dependent RNA polymerase, and synthesis of full-length minus-strand RNA. A nested set of subgenomic mRNAs that contain 5Ј and 3Ј ends identical to those on the genomic RNA are also synthesized during infection (reviewed in reference 27). A minusstrand RNA complement to each subgenomic RNA is present in infected cells (17,18,48). The mechanism for the synthesis of the subgenomic RNAs is not fully understood, but several models have been proposed to explain mRNA transcription (2,24,47,58).Previous reports have implicated a role for the coronavirus UTRs in genome replication. Mouse hepatitis coronavirus (MHV) defective genomes lacking the 3Ј-terminal 55 nucleotides (nt) of the 3Ј UTR and the poly(A) tail were unable to serve as templates for minus-strand synthesis (33). In addition, UTRs from both the 5Ј and 3Ј ends of the genome were necessary for defective genome plus-strand synthesis (23, 32). These UTRs must therefore serve as cis-acting signals for defective genome replication and, in this capacity, recruit viral factors and possibly cellular proteins for formation of the replication complex.Several studies have investigated whether host proteins specifically bind to the UTRs of MHV (10,64). Proteins with molecular masses of 142, 120, 100, 103, 81, 70,55, and 33 kDa bound the MHV 3Ј UTR (64). To date, none of these proteins have been identified. However, two cellular proteins that bind to the 5Ј end of the MHV genome have been identified. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a cellular protein involved in alternative splicing of cellular mRNAs, binds the MHV minus-strand complement of the leader sequence (30). Polypyrimidine tract binding protein (PTB), also known as heterogeneous nuclear RNP I (hnRNP I), intera...

Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase-polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.

The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N-viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3' end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949-4524), as well as a region at the 3' end of the MHV N ORF (nts 4837-5197) within MIDI-C. Binding to the full-length MIDI-C transcript (approximately 5500 nts) and to an approximately 1-kb transcript from the gene 1a region (nts 935-1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3 end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65-to 70-kDa host protein, in lysates of virus-infected human cells.Many positive-sense single-stranded RNA viral genomes are relatively small templates that encode information for complex viral replication cycles and thus require highly efficient utilization of limited coding capacity. The number of functional activities expressed from any genome can be expanded by utilizing both precursor polypeptides and their processed cleavage products. For example, poliovirus protein 3D is an RNA-dependent RNA polymerase, while its presumed precursor, 3CD, which is a fusion between the 3C protease and 3D polymerase, manifests no polymerase activity but functions as a specific protease with substrate recognition properties different from its cleavage product, 3C protease (29, 68). Another mechanism that expands coding capacity is the utilization of the same polypeptide for multiple functions. For example, in addition to proteolytic activity, 3CD also functions as a specific RNAbinding protein with crucial roles in viral RNA replication. It binds the 5Ј-terminal RNA cloverleaf structure (2, 3, 23, 49) as well as an internal stem-loop structure in the 2C coding region (66, 67); both interactions are required for the initiation of RNA replication. Finally, posttranslational modifications can further modify the function of viral proteins; for example, the covalent myristoylation of viral capsid protein VP0 facilitates its transition from a precursor protein to a component of an assembled capsid (4, 42).The poliovirus genome contains a single open reading frame that c...