Intracellular Restriction of a Productive Noncytopathic Coronavirus Infection

Abstract

Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 10 7 to 10 8 PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition.Coronaviruses belong to the Coronaviridae family in the Nidovirales order. Coronavirus infections can follow different scenarios in vivo, as well as in vitro, suggesting complex and diverse interaction with their hosts. Viral genome expression and molecular mechanisms of virus-host interaction have mostly been studied in the context of efficient coronavirus replication in a lytic infection of cultured, transformed cells. Cytopathic infection with one of the best-studied coronaviruses, murine hepatitis virus (MHV), causes drastic changes in the cellular infrastructure (20, 36) and metabolism (14,37,44) and results in cell death. Coronavirus infection in vitro can take a different course and exhibit limited or no cytopathic effect (CPE) or virus-induced cell death. Productive noncytopathic infections have been described for primary cells or relatively differentiated cell lines infected with coronaviruses MHV (19,21,27), human coronavirus OC43 (1), human coronavirus 229E (2), and severe acute respiratory syndrome (SARS) coronavirus (5). Sturman and Takemoto reported that MHV infection of the mouse BALB/3T3 cell line produced high yields of virus but showed little CPE within 1 day postinfection (p.i.) (40). Mouse embryo fibroblast BALB/3T3 cell lines are obtained in such a way that the cells exhibit the contact inhibition that is characteristic of nontransformed cells in vivo (41). The coronavirus CPE became pronounced when BALB/3T3 cells were transformed, either spontaneously or by infection with oncogenic viruses (40). Which mechanisms or host factors prevent CPE and virus-induced cell death in nontransformed ...

Organ tropism of murine coronavirus does not correlate with the expression levels of the membrane-anchored or secreted isoforms of the carcinoembryonic antigen-related cell adhesion molecule 1 receptor

“…Cells harvested from one dish were divided into two parts, one used for RNA extraction and transcript analysis and the other for the flow-cytometrical analysis of the expression level of membrane-anchored CEACAM1. Compared to earlier results (Slobodskaya et al, 2008), the use of a second antibody conjugated to allophycocyanin instead of fluorescein-isothiocyanate markedly improved the signal to noise ratio during flow cytometry. With the improved resolution between CEACAM1-positive and autofluorescent control cells, it became obvious that in all but one of the cell lines the majority of the cells (60-100 %) were truly CEACAM1-positive ( Fig.…”

“…The L929 cell line was represented by a culture passaged less than six times after the delivery from the American type culture collection (L929) and a L929-descendant (L) with a long passage history originating from Dr P. Rottier’s laboratory. All cell lines were susceptible to productive MHV-A59 infection, which was non-cytopathogenic in C2C12 and NIH 3T3 cells and cytopathogenic in the others (Slobodskaya et al , 2008).…”

“…The extent of receptor exposure and its involvement in interactions with neighbouring cells will influence the actual number of sites available for virus binding. This factor, which is determined by the specific architecture of an organ, together with the other, yet unknown, factors modulating virus entry and the intracellular phase of virus reproduction (Bender et al , 2010; Slobodskaya et al , 2008; Taguchi & Hirai-Yuki, 2012) plus the local innate immunity profiles (Zhao et al , 2011) most probably define the tissue and organ tropism of MHV.…”