The capsid five-fold cylinder and the VP1 N-terminal unique region are critical components of the parvoviral 'entry machine'

The atomic structure of the MVM virion reveals cylindrical projections at each five-fold symmetry axis. Ten amino acids from a single VP2 N-terminus have been modeled running through the cylinder indicating that they are the extrusion portals for the VP2 N-termini. L172 projects into the center of this pore, at its inner end, from five independent VP2 molecules, forming its tightest constriction. Suggesting that L172 modulates the extrusion of the VP N-termini, we targeted this residue for mutagenesis. A complete 20 amino acid substitution library at this position has been constructed, within which; four major phenotypic classes have been identified. Two mutants are able to infect cells indistinguishably from WT. A group of five mutants produce levels of capsids equivalent to WT at 32°C but are significantly defective for assembly at 39°C. One mutant, L172W, was found to be unable to package DNA at 32°C. Finally, a series of mutants were found that produce virions effectively yet are unable to establish infection. In depth analysis of one such mutant, L172T, demonstrated that both cell binding and entry occurred similarly to wildtype. In vivo and in vitro evidence suggests that L172T is inactivated early during trafficking through the degradation of the VP1 N-terminus.; The VP1 protein itself is dispensable for both assembly and DNA packaging but it is required for the production of an infectious virion. The VP1 specific region contains both nuclear localization signals and an active phospholipase A2 (PLA2) enzymatic core. The cis/trans relationship of parvoviral PLA2 was studied in detail as well as the ability of endosome disrupting reagents to rescue PLA2 mutant virions. Our results indicate that PLA2 activity can indeed function in trans. Endosomolytic reagents were also effective in rescuing mutant infectivity with adenovirus co-infection being the most effective. The rescue of PLA2 mutants with endosomolytic reagents indicates that these virions are not defective for the establishment of infection after release from endosomes and suggests that the escape from endosomes is the primary role of the parvoviral PLA2 enzyme.