Encapsidation of minute virus of mice DNA: aspects of the translocation mechanism revealed by the structure of partially packaged genomes

Abstract

Minute virus of mice (MVM) packages a single, negative-sense copy of its linear single-stranded DNA genome, but a chimeric virus, MML, in which >95% MVM sequence was fused to the right-hand terminus of LuIII, packages >40% positive-sense DNA. While encapsidation of both MML strands begins efficiently, genome translocation frequently stalls at specific sites in positive-sense DNA. Internalized sequences, derived from the 3′ end of the strand, ranged from 1 to 5 kb in length, with species of around 2 kb predominating. When nuclease activity during isolation was minimized, these truncated species were found to be part of pre-excised 5 kb single-strands. Similarly, some partially encapsidated negative-sense DNAs were observed, forming a continuum of protected 3′ sequences between 1 and 3 kb in length, but these were less abundant and more uniformly distributed than their positive-sense counterparts, indicating that the negative strand has evolved for efficient internalization. The paucity of protected DNAs shorter than 1–2 kb suggests that translocation is biphasic, proceeding efficiently through the first (3′) third of the genome, but prone to stall thereafter. Sequences with conspicuous secondary structure, including stem–loop and guanidine rich regions, were found to interrupt packaging, especially when positioned near the 5′ end of the strand. Since VP2 amino-terminal peptides were exposed at the particle surface in all packaging intermediates, extrusion of this peptide precedes translocation of the full-length strand.