Persistent infection of cultured cells with mouse hepatitis virus (MHV) results from the epigenetic expression of the MHV receptor

Abstract

The A59 strain of murine coronavirus mouse hepatitis virus (MHV) can cause persistent infection of 17C1-1 cells and other murine cell lines. Persistently infected cultures released large amounts of virus (10 7 to 10 8 PFU/ml) and were resistant to superinfection with MHV but not to infection with unrelated Semliki Forest and vesicular stomatitis viruses. The culture medium from persistently infected cultures did not contain a soluble inhibitor such as interferon that protected uninfected cells from infection by MHV or vesicular stomatitis virus. The persistent infection was cured if fewer than 100 cells were transferred during subculturing, and such cured cultures were susceptible to reinfection and the reestablishment of persistent infection. Cultures of 17C1-1 cells that had been newly cloned from single cells consisted of a mixture of MHV-resistant and -susceptible cells. 17C1-1/#97 cells, which were cured by subcloning after 97 passages of a persistently infected culture over a 1-year period, contained 5 to 10% of their population as susceptible cells, while 17C1-1/#402 cells, which were cured by subcloning after 402 passages over a 3-year period, had less than 1% susceptible cells. Susceptibility to infection correlated with the expression of MHV receptor glycoprotein (MHVR [Bgp1 a ]). Fluorescenceactivated cell sorter analysis with antibody to MHVR showed that 17C1-1/#97 cells contained a small fraction of MHVR-expressing cells. These MHVR-expressing cells were selectively eliminated within 24 h after challenge with MHV-A59, and pretreatment of 17C1-1/#97 cells with monoclonal antibody CC1, which binds to the N-terminal domain of MHVR, blocked infection. We conclude that the subpopulation of MHVR-expressing cells were infected and killed in acutely or persistently infected cultures, while the subpopulation of MHVRnonexpressing cells survived and proliferated. The subpopulation of MHVR-negative cells produced a small proportion of progeny cells that expressed MHVR and became infected, thereby maintaining the persistent infection as a steady-state carrier culture. Thus, in 17C1-1 cell cultures, the unstable or epigenetic expression of MHVR permitted the establishment of a persistent, chronic infection. MATERIALS AND METHODSCells and viruses. The spontaneously transformed mouse BALB/c 3T3 cells, 17C1-1 cells (28), and the A59 strain of MHV were kindly provided by L. Sturman. The 17C1-1 cells used in this study were cloned from a single cell and grown at 37ЊC and 7.5% CO 2 in Dulbecco's modified Eagle medium containing 6% fetal bovine serum (FBS), 5% tryptose phosphate broth (TPB), 500 U of penicillin per ml, and 100 U of streptomycin per ml (neutral pH growth medium). The cultures were routinely passaged every 3 days at a dilution of 1:60. The cells were detached from the monolayer by incubation in Hanks' balanced salt * Corresponding author.

Establishment of Vero E6 cell clones persistently infected with severe acute respiratory syndrome coronavirus

“…Interestingly, the cultured cells persistently infected with MHV were shown to be resistant to superinfection with MHV but not to infection with other unrelated viruses [51]. When the culturing of cells persistently infected with MHV was carried out for >400 passages, the yield of released virus gradually decreased and only~10% of the cells were viral antigen-positive at passage 97 [51], which was similar to the pattern we observed for SARS-CoV in this study. In the case of MHV persistence, if fewer than 100 cells were subcultured, then the persistent infection ended and the Fig.…”

“…resulting cultures no longer produced virus and became susceptible to MHV infection [51]. In contrast, we were able to obtain several virus-positive cell clones by culturing a single Vero/SARS-CoV cell in the wells of 96-well microplates.…”

“…Similar observations have been reported for other CoVs: a number of MHV variants with deletions in the S glycoprotein have been isolated from persistently MHV-infected animals; however, the significance of generating and potentially accumulating deletion variants in the persistent viral population remains unclear, although it has been proposed that the complexity of the population of persisting viral RNAs may contribute to chronic disease [49,50]. Interestingly, the cultured cells persistently infected with MHV were shown to be resistant to superinfection with MHV but not to infection with other unrelated viruses [51]. When the culturing of cells persistently infected with MHV was carried out for >400 passages, the yield of released virus gradually decreased and only~10% of the cells were viral antigen-positive at passage 97 [51], which was similar to the pattern we observed for SARS-CoV in this study.…”

Contributions of the S2 spike ectodomain to attachment and host range of infectious bronchitis virus

“…Many viruses that have been serially passaged, or isolated from persistently infected cells, have acquired the ability to infect previously non-susceptible cells. For coronaviruses, the extended host range of such viruses has not only been described for IBV (Otsuki et al, 1979;Tay et al, 2012;), but also for MHV (Baric et al, 1997(Baric et al, , 1999McRoy and Baric, 2008;Sawicki et al, 1995;Schickli et al, 1997Schickli et al, , 1998. Cells persistently infected with MHV and passaged multiple times generated a virus that gained the ability to enter cells independent of its natural protein receptor CEACAM, but rather use heparan sulfate, explaining their extended cell tropism (de Haan et al, 2005).…”

Down-regulation of transcription of the proapoptotic gene BNip3 in cultured astrocytes by murine coronavirus infection

“…In the past, most studies have been focused on how MHV has evolved during persistent infection in the CNS and in cultured cells (Adami et al, 1995;Rowe et al, 1998). Information on coevolvement of the host has been limited to the alteration of receptors by MHV infection (Chen and Baric, 1996;Sawicki et al, 1995).…”