C-Terminal nsP1a Protein of Human Astrovirus Colocalizes with the Endoplasmic Reticulum and Viral RNA

Guix, Susana; Caballero, Santiago; Bosch, Albert; Pintó, Rosa M.

doi:10.1128/jvi.78.24.13627-13636.2004

Cited by 46 publications

(50 citation statements)

References 60 publications

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“…Transcription of the negativestrand genome yields the genomic and subgenomic RNA. Human astrovirus (HAstV) proteins have been associated with membranes in infected cells likely serving as the site for replication and assembly (13)(14)(15). After assembly, the progeny virions egress from the cell, a process promoted by caspase activation (16).…”

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confidence: 99%

Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo

Marvin

Huerta

Sharp

et al. 2016

J Virol

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Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-␤]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results in vivo using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis in vivo. IMPORTANCEHuman astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both in vitro and in vivo. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis. The successful replication and spread of many enteric viruses depend upon modulating immune factors produced by intestinal epithelial cells (IECs) including interferons (IFNs) (1, 2). For instance, enteric adenoviruses are sensitive to IEC-produced type I IFNs, unlike respiratory adenoviruses (3), while rotavirus exploits type I IFN signaling in IECs to promote early viral replication (4). However, nothing is known about the impact of IFN on astrovirus infection.Astroviruses are small, nonenveloped, RNA viruses that are one of the most important causes of pediatric acute gastroenteritis worldwide (5-8). Infection begins by binding to an unidentified receptor(s) on epithelial cells after fecal-oral transmission followed by entry via endosomes (9). After viral uncoating, the positive-sense, single-stranded RNA genome is translated into a polyprotein precursor that is subsequently cleaved into proteins required for replication and the assembly of progeny virions. The genome contains three open reading frames: ORF1a, ORF1b, and ORF2. ORF1a and ORF1b encode nonstructural proteins involved in transcription and replication of the virus, while ORF2 encodes the capsid protein (10, 11). Negative-strand RNA is produced from the positive genomic strand, which can be detected 6 to 12 h postinfection (hpi) (12). Transcription of the negativestrand genome yields the genomic and subgenomic RNA. Human astrovirus (HAstV) proteins have been associated with membranes in infected cells likely serving as the site for replication and assembly (13-15). After assembly, the progeny virions egress from the cell, a process promoted by caspase activation (16).Recently, Guix et al. found that HAstV type 4 (HAstV-4) replication induces type I IFN production and that pretreatment of Caco2 cells with beta interferon (IFN-␤) reduced HAstV-4 capsid protein synthesis and progeny virion production (17). However, whether the IFN-␤ produced during astrovirus infection is sufficient to limit astrovirus replication, and at what step in the viral life cycle IFN-␤ affects astrovirus, remains...

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confidence: 99%

Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo

Marvin

Huerta

Sharp

et al. 2016

J Virol

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“…Although the exact boundaries of the proteolytical cleavages of the ORF1a are not well identified, our initial working hypothesis was that a first cleavage at position Gln-567/Thr-568 (14) would render the nsP1a/4 protein of around 40 kDa and that further unknown processes of this protein would render smaller proteins. Some of these proteins are in the range of 21 to 27 kDa and include phosphorylated forms (7). The crystal structure of the astrovirus protease has very recently been described (27), suggesting a high preference for Glu and Asp at the P1 substrate position and consequently a preference for the Glu-654/Ile-655 cleavage rather than the Gln-567/Thr-568 cleavage and even suggesting that these latter residues are located in the S1 pocket.…”

Section: Discussionmentioning

confidence: 99%

“…nsP1a/4 protein interacts with the viral polymerase. The involvement of nsP1a/4 in viral RNA replication as well as its interaction with the endoplasmic reticulum (7,8) suggests that this protein may be an integral part of the viral replication complex interacting with the RNA polymerase. The potential interaction between nsP1a/4 and RNA polymerase was studied through coimmunoprecipitation of these proteins from ex- ) was used as a negative control.…”

Section: Production Of Astrovirus Nsp1a/4 and Rdrp In Insect Cellsmentioning

confidence: 99%

“…In addition, computational analyses of the nsP1a/4 coding region performed in our laboratory revealed the presence of acidic regions and of glutamine-and proline-rich regions, a death domain, and several putative O-glycosylation and phosphorylation sites (6)(7)(8). Biological and functional analyses also showed a colocalization of the nsP1a/4 with the endoplasmic reticulum and viral RNA and revealed a role of this protein in RNA replication, as well as a relationship between the genetic diversity within the HVR of the nsP1a/4 coding region and the virus replication phenotype (7,8). Two patterns of replication that are associated with the nsP1a/4 genotype have been described: genotypes IV and V show higher levels of subgenomic RNA and lower levels of antigenomic RNA than genotypes VI and XII (8).…”

mentioning

confidence: 99%

“…Based on the HVR nucleotide genetic variability, 15 different nsP1a/4 protein HVR-derived genotypes (named using Roman numerals) have been established, and a restriction fragment length polymorphism (RFLP) typing method has been developed to consistently distinguish between genotypes (9). In addition, computational analyses of the nsP1a/4 coding region performed in our laboratory revealed the presence of acidic regions and of glutamine-and proline-rich regions, a death domain, and several putative O-glycosylation and phosphorylation sites (6)(7)(8). Biological and functional analyses also showed a colocalization of the nsP1a/4 with the endoplasmic reticulum and viral RNA and revealed a role of this protein in RNA replication, as well as a relationship between the genetic diversity within the HVR of the nsP1a/4 coding region and the virus replication phenotype (7,8).…”

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confidence: 99%

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The C-Terminal nsP1a Protein of Human Astrovirus Is a Phosphoprotein That Interacts with the Viral Polymerase

Fuentes

Guix

Bosch

et al. 2011

J Virol

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Human astrovirus nonstructural C-terminal nsP1a protein (nsP1a/4) colocalizes with the endoplasmic reticulum and viral RNA. It has been suggested that nsP1a/4 protein is involved in the RNA replication process in endoplasmic reticulum-derived intracellular membranes. A hypervariable region (HVR) is contained in the nsP1a/4 protein, and different replicative patterns can be distinguished depending on its variability. In the present work, both the astrovirus RNA-dependent RNA polymerase and four types (IV, V, VI, and XII) of nsP1a/4 proteins have been cloned and expressed in the baculovirus system to analyze their interactions. Different isoforms of each of the nsP1a/4 proteins exist: a nonphosphorylated isoform and different phosphorylated isoforms. While the polymerase accumulates as a monomer, the nsP1a/4 proteins accumulate as oligomers. The oligomerization domain of nsP1a/4-V is mapped between residues 176 and 209. For all studied genotypes, oligomers mainly contain the nonphosphorylated isoform. When RNA polymerase is coexpressed with nsP1a/4 proteins, they interact, likely forming heterodimers. The polymerase binding region has been mapped in the nsP1a/4-V protein between residues 88 and 176. Phosphorylated isoforms of nsP1a/4 type VI show a stronger interactive pattern with the polymerase than the nonphosphorylated isoform. This difference is not observed in genotypes IV and V, suggesting a role of the HVR in modulating the interaction of the nsP1a/4 protein with the polymerase through phosphorylation/dephosphorylation of some critical residues.

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Genetic analysis of the hypervariable region of the human astrovirus nsp1a coding region: Design of a new RFLP typing method

Guix

Caballero

Fuentes

et al. 2007

Journal of Medical Virology

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Human astroviruses (HAstV) are causative agents of viral gastroenteritis worldwide. A hypervariable region (HVR) is located close to the C-terminus of the nsP1a, and recent data support the involvement of the HVR-containing nonstructural protein in viral RNA replication processes, suggesting a correlation between variability in this region and pathogenic properties. The HVR of the C-terminal nsP1a coding region of 104 wild-type and reference isolates of HAstV was sequenced. A phylogenetic analysis was performed to identify different genotypes, and a restriction fragment length polymorphism (RFLP) method was designed. An extensive nucleotide and deduced amino acid sequence variability was observed, as well as many insertions and deletions that retained the reading frame. The resultant phylogenetic tree supported the subdivision of HAstV into the two previously described major genetic groups, genogroup A and B, and the identification of 12 genotypes (9 within genogroup A, and 3 within genogroup B), which could be identified by RFLP. A correlation analysis was performed between genotype information and viral load using information from 35 clinical samples. Significant differences were observed between the viral load in clinical samples and certain HAstV genotypes that belonged to the same serotype, confirming the influence of C-terminal nsP1a variability on the viral replication phenotype. The use of the new RFLP typing method based on the HVR of the C-terminal nsP1a coding region by diagnosticians would help to understand the relationship between different genotypes and the severity of the gastroenteritis.

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C-Terminal nsP1a Protein of Human Astrovirus Colocalizes with the Endoplasmic Reticulum and Viral RNA

Cited by 46 publications

References 60 publications

Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo

Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo

The C-Terminal nsP1a Protein of Human Astrovirus Is a Phosphoprotein That Interacts with the Viral Polymerase

Genetic analysis of the hypervariable region of the human astrovirus nsp1a coding region: Design of a new RFLP typing method

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