Infectionwith BKvirus in man is common in England (Gardner, 1973); haemagglutinationinhibiting (HI) and complement-fixing antibodies were found in all age gropps. We investigated 453 healthy people in Italy by the HI test, and also by the fluorescent-antibody (FA) technique to detect antibodies to other structural components of the virion. MATERIALS AND METHODS Virus and cells. BK virus was kindly supplied by Dr S. D. Gardner and was grown inVero cells. Growth and maintenance media were Eagle's minimum essential medium supplemented with 2.5 % foetal bovine serum. One week after infection of the cultures the medium was changed and after a further 7-10 days' incubation, when cytopathic effects were clearly evident, cells and medium were frozen and thawed once, treated for 2 min. in a Branson ultrasonic disintegrator, and centrifuged at 800 g for 10 min. to sediment cellular debris. The supernatant fluid had a titre of 2048 to 40,768 haemagglutinating units (HAU) per ml, and was used as antigen in the haemagglutination (HA) and HI antibody tests. SV40 was grown in Vero cells in the same way as BK virus.Sera. Serum specimens were obtained from healthy donors, ages 19-65 years, in the Blood Centre of the University Hospital, Bologna. Other sera were collected from healthy children and young people, aged 6 months-18 years, in Milan.Haemagglutination. HA and HI antibody tests were performed in disposable plates by the microtitre method (Sever, 1962). Serial two-fold dilutions of BK virus were made in 0.05-ml amounts of phosphate-buffered saline (PBS), pH 7.2. Human type-0 erythrocytes, from healthy donors, were washed three times and suspended to a concentration of 0-5 % in PBS. One volume of PBS and two volumes of 0.5% erythrocytes were added to each virus dilution. Plates were incubated at +4"C and the HA titre was read 4 hours later, when control erythrocytes in PBS only had completely sedimented. The highest dilution of antigen giving complete haemagglutination was considered to contain 1 HAU. Haemagglutination-inhibition. Sera were heated at 56°C for 30 min. and treated with NaI04 to remove non-specific inhibitors. Serial, two-fold dilutions of serum, from 1 in 4 to 1 in 8192, were made in 0.05 ml amounts of PBS. One volume of PBS containing 8 HAU of antigen was added to each serum dilution and the mixtures were kept at room temperature for 1 hour. After this time, two volumes of 05% erythrocytes were added, the plates were incubated at + 4°C for 4 hours, and the HI-antibody titres were read. From the correlation

The stromal interaction molecular 1 gene (STIM1) encodes a type I trans-membrane protein of unknown function, which induces growth arrest and degeneration of the human tumor cell lines G401 and RD but not HBL100 and CaLu-6, suggesting a role in the pathogenesis of rhabdomyosarcomas and rhabdoid tumors. Here, we describe the STIM1 genomic organization including the identification of the promoter region. The gene consists of 12 exons that span a region larger than 250 kb between the genes RRM1 and NUP98. Nucleotide sequences of all exon-intron boundaries were determined and oligonucleotide primers for the amplification of individual exons were designed. The promoter region was identified within a 1.8-kb SacI fragment at the 5′ end of the gene. In vitro CpG methylation of the promoter region indicated that transcription can be downregulated by this mechanism. The genetic tools developed in the present work will help to determine whether pathogenetic mechanisms that associate STIM1 with tumorigenesis involve mutations in coding sequences and/or promoter, and whether methylation could determine STIM1 transcriptional down-regulation in tumor samples.

Primary hamster kidney cells were transformed by BK virus, a new human papovavirus. Transformed (HKBK) cells produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.

BK virus (BKV) DNA was detected by blot hybridization in a human adenoma of pancreatic islets from patient I.R. BKV DNA was free, and no evidence was found of viral sequences integrated into cellular DNA. Virus was rescued by transfection of human embryonic fibroblasts with tumor DNA. The DNA from rescued virus (BKV-IR) was different from wild-type BKV DNA by restriction endonuclease mapping. The genome of BKV-IR is 235 base pairs (bp) shorter than the genome of wild-type BKV. This alteration originates from a deletion of approximately 300 bp involving HindIII fragments B and D, and an insertion of 70 bp in the region of HindIII fragment C. Transformation of hamster kidney cells was induced by total tumor DNA as well as by BKV-IR and BKV-IR DNA. No antibodies to BKV tumor (T) antigen were detected in the patient's serum by immunofluorescence. The significance of episomal BKV DNA in a human tumor is discussed.

Ependymomas were produced in 44 of 50 Syrian golden hamsters and in 9 of 31 outbred Swiss mice inoculated intracerebrally with high-titer, purified BK virus (BKV). Tumors contained a T-antigen that reacted with BKV-specific T-antibody in immunofluorescence and complement-fixation tests. A proportion of tumor-bearing animals had antibodies to BKV T-antigen in their sera. BKV could be rescued from two tumor cell lines by Sendal virus-mediated fusion with Vero cells. A low, or lack of, oncogenic activity was displayed by BKV inoculated sc, ip, or iv.

SUMMARYAfter exposure of human embryonic fibroblasts to BK virus, virus particles adsorbed to the plasma membrane were engulfed by pinocytosis or captured by vesicles, possibly originating from the endoplasmic reticulum, within 2 h after infection. Most of the virus particles were then transported into lysosomes or into the nucleus, while a small amount of virus was found free in the cytoplasm. Virus particles entered the nucleus between ~ and 12 h after infection, were still detectable in the nucleus at 24 h after infection and became morphologically undiscernible at 3o h after infection, suggesting that a nuclear uncoating mechanism was active between 24 and 30 h after infection. Virus progeny started to appear in the nucleus of infected cells at 4 days after infection, but not until 7 to 8 days after infection did the virus escape into the cytoplasm and cell degeneration became evident. The possible explanations for the long replicative cycle of BK virus are discussed.

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.

We have localized a human homolog, REV3L, of the Saccharomyces cerevisiae REV3 gene on chromosome region 6q21. The full-length cDNA consists of 10,919 nucleotides, with a putative open reading frame of 9,159 bp for a predicted protein of 3,053 amino acids. The gene contains 33 exons in about 200 kb of genomic DNA. In contrast to the previously reported sequence, an additional exon and an alternative splicing site are demonstrated.