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The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm3. The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV.

DIs is a restrictive host range mutant of vaccinia virus strain DIE that grows well only in chick embryo fibroblast cells but is unable to grow in most mammalian cells. In this study, we identified one major deletion (15.4 kbp) which results in the loss of 19 putative open reading frames in the left end of the genome. We then established a system to express foreign genes by inserting them into the deleted region of DIs. We constructed rDIs to express the bacteriophage T7 polymerase (T7pol) gene and showed the expression in various mammalian cell lines by reporter luciferase gene expression under the T7 promoter. We also expressed the full-length human immunodeficiency virus (HIV)-1 NL432 gag gene. The expressed gag gene product induced high levels of cytotoxic T lymphocytes in immunized mice. These data suggest that DIs is useful as an efficient, transient replication-deficient viral vector.

The three-dimensional structure of a self-assembled, recombinant hepatitis E virus particle has been solved to 22-Å resolution by cryo-electron microscopy and three-dimensional image reconstruction. The single subunit of 50 kDa is derived from a truncated version of the open reading frame-2 gene of the virus expressed in a baculovirus system. This is the first structure of a T = 1 particle with protruding dimers at the icosahedral two-fold axes solved by cryo-electron microscopy. The protein shell of these hollow particles extends from a radius of 50 Å outward to a radius of 135 Å. In the reconstruction, the capsid is dominated by dimers that define the 30 morphological units. The outer domain of the homodimer forms a protrusion, which corresponds to the spike-like density seen in the cryo-electron micrograph. This particle retains native virus epitopes, suggesting its potential value as a vaccine.

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.

Virions resembling papovavirus were demonstrated in glial cells in the brain of an aged patient without overt progressive multifocal leukoencephalopathy. The patient was not in a severely immunocompromised state. On histological examination, only a few tiny incomplete necrotic foci were found in the subcortical area. These foci were widely dispersed. Rare, swollen oligodendroglial cells and astrocytes in which papovavirus capsid protein (VP-1) was demonstrated immunohistochemically were present around the foci. The two typical types of virus particles i.e. 35 to 40 nm round particles and elongated particles, were observed in the nuclei of the swollen glial cells. The latter were in the minority. Distinct crystals were also found in the nuclei. The centre-to-centre distance of the particles in the crystals, about 40 nm, and the electron-opaque spots of the round-shaped virions and of the elongated particles, were indicative of structural subunits of papovavirus capsids. This case provides further evidence that papovavirus, possibly JC virus, may be reactivated in the brains of aged patients who are not in an immunocompromised state.

We examined the phylogenetical correlation between two types of JC virus (JCV) isolates, archetypes derived from the urine of nonimmunocompromised individuals and PML-types derived from the brain of patients with progressive multifocal leukoencephalopathy (PML) in Japan. A phylogenetic tree was constructed for eight JCV isolates, five PML-types obtained in this and previous studies and three representative archetypes, from DNA sequence data on the VP1 (major capsid protein) gene. The eight isolates were divided into two major groups, named subtypes MY and CY after the representative archetypal isolates. Four of five PML-type isolates belonged to subtype MY, and the other one to subtype CY. Isolates belonging to subtype MY were further divided into two groups; one group containing archetype MY and three PML-types and the other one containing archetype YI and a PML-type. These findings, together with those in our previous study that correlated various JCV isolates in the world provide evidence for the hypothesis that JCVs associated with PML may have been generated from archetypal JCVs persisting in the patients.

We performed immunohistochemical staining on the nontumorous renal tissue of 45 patients with renal cancer but without progressive multifocal encephalopathy using JCV-specific antibody. For one patient we found positive staining of the nuclei of the renal collecting ducts. Immunoelectron microscopic examination of the positive cell nuclei revealed electron-dense polyomavirus-like particles.

Ninety-one brain tissue sections taken at autopsy from 33 elderly patients (63-100 years old) without progressive multifocal leukoencephalopathy were examined for the presence of JC virus DNA by the polymerase chain reaction and Southern hybridization analysis after DNA extraction. JC virus DNA was detected in 15 sections from 10 patients. These results suggest that JC virus is frequently present in the brains of aged patients.

1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy was used to study aerobic glucose metabolism in eggs of Angiostrongylus cantonensis in an NCTC-109 medium supplemented with fetal calf serum. Without any pretreatment of the spent medium, we were able to identify and quantitate, by NMR, the end-products of glucose metabolism in eggs after cultivation for 2, 4, and 8 days. We demonstrated that A. cantonensis eggs took up glucose rapidly; among the major end products were found lactic acid, acetic acid and alanine. The eggs are parasitic in a sense that the energy metabolism in them is dependent mainly upon the energy source present in outer medium.

Analysis of cytosol proteins by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis revealed a prominent increase in the amount of a cytosol protein with molecular weight of 88 000 in transformed human adult, human embryo, mouse adult, and hamster embryo fibroblasts as compared with normal fibroblasts. The cytosol protein with M r 88 000 is also increased in the cytosol of four kinds of rat ascites hepatoma cell as compared to normal and regenerating liver. The protein with M r 88 000 exists as one of the major cytosol proteins in transformed fibroblasts, hepatoma cells and HeLa cells, constituting 7–10% of total cytosol proteins. The data suggest that the cytosol protein with M r 88 000 is associated with certain growth characteristics of cells.

The tumor-associated cell-surface antigen (TSSA) on simian virus 40 (SV40)-transformed hamster cells was studied serologically by a complement-dependent cytotoxicity test. An antiserum was obtained from guinea pigs inoculated with SV40-transformed hamster cells. The serum was cytotoxic to SV40-transformed hamster cells after absorption with 15-day hamster embryo cells, hamster cells transformed either by polyoma virus or adenovirus 12, various tissues of hamster origin (brainliver, spleen, and kidney), or sheep red blood cells. These results indicated that the major TSSA induced specifically by SV40 was similar or identical to the antigen present during early stages of embryogenesis.

1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy were used to study metabolites excreted by young adult Angiostrongylus cantonensis maintained aerobically in the presence of D-[13C6]glucose. End-products of glucose metabolism identified and quantitated by means of 1H-NMR were lactate, acetate and alanine, in the molar ratio of 1:0.13:0.05 for males and 1:0.07:0.04 for females. 13C-NMR analyses proved that all the three products originated from the glucose present in the medium.

Guinea pig (strain No13) kidney cells were transformed with SV40 (SVNo13). When this transformant was injected subcutaneously into syngeneic irradiated guinea pigs, tumors were produced in some animals. One of them became transplantable (ST13). Clones were obtained from cultured tumor cells (STC13). About one copy of the viral genome appeared to exist in both SVNo13 and STC13 clones as determined by reassociation kinetics. We have examined the distribution of integrated viral sequences in the host DNA by blot hybridization. In SVNo13, the blotting pattern with BamHI showed two main bands of 6.9 and 4.3 kilobases (kb), whereas in STC13 clones the 6.9-kb band was replaced with a shorter one of 6.1-kb. The blotting pattern with EcoRI also showed distinct changes between SVNo13 and STC13 clones. These results suggest either the occurrence of some mutations or deletions in the cellular DNA adjacent to the integrated viral genome or the transposition of viral DNA sequences during tumor development.

The effect of ethidium bromide (EB) on the synthesis of circular DNA of mammalian cells was studied by isopycnic centrifugation in a CsCl-EB solution. EB (0.1—0.5 μg/ml) interferes with the synthesis of newly-formed circular DNA of HeLa cell mitochondria and causes degradation of the pre-existing circular DNA, as well. Under the same conditions, nuclear DNA synthesis was not inhibited. This effect was not reversible at a concentration of 0.5 μg EB/ml or more. Cytosine arabinoside (ara-C) did not exhibit an effect similar to that of EB.