title: During Infection, Theiler's Virions Are Cleaved by Caspases and Disassembled into Pentamers.
abstract: Infected macrophages in spinal cords of mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV) undergo apoptosis, resulting in restricted virus yields, as do infected macrophages in culture. Apoptosis of murine macrophages in culture occurs via the intrinsic pathway later in infection (>10 h postinfection [p.i.]) after maximal virus titers (150 to 200 PFU/cell) have been reached, with loss of most infectious virus (<5 PFU/cell) by 20 to 24 h p.i. Here, we show that BeAn virus RNA replication, translation, polyprotein processing into final protein products, and assembly of protomers and pentamers in infected M1-D macrophages did not differ from those processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the initial difference from BHK-21 cell infection was seen at 10 to 12 h p.i., where virions from the 160S peak in sucrose gradients had incompletely processed VP0 (compared to that in infected BHK-21 cells). Thereafter, there was a gradual loss of the 160S virion peak in sucrose gradients, with replacement by a 216S peak that was observed to contain pentamers among lipid debris in negatively stained grids by electron microscopy. After infection or incubation of purified virions with activated caspase-3 in vitro, 13- and 17-kDa capsid peptide fragments were observed and were predicted by algorithms to contain cleavage sites within proteins by cysteine-dependent aspartate-directed proteases. These findings suggest that caspase cleavage of sites in exposed capsid loops of assembled virions occurs contemporaneously with the onset and progression of apoptosis later in the infection.
  
  
  OBJECTIVE
  Theiler's murine encephalomyelitis virus (TMEV) infection in mice results in establishment of virus persistence in the central nervous system and chronic inflammatory demyelinating disease, providing an experimental animal model for multiple sclerosis. Virus persistence takes place primarily in macrophages recruited into the spinal cord that undergo apoptosis and in turn may facilitate viral spread via infected apoptotic blebs. Infection of murine macrophages in culture results in restricted virus yields late in infection. Here it is shown that the early steps of the virus life cycle in infected macrophages in vitro do not differ from these processes in TMEV-infected BHK-21 cells, which undergo necroptosis. However, the findings late in infection suggest that caspases cleave sites in exposed capsid loops and possibly internal sites of assembled virions occurring contemporaneously with onset and progression of apoptosis. Mechanistically, this would explain the dramatic loss in virus yields during TMEV-induced apoptosis and attenuate the virus, enabling persistence.
authors.affiliation: Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois, USA The Graduate School, University of Illinois at Chicago, Chicago, Illinois, USA.
authors.forename: Sevim Yildiz
authors.initials: SY
authors.lastname: Arslan
authors.affiliation: Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois, USA.
authors.forename: Kyung-No
authors.initials: KN
authors.lastname: Son
authors.affiliation: Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois, USA hlipton@uic.edu.
authors.forename: Howard L
authors.initials: HL
authors.lastname: Lipton
mesh_terms: D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D008264:Macrophages / Q000821:virology*; D051379:Mice; D059748:Proteolysis; D017945:Theilovirus / Q000502:physiology*; D019562:Viral Load; D014771:Virion / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D020169:Caspases