Studies of the capsid determinants of canine parvovirus host range and mechanisms of virus uptake and infectious entry into cells in vitro

Viruses can infect a range of different hosts and cells in vivo and in vitro. This property is determined by both viral and host factors. For canine parvovirus (CPV) host range is primarily determined by changes in the capsid protein of the virus. One objective of these studies was to understand how structural changes in a defined region of the capsid resulted in loss of the ability to replicate in canine cells. A second objective was to characterize the early events during CPV infectious cellular entry.; Analysis of mutants containing defined mutations in the shoulder region of the CPV capsid revealed that a specific conformation of this region determined the ability to replicate in canine cells. The formation of a new salt bridge caused both the loss of the canine host range and increased capsid stability. The capsid stability of 2 mutants and feline panleukopenia virus (FPV) was greater than CPV against urea denaturation. There was no difference between the binding of CPV and two host range mutants to the plasma membrane of canine cells and wild type and mutant infectious plasmids replicated similarly when transfected into canine cells.; CPV most likely enters and infects cells by clathrin-mediated endocytosis. I found that CPV particles bound to electron-dense coated pits, and overexpression of a dominant negative mutant of dynamin (dynamin K44A) in cells, delayed the uptake of virions, inhibited infection by 40%, caused a 40% decrease in the number of cells in S-phase of the cell cycle and lead to increased binding of CPV to the cell surface. Viral particles and transferrin were found to co-localize in perinuclear vesicles, probably within the perinuclear recycling endosome. Transport of virions to perinuclear vesicles and cell infection was inhibited by 20 nM bafilomycin added before or 90 minutes after virus inoculation. High concentrations of CPV particles failed to permeabilize membranes and allow co-entry of the translation-inhibiting toxin a -sarcin. In contrast, canine adenovirus particles permeabilized cellular membranes and allowed toxin entry.