Electron and confocal microscopy were used to observe the entry and the movement of polyomavirus virions and artificial virus-like particles (VP1 pseudocapsids) in mouse fibroblasts and epithelial cells. No visible differences in adsorption and internalization of virions and VP1 pseudocapsids ("empty" or containing DNA) were observed. Viral particles entered cells internalized in smooth monopinocytic vesicles, often in the proximity of larger, caveola-like invaginations. Both "empty" vesicles derived from caveolae and vesicles containing viral particles were stained with the anti-caveolin-1 antibody, and the two types of vesicles often fused in the cytoplasm. Colocalization of VP1 with caveolin-1 was observed during viral particle movement from the plasma membrane throughout the cytoplasm to the perinuclear area. Empty vesicles and vesicles with viral particles moved predominantly along microfilaments. Particle movement was accompanied by transient disorganization of actin stress fibers. Microfilaments decorated by the VP1 immunofluorescent signal could be seen as concentric curves, apparently along membrane structures that probably represent endoplasmic reticulum. Colocalization of VP1 with tubulin was mostly observed in areas close to the cell nuclei and on mitotic tubulin structures. By 3 h postinfection, a strong signal of the VP1 (but no viral particles) had accumulated in the proximity of nuclei, around the outer nuclear membrane. However, the vast majority of VP1 pseudocapsids did not enter the nuclei.Structural proteins of nonenveloped viruses are selected by evolution for the efficient delivery of genetic information via plasma membranes into cells for its expression. Hence, studying the properties of viral coat structures and detailed understanding of early steps of viral infection (entry, movements toward the cell nuclei, and uncoating) could help to solve an important aspect of gene therapy: the development of efficient systems for the transfer of exogenous genetic information into target cells.Polyomaviruses, a member of the Papovaviridae family, have a wide range of hosts and different pathogenic responses in infected organisms. Despite this variation, the structures of the virions and genomic organizations of these viruses are very similar. Genomic circular double-stranded DNA (5.3 kbp) of the mouse polyomavirus encodes three early antigens (large, middle, and small T antigen) and three late structural proteins, VP1, VP2, and VP3. The late proteins, together with viral DNA and cellular histones (except H1), are assembled into virions in the cell nuclei. Neither VP2 nor VP3 is required for assembly of the capsid-like structure, and their functions in the viral replicative cycle are still unclear. The multifunctional VP1 can self-assemble into capsid-like particles (VP1 pseudocapsids) and is responsible for interaction with the sialic acid of an as-yet-unknown receptor (15, 37). Moreover, it has a nonspecific DNA binding activity (23), suggesting a role in nucleocore assembly. The problem is that li...

Abstract`Empty' polyomavirus pseudocapsids, self-assembled from the major structural protein VP1, bind DNA nonspecifically and can deliver it into the nuclei of mammalian cells for expression [Forstova è et al. (1995) Hum. Gene Ther. 6, 2973 06]. Formation of suitable VP1-DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1-DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double-stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain.z 1999 Federation of European Biochemical Societies.