We have compared HMG1 with the product of tryptic removal of its acidic C-terminal domain termed HMG3, which contains two 'HMG-box' DNA-binding domains. (i) HMG3 has a higher affinity for DNA than HMG1. (ii) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (iii) Electron microscopy shows that HMG3 at a low protein:DNA input ratio (1:1 w/w; r = 1), and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (iv) HMG3 at high input ratios (r = 6), but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties.

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...

Mouse polyomavirus (PyV) virions enter cells by internalization into smooth monopinocytic vesicles, which fuse under the cell membrane with larger endosomes. Caveolin-1 was detected on monopinocytic vesicles carrying PyV particles in mouse fibroblasts and epithelial cells (33). Here, we show that PyV can be efficiently internalized by Jurkat cells, which do not express caveolin-1 and lack caveolae, and that overexpression of a caveolin-1 dominant-negative mutant in mouse epithelial cells does not prevent their productive infection. Strong colocalization of VP1 with early endosome antigen 1 (EEA1) and of EEA1 with caveolin-1 in mouse fibroblasts and epithelial cells suggests that the monopinocytic vesicles carrying the virus (and vesicles containing caveolin-1) fuse with EEA1-positive early endosomes. In contrast to SV40, PyV infection is dependent on the acidic pH of endosomes. Bafilomycin A1 abolished PyV infection, and an increase in endosomal pH by NH 4 Cl markedly reduced its efficiency when drugs were applied during virion transport towards the cell nucleus. The block of acidification resulted in the retention of a fraction of virions in early endosomes. To monitor further trafficking of PyV, we used fluorescent resonance energy transfer (FRET) to determine mutual localization of PyV VP1 with transferrin and Rab11 GTPase at a 2-to 10-nm resolution. Positive FRET between PyV VP1 and transferrin cargo and between PyV VP1 and Rab11 suggests that during later times postinfection (1.5 to 3 h), the virus meets up with transferrin in the Rab11-positive recycling endosome. These results point to a convergence of the virus and the cargo internalized by different pathways in common transitional compartments.Adsorption of mouse polyomavirus (PyV) on the host cell surface is mediated by the interaction of its major structural protein, VP1, with sialic acid. Recently, anionic glycosphingolipids GD1a and GT1b, which are heavily glycosylated gangliosides carrying sialic acid residues, were identified as specific receptors for PyV (37). Integrin ␣4␤1 (also sialyated) has been implicated as a possible coreceptor in mouse cells (9). For simian virus 40 (SV40), another member of the Polyomaviridae, the major histocompatibility complex class I molecule was described as a receptor (8). However, it was later shown that the major histocompatibility complex class I molecule is not endocytosed together with the virus (2). Tsai et al. (37) previously demonstrated that ganglioside GM1 can serve as a functional receptor for SV40. This virus enters cells via caveola invaginations that fuse with larger peripheral organelles (called caveosomes) enriched by caveolin-1. In the steps that followed, SV40 was detected in tubular, caveolin-free membrane vesicles that move along microtubules and deliver virions to the smooth endoplasmic reticulum (ER) (29). The import of SV40 into the ER was found to be brefeldin A sensitive and thus mediated by the ER-Golgi-intermediate compartment represented by COPIcoated vesicles (25, 32). The endocytic pat...