Apoptosis in astrovirus-infected CaCo-2 cells

Abstract

Cell death processes during human astrovirus replication in CaCo-2 cells and their underlying mechanisms were investigated. Morphological and biochemical alterations typical of apoptosis were analyzed in infected cells using a combination of techniques, including DAPI staining, the sub-G(0)/G(1) technique and the TUNEL assay. The onset of apoptosis was directly proportional to the virus multiplicity of infection. Transient expression experiments showed a direct link between astrovirus ORF1a encoded proteins and apoptosis induction. A computer analysis of the astrovirus genome revealed the presence of a death domain in the nonstructural protein p38 of unknown function, encoded in ORF1a. Apoptosis inhibition experiments suggested the involvement of caspase 8 in the apoptotic response, and led to a reduction in the infectivity of the virus progeny released to the supernatant. We conclude that apoptotic death of host cells seems necessary for efficient human astrovirus replication and particle maturation.

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

“…For transmission electron microscopy studies, approximately 10 7 infected (multiplicity of infection of 5) and mock-infected CaCo-2 cells were processed at 48 h postinfection as previously described (15). For immunoelectron microscopy, samples were fixed with 4% (vol/vol) paraformaldehyde and 0.2% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2.…”

“…Recently, computer analysis of nsP1a revealed the presence of two coiled-coil regions common to all known human astrovirus (22), and a putative death domain (15). In addition, a similarity between the putative astrovirus nuclear localization signal and calicivirus genome-linked proteins has also been described (22).…”

New sustained release of Zidovudine Matrix tablets − cytotoxicity toward Caco-2 cells

“…Since the newly established protocol ruling toxicological tests 10,11 severely limits the use of animals in lethal experiments, there has been an increased interest in in vitro techniques, for obtaining reliable biological data 12,13 . The human cell line Caco-2, in particular, has shown to be a suitable and reliable model for this kind of studies 14 , as it is derived from human colon adenocarcinoma cells and exhibits several classes of different markers typically found in adult's intestinal differentiated cells (e.g., alkaline phosphatase and microvilli in the brush border) [15][16][17][18][19] . In fact, the use of Caco-2 cells in cytotoxicity evaluation experiments presents several advantages: (i) there are no significant differences in either the morphological or physiological characteristics of intestinal cancer cells compared to normal cells 15,20 ; (ii) it allows to gather information on the potential drug's toxicity toward the mucosa [21][22][23] ; (iii) it provides information, at the cellular level, on drug's absorption, metabolism, and transport through the intestinal wall [24][25][26][27][28][29] .…”

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

“…Sequence analysis has predicted four transmembrane domains and a helicase conserved motif close to the nsP1a N-terminal end (1,11). In the nsP1a C-terminal end, named here the nsP1a/4 protein, several domains have been described: two coiled-coil regions, one coinciding with a death domain (DD), a nuclear localization signal (NLS), a putative viral genome-linked protein (VPg), and a hypervariable region (HVR) (1,6,11,13,22,25,29). 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).…”

“…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).…”