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...

Rotavirus strains differ in their need for sialic acid (SA) for initial binding to the cell surface; however, the existence of a postattachment cell receptor, common to most, if not all, rotavirus strains, has been proposed. In the present study, antibodies to the ␣v and ␤3 integrin subunits, and the ␣v␤3 ligand, vitronectin, efficiently blocked the infectivity of the SA-dependent rhesus rotavirus RRV, its SA-independent variant nar3, and the neuraminidase-resistant human rotavirus strain Wa. Vitronectin and anti-␤ 3 antibodies, however, did not block the binding of virus to cells, indicating that rotaviruses interact with ␣v␤3 at a postbinding step, probably penetration. This interaction was shown to be independent of the tripeptide motif arginine-glycine-aspartic acid present in the natural ligands of this integrin. Transfection of CHO cells with ␣v␤3 genes significantly increased their permissiveness to all three rotavirus strains, and the increment of virus infectivity was reverted by incubation of these cells either with antibodies to ␤3 or with vitronectin. These findings implicate ␣v␤3 integrin as a cellular receptor common to neuraminidase-sensitive and neuraminidase-resistant rotaviruses, and support the hypothesis that this integrin could determine, at least in part, the cellular susceptibility to rotaviruses.

In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.

Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.Human astroviruses (HAstV) have been found to be a frequent cause of gastroenteritis among young children worldwide (5,7,8,15). The virions are formed by a nonenveloped protein capsid and a positive-stranded RNA genome of approximately 7 kb (9, 22). The RNA genome has three open reading frames (ORFs) (ORF1a, -1b, and -2), each encoding at least one polyprotein. The ORF1a contains viral serine protease and nuclear localization signal motifs, whereas the ORF1b has an RNA-dependent RNA polymerase motif (9,22). The products of ORF1a and ORF1b are synthesized from the genomic RNA as two polyproteins, with the latter being produced as a polyprotein 1a-1b (approximately 160 kDa) through a frameshift translational mechanism (12)(13)(14). It is believed that the products of these ORFs, processed to smaller polypeptides by the viral protease, are involved in the viral RNA replication. ORF2, of approximately 780 amino acid residues, depending on the strain (21), codes for the structural virus polypeptides (18). The structural polyprotein is translated from a polyadenylated subgenomic RNA produced at high levels during infection, which is 3Ј-colinear with the genomic RNA (17). Based on the homology among strains belonging to different serotypes, at least two domains in the product of this ORF have been predicted (16,21). The first domain includes amino acid residues 1 to 415, and it is highly conserved among all the human serotypes and some viruses from animal origin; the second domain (amino acid 416 to the end) is highly divergent among human serotypes (10,16,21,23). Neutralizing epitopes have been mapped to the second domain (3, 20); therefore, it is likely that this hypervariable region is exposed on the viral particle. It is known that astrovirus in...

Astroviruses are single-stranded, plus-sense RNA viruses that infect both mammals and birds, causing gastroenteritis and other extraintestinal diseases. Clinical studies have established astroviruses as the second leading cause of viral diarrhea in young children. Here we report the crystal structure of the human astrovirus dimeric surface spike determined to 1.8-Å resolution. The overall structure of each spike/projection domain has a unique three-layered β-sandwiches fold, with a core, six-stranded β-barrel structure that is also found in the hepatitis E virus capsid protrusions, suggesting a closer phylogenetic relationship between these two viruses than previously acknowledged. Based on a hepatitis E virus capsid model, we performed homology modeling and produced a complete, T ¼ 3 astrovirus capsid model with features remarkably similar to those observed in a cryoelectron microscopy reconstruction image of a human astrovirus. Mapping conserved residues onto the astrovirus projection domain revealed a putative receptor binding site with amino acid compositions characteristic for polysaccharide recognition. Our results will have an important impact on future characterization of astrovirus structure and function, and will likely have practical applications in the development of vaccines and antivirals.structural protein | naked virus | attachment domain A stroviruses are small, nonenveloped, icosahedral viruses with a positive-sense, single-stranded RNA genome of approximately 7 kb in size. With members infecting mammals and avian species, the family of Astroviridae consists of two genera, Mammastrovirus and Avastrovirus (1, 2). Human astroviruses, eight serotypes in total, are one of the leading causes of gastroenteritis in young children, elderly people, and immunocompromised adults. Sporadic astrovirus infections as well as large-scale outbreaks in susceptible populations have been described (2). In addition to humans, astroviruses can infect a wide range of wild and domestic animals, causing gastroenteritis in most mammals and both intestinal and extraintestinal diseases in birds (1).The genomic RNA of astroviruses is polyadenylated with three ORFs. ORF1a and the downstream overlapping ORF1b encode two nonstructural polyproteins, nsp1a and nsp1ab. The nsp1ab protein is produced from both ORF1a and ORF1b through a translational frame-shift mechanism (3). Polyproteins nsp1a and nsp1ab are processed by a viral 3C-like serine protease and other cellular proteases into several proteins (e.g., the RNAdependent RNA polymerase and the serine protease) that are likely to function in the replication of the viral genome (4). ORF2 at the 3′ end of the viral genome encodes the viral capsid protein (CP) (5) and is found in both genomic and subgenomic RNAs. It is unclear whether the astrovirus RNA contains a 5′ cap. Viral capping enzymes such as guanylyltranferase and methyltransferases have not been identified in the astrovirus genome.When overexpressed in eukaryotic hosts, the 796-aa CP of the serotype 2 astrovirus was ...

In this work we have shown that astrovirus infection induces apoptosis of Caco-2 cells, since fragmentation of cellular DNA, cleavage of cellular proteins which are substrate of activated caspases, and a change in the mitochondrial transmembrane potential occur upon virus infection. The human astrovirus Yuc8 polyprotein capsid precursor VP90 is initially processed to yield VP70, and we have shown that this processing is trypsin independent and occurs intracellularly through four cleavages at its carboxy-terminal region. We further showed that VP90-VP70 processing is mediated by caspases, since it was blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk), and it was promoted by the apoptosis inducer TNF-related apoptosis-inducing ligand (TRAIL). Although the cell-associated virus produced in the presence of these compounds was not affected, the release of infectious virus to the cell supernatant was drastically reduced in the presence of z-VAD-fmk and increased by TRAIL, indicating that VP90-VP70 cleavage is important for the virus particles to be released from the cell. This is the first report that describes the induction and utilization of caspase activity by a virus to promote processing of the capsid precursor and dissemination of the viral particles.

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Objective. Limited educational and job opportunities for youth has led to a phenomenon termed NEET (not in education, employment or training). The objective is to estimate the prevalence of psychiatric disorders, substance use and suicidal behavior in youth classified as NEET and to compare with those who study only, work only or do both. Material and Methods. 3 005 12-to-17 year-olds in Mexico City were evaluated in 2005 with the Composite International Diagnostic Interview. Descriptive and logistic regression analyses considered the multistage weighted sample design. Results. NEET youth as well as those who work only or study and work simultaneously have greater odds of psychiatric disorder, substance use and suicidal behavior compared to those who study exclusively even after controlling for social disadvantage. Conclusion. Vulnerability is not circumscribed to NEET adolescents, but to all teens who are not exclusive students. Supporting youth to continue studying exclusively may buffer negative mental health outcomes.