Temporal synthesis of proteins and RNAs during human astrovirus infection of cultured cells

Abstract

Astroviruses are nonenveloped particles with a distinctive star-shaped surface structure that have been detected by electron microscopy in stool samples from humans and animals with gastroenteritis. We examined the patterns of macromolecular synthesis in astrovirus-infected cells with a goal of establishing a molecular basis for taxonomic classification. Trypsin is required for continuous replication of astrovirus in cultured cells; however, during a single cycle of infection, astrovirus antigen was synthesized earlier and at higher levels when serum, rather than trypsin, was included in the growth medium. This enhanced production of antigen, as measured by enzyme immunoassay, was accompanied by the appearance of aggregates of virus particles in the cytoplasm of infected cells. During astrovirus replication in cells cultured in the presence of serum, we detected a single infection-specific protein (90 kDa) beginning at 12 h postinfection. This protein was recognized by antiastrovirus rabbit serum and was sensitive to trypsin digestion in vitro, with the concomitant appearance of three smaller immunoreactive proteins (31, 29, and 20 kDa). We also detected two dactinomycin-resistant RNAs (7.2 and 2.8 kb), both of which were polyadenylated, in the cytoplasm of astrovirus-infected cells. The larger of these two RNAs is presumably the viral genome, whereas the smaller species may be a subgenomic messenger. Comparison of the proteins and RNAs synthesized in astrovirus-infected cells with those of the recognized families of nonenveloped single-stranded RNA animal viruses suggests that astroviruses should not be classified as members of either Caliciviridae or Picornaviridae.

Apoptosis in astrovirus-infected CaCo-2 cells

“…However, in the apoptosis studies, no trypsin was added to the p.i. medium to ensure a single cycle of virus replication (Bass and Qiu, 2000;Méndez et al, 2002;Monroe et al, 1991). Briefly, 3day-old cell monolayers were washed twice with phosphatebuffered saline (PBS) and inoculated with viral stocks pretreated with 10 Ag/ml trypsin (GIX Sigma) for 30 min at 37 jC.…”

“…The astrovirus genome consists of a 6.8 kb single-stranded, polyadenylated positive-sense RNA, which is infectious when transfected into permissive cells (Geigenmüller et al, 1997). HAstV can be isolated and propagated in human intestinal CaCo-2 continuous cell line in the presence of trypsin, which is involved in the capsid protein maturation (Monroe et al, 1991).…”

“…Both proteins are proteolytically processed giving rise to a variety of proteins, although it is not clear whether the viral protease is responsible for all the cleavages (Geigenmüller et al, 2002a(Geigenmüller et al, , 2002bKiang and Matsui, 2002). A subgenomic RNA that contains ORF2 is detected in the cytoplasm of infected cells (Monroe et al, 1991). This subgenomic RNA is translated as a 87-kDa capsid precursor which gives raise to mature capsid proteins in a process that involves trypsin and a putative cellular protease (Bass and Qiu, 2000;Méndez et al, 2002;Monroe et al, 1991) and it has been described by different laboratories that viral particles grown in the absence of trypsin show an importantly reduced infectivity titer (Bass and Qiu, 2000;Méndez et al, 2002;Monroe et al, 1991).…”

V, 2.Ribosomal frameshifting in astroviruses

“…Human astrovirus serotype 1 (HAstV-1; Willcocks et al, 1994), like all astroviruses, contains three sequential open reading frames (ORFs) designated ORFs la, lb and 2. The most 3' coding region, ORF 2, encodes the viral structural proteins (Matsui et al, 1993;Lewis et al, 1994) and is expressed from a sub-genomic mRNA (Monroe et al, 1991;Matsui et al, 1993;Geigenm/iller et al, Section V, Chapter 1 of this book). ORFs la and lb contain amino-acid motifs indicative of non-structural proteins (reviewed in Cubitt, 1996), and a characteristic YGDD motif found in the RNA-dependent RNA polymerases of a variety of RNA viruses (Kamer and Argos, 1984) is located in ORF lb.…”

Astrovirus evolution and emergence

“…This review provides an update on the evolution of astroviruses, especially as it relates to the potential emergence of novel human astroviruses. subgenomic (sg) RNA (Monroe et al, 1991). Assuming astroviruses replicate and transcribe their RNA in a similar mechanism as alphaviruses, full-length genomic RNA is used to synthesize negative-sense full-length RNA, which in turn serves as a template for the transcription of genomic RNA and sgRNA .…”