Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses

Abstract

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10 5 PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.

Loss of IKK subunits limits NF-κB signaling in reovirus infected cells

“…Polyclonal antisera raised against T3D, T1L that have been described (42) were used to detect viral proteins in T3A infected cells. Rabbit antisera specific for IKKβ and p65 Ser536 phosphorylation specific antibody were purchased from Cell Signaling (catalog # 8943, 3033), rabbit antisera specific for p65 and NEMO were purchased from Santa Cruz Biotechnology (catalog # sc-372, sc-8330).…”

Lipids Cooperate with the Reovirus Membrane Penetration Peptide to Facilitate Particle Uncoating

“…6B). To ensure that intact T3D ISVPs did not contaminate the transferred ISVP* supernatant, aliquots of the no spin and spin reactions were analyzed for residual infectivity by plaque assay and for the presence of the 1 ␦ fragment by Western blotting using a rabbit, ␣-reovirus polyclonal antibody (63).…”

“…Equal volumes from each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of the 1 ␦ fragment by Western blotting using a rabbit, ␣-reovirus polyclonal antibody (63).…”

Reovirus Activates a Caspase-Independent Cell Death Pathway

“…Antibodies and reagents. Antisera raised against T3D, T1L, and NS have been described (82,83). Rabbit antisera specific for cleaved caspase-3 and HMGB1 were purchased from Cell Signaling, rabbit antisera specific for RelA and IB␣ were purchased from Santa Cruz Biotechnology, and rabbit antiserum against poly(ADP-ribose) polymerase (PARP) was purchased from Roche.…”