Studies of human polyomaviruses, with HPyV7, BKPyV, and JCPyV present in urine of allogeneic hematopoietic stem cell transplanted patients with or without hemorrhagic cystitis.

BACKGROUND: BK polyomavirus (BKPyV) can cause hemorrhagic cystitis (HC) in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients and polyomavirus-associated nephritis in renal transplant patients, while JC polyomavirus (JCPyV) can generate progressive multifocal leukoencephalopathy in immunocompromised individuals. Since 2007, additional human polyomaviruses (HPyVs) have been identified. In this study, we examined the urines of allo-HSCT patients for possible presence of polyomaviruses BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, and HPyV10 (MWPyV). METHODS: A total of 185 urinary samples obtained 2002-2007 from 105 allo-HSCT patients, 32/105 with HC, were tested for the above-listed HPyVs by a bead-based multiplex assay. Of these, 142 urine samples had previously been tested for BKPyV and JCPyV by nested polymerase chain reaction (PCR). RESULTS: Aside from BKPyV and JCPyV, which dominated, HPyV7 was detected in 5 BKPyV-positive urinary samples from 1 patient. The multiplex assay was more sensitive and specific than the nested PCR. BKPyV and/or JCPyV were found in all but 1 of the previously BKPyV- or JCPyV-positive samples, although 6 previously BKPyV-positive cases were now JCPyV-positive or the reverse. Furthermore, 18/79 previously negative samples were found to be BKPyV and/or JCPyV positive, and a total of 21 double infections were found. Lastly, in 1/29 HC patients, only JCPyV was detected. CONCLUSION: HPyV7 was found for the first time in urine of an allo-HSCT patient, and BKPyV and JCPyV were more commonly found in urine samples using the bead-based assay compared to testing by nested PCR. Finally, only JCPyV was detected in the urine of 1 HC patient.

No detection of BK virus, JC virus, KI, WU and Merkel cell polyomaviruses in cerebrospinal fluid of patients with neurological complications after hematopoetic stem cell transplantation.

Neurological complications, often due to viral reactivation, after allogeneic hematopoetic stem cell transplantation (HSCT) are associated with increased mortality. Here, cerebrospinal fluid from 20 HSCT patients with neurological symptoms were analyzed and found to be negative by PCR for BK virus, JC virus, KI, WU and Merkel cell polyomavirus DNA.

Murine pneumotropic virus VP1 virus-like particles (VLPs) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus VP1 VLPs.

The ability of murine pneumotropic virus (MPtV) major capsid protein VP1 to form virus-like particles (VLPs) was examined. MPtV-VLPs obtained were used to estimate the potential of MPtV to attach to different cells and to assess some characteristics of the MPtV cell receptor. Furthermore, to evaluate if MPtV-VLPs could potentially complement murine polyomavirus (MPyV) VP1 VLPs (MPyV-VLPs) as vectors for prime-boost gene therapy, the capability of MPtV-VLPs to serologically cross react with MPyV-VLPs and to transduce DNA into cells was examined. MPtV VP1 obtained in a recombinant baculovirus system formed MPtV-VLPs readily. MPtV-VLPs were shown by FACS analysis to bind to different cells, independent of MHC class I antigen expression. In addition, MPtV-VLPs did not cause haemagglutination of red blood cells and MPtV-VLP binding to cells was neuraminidase resistant but mostly trypsin and papain sensitive, indicating that the MPtV receptor lacks sialic acid components. When tested by ELISA and in vivo neutralization assays, MPtV-VLPs did not serologically cross react with MPyV-VLPs, suggesting that MPtV-VLPs and MPyV-VLPs could potentially be interchanged as carriers of DNA in repeated gene therapy. Finally, MPtV-VLPs were shown to transduce foreign DNA in vitro and in vivo. In conclusion, the data suggest that MPtV-VLPs, and possibly also MPtV, bind to several different cell types, that binding is neuraminidase resistant and that MPtV-VLPs should potentially be able to complement MPyV-VLPs for prime-boost gene transfer in vivo.

Genetic immunization is augmented by murine polyomavirus VP1 pseudocapsids.

To improve immune responses induced by DNA immunization, murine polyomavirus major capsid protein (VP1) pseudocapsids were complexed with a DNA plasmid encoding the p37 (p24 and p17) nucleocapsid proteins of the human immunodeficiency virus type 1 (HIV-1). A 10-fold increase in antibody titer was noted in mice given DNA plasmid together with VP1 pseudocapsids in comparison to animals that received DNA plasmid alone. Cell mediated responses to HIV-1 p24 occurred, but were not significantly augmented by delivering the DNA as a VP1 complex. We have consequently for the first time shown a carrier/adjuvant effect of polyomavirus pseudocapsids that strongly increased the humoral immune response in DNA immunization.

v-Myb of E26 leukemia virus up-regulates bcl-2 and suppresses apoptosis in myeloid cells.

Many oncogenes have been shown to be deregulated transcription factors, yet direct target genes mediating cell transformation remain elusive. Here we describe such a target for v-Myb by exploiting a temperature-sensitive mutant of the E26 avian leukemia virus encoding Myb-Ets. Myeloblasts transformed by the mutant differentiate into macrophages or die by apoptosis when shifted to the nonpermissive temperature as a result of inactivation of v-Myb. During this process mRNA of the antiapoptotic oncoprotein Bcl-2 is down-regulated with kinetics similar to those of Mim-1, a differentiation-related protein whose expression is directly regulated by Myb. Forced expression of bcl-2 rescues the cells from apoptosis, without preventing either their withdrawal from the cell cycle or their differentiation. v-Myb appears to act directly on the bcl-2 gene, because a bcl-2 promoter-driven reporter is activated by Myb-Ets and v-Myb-VP16 and requires intact Myb binding sites within the promoter. Surprisingly, inactivation of v-Myb in multipotent progenitors transformed by E26 virus does not induce apoptosis, indicating that bcl-2 regulation by the oncoprotein is required for the transformation of some cell types but not others.

Regulation of G1 progression by E2A and Id helix-loop-helix proteins.

In NIH3T3 fibroblasts, the ubiquitous helix-loop-helix (HLH) protein E2A (E12/E47) and the myogenic HLH proteins MyoD, MRF4 and myogenin are growth-inhibitory, while two ubiquitous Id proteins lacking the basic region are not. The dimerization domain mediates inhibition. However, in addition to the HLH region, E2A contains two inhibitory regions over-lapping with the main transcriptional activation domains. The growth-suppressive activity of the intact E47 as well as MyoD was counteracted by the Id proteins. When E47 lacking the HLH domain was overexpressed, Id could no longer reverse growth inhibition. By increasing the amount of E47 with an inducible system or neutralizing the endogenous Id with microinjected anti-Id antibodies, withdrawal from the cell cycle occurred within hours before the G1-S transition point. The combined results suggest that the Id proteins are required for G1 progression. The antagonism between the E2A and Id proteins further suggests that both are involved in regulatory events prior to or near the restriction point in the G1 phase of the cell cycle.

Wild-type p53 induces apoptosis in a Burkitt lymphoma (BL) line that carries mutant p53.

All Burkitt lymphoma (BL) biopsies and cell lines carry a c-myc/Ig translocation. The resulting constitutive activation of c-myc is regarded as an essential factor for the progressive growth of the tumor cells. At least 60% of BL cell lines carry a mutated p53 gene as well. It has been shown that the growth of mutant p53 carrying tumor cells could be inhibited by the introduction of wild-type p53. In order to examine whether this also applies to the presumably 'myc-driven' BL cell, we have transfected the Epstein-Barr virus (EBV) negative BL41 cell line with a temperature sensitive p53 mutant (p53-Val135) that expresses p53 with a largely mutant conformation at 37.5 degrees C and mostly wild-type conformation at 32 degrees C. At 37.5 degrees C, the p53-Val135 transfected cells behaved like the parental or neo transfected control cells. However, expression of exogenous wild-type p53 at 32 degrees C resulted in a rapid reduction of the number of viable cells while the parental and neo control cells remained unaffected. Cell death was due to apoptosis as shown by chromatin and nuclear condensation and specific DNA fragmentation. The first signs of apoptosis were evident after 10 h at 32 degrees C and after 3 days 90-100% of the cells had undergone apoptosis. These findings indicate an incompatibility between expression of wild-type p53 and progressive growth of BL cells if their neoplastic development has included a p53 mutation. The question whether apoptosis was induced in by the wild-type protein per se or by the contradictory signals of a constitutively activated c-myc and wild-type p53 needs further investigation.

Reconstitution of wild-type p53 expression triggers apoptosis in a p53-negative v-myc retrovirus-induced T-cell lymphoma line.

Inactivation or mutation of the p53 tumor suppressor gene has been observed in a wide variety of human and murine tumors. We have found that a v-myc retrovirus (J3)-induced T-cell lymphoma line (J3D) has lost one of its p53 alleles, whereas the other has become inactivated due to the insertion of a Moloney murine leukemia provirus in intron 4 with an opposite transcriptional orientation. No p53 protein could be detected by immunoprecipitation with monoclonal anti-p53 antibodies. We have transfected this line with the temperature-sensitive murine Val135 construct that is expressed as mutant p53 at 37 degrees C and largely wild-type p53 at 32 degrees C. There was no difference in the number of viable cells among the p53 transfectants, the parental cells, and neomycin vector-transfected control cells at 37 degrees C. Following a temperature shift to 32 degrees C, the p53 transfectants rapidly lost viability, and 95-100% of the cells were dead by 3 days, whereas the control cells remained unaffected. Examination of DNA isolated from p53-transfected cells grown at 32 degrees C revealed nucleosomal fragmentation, indicating cell death by apoptosis. It is suggested that apoptosis is triggered by contradictory signaling. Constitutively expressed v-myc can stimulate cell proliferation, whereas expression of wild-type p53 in cells that have lost endogenous p53 expression in the course of their neoplastic development may suppress growth.

Philadelphia chromosome as the sole abnormality and p210 bcr-abl chimeric protein expression in an Epstein-Barr virus-transformed B cell line from a patient with chronic myeloid leukemia.

A 'B' cell line, originating from a patient with chronic myeloid leukemia and containing the Philadelphia chromosome, was established after Epstein-Barr virus transformation. The Philadelphia chromosome was the sole chromosomal abnormality in this line, designated as PhB1 cell line. In DNA hybridization studies we detected rearrangements in the bcr gene and in the immunoglobulin heavy chain joining region. The phenotypes of the cells were typical of mature B cells expressing antigens CD19, CD20, CD22, CD23, CD39, HLADR, IgM, and kappa. The expression of the 210 bcr-abl chimeric protein was detected by means of an immunoprecipitate assay.

Immunization against polyoma tumors with synthetic peptides derived from the sequences of middle- and large-T antigens.

We have used 9 synthetic peptides corresponding to sequences of polyoma virus small-T, middle-T and large-T antigens as immunogens in order to map antigenic epitopes that can induce polyoma-tumor-specific immunity in different mouse strains. We found that immunization of mice with synthetic peptides derived from amino acid (aa) sequences common to all 3 T-antigens (aa 1-19), or sequences common to only middle-T and small-T (aa 162-176), as well as synthetic peptides unique for middle-T (aa 269-282 and 371-381), or unique for large-T (aa 108-124, 316-333 and 436-449) can induce immunity against polyoma tumors. The synthetic peptides can be divided into 3 types with regard to immunogenicity; (i) peptides that immunize in more than one mouse strain and may represent immunodominant sites, (ii) peptides that may be immunogenic in only one strain, and thus strain-specific, and finally (iii) peptides that do not immunize in the strains tested so far.

Tumorigenicity of EBV-carrying lymphoblastoid cell lines (LCLs): distinctive grading in SCID mice.

The in vitro and in vivo growth of lymphoblastoid cell lines (LCLs), Burkitt lymphomas (BLs) and PBL-derived immunoblastomas in SCID mice was studied in parallel. Most, but not all, of the LCLs tested were tumorigenic in SCID mice. Long-term culturing in vitro was not a necessary prerequisite for tumorigenicity. There was a good correlation between in vivo and in vitro growth. Three major classes of cells could be distinguished on the basis of their growth properties.

Mutant p53 detected in a majority of Burkitt lymphoma cell lines by monoclonal antibody PAb240.

The status of the p53 gene in lymphoblastoid cell lines (LCLs) and Burkitt lymphoma cell lines (BLs) was investigated. Southern blot analysis demonstrated that no major deletions or rearrangements had occurred in the p53 gene in any of the cell lines. The p53 protein was examined by immunoprecipitation using two monoclonal anti-p53 antibodies. PAb1801 recognizes both wild-type and mutant p53. PAb240 reacts exclusively with mutant p53. Fourteen LCLs reacted with PAb1801, but not with PAb240, suggesting that none of them expressed mutant p53. However, one LCL had mutant p53. This LCL differs from other LCLs in that it grows to higher cell densities and has a higher agarose clonability. All BLs expressed p53. Out of 15 BLs, nine (60%) carried mutant p53, as indicated by their reactivity with PAb240. Among the nine BLs with mutant p53, eight Epstein-Barr virus (EBV)-positive. Three out of the six BLs with wild-type p53 were EBV-positive. Multiple EBV-converted sublines all exhibited the same p53 status as the parental line. Our results indicate that the p53 gene is mutated in a majority of Burkitt lymphoma cell lines (BLs), and suggest that p53 mutation contributes to the malignant phenotype of these cell lines.

Complete transformation of embryonal rat fibroblasts by polyomavirus occurs during passage in vitro.

The tumorigenicity of secondary rat embryo fibroblasts transfected with a plasmid harboring a replication origin-defective polyomavirus was found to increase during in vitro propagation. Thus, polyomavirus-transfected cells were found to be more than 10,000-fold more tumorigenic when injected into syngenic rats at 3 months after transfection compared to those injected at an earlier time point. Furthermore, most clones of polyomavirus-transfected cells did not grow in semisolid medium at 52 days after transfection but did grow at 95 days. Addition of glucocorticoid hormones, but not of 25% fetal calf serum, to the growth medium of the early passage cells resulted in limited anchorage-independent growth. An altered level of expression of a number of proteins was found in cells analyzed at different times after transfection. Notably, the expression of a component of the actin filament system, tropomyosin 2, was shown to decrease during growth in vitro. The development of a more fully transformed phenotype at late passages correlated with loss of the requirement for large T-antigen for growth. Thus, cells transfected with a polyomavirus mutant encoding a thermolabile large T-antigen did not grow at the restrictive temperature at 6 weeks after transfection, but grew well at 5 months after transfection. We suggest that these phenomena may be explained by assuming that establishment of rodent fibroblasts, and thereby sensitivity to transformation by middle T-antigen, is not an immediate consequence of expression of large T-antigen but occurs after a period of growth in vitro.

A single peptide derived from the sequence common to polyoma small and middle T-antigen induces immunity against polyoma tumors.

Polyoma virus, an oncogenic virus, fails to induce tumors in immunocompetent rodents due to T cell-dependent mechanisms. The target recognized by the immune system has been functionally defined as polyoma tumor-specific transplantation antigen (TSTA) and has been postulated to be related to the virus three early proteins small T (ST), middle T (MT), and large T (LT) antigens. We show here that immunization with a synthetic peptide corresponding to amino acids 162-176 of polyoma MT and ST was able to decrease tumor progression of polyoma tumors, but not of nonpolyoma tumors. This indicated that these amino acids constitute an epitope of the polyoma tumor-specific transplantation antigen.

Polyoma T-antigen-derived synthetic peptides induce polyoma-virus-specific macrophage migration inhibition.

Synthetic peptides, 2 derived from the sequence common to small, middle and large T-antigen, and I derived from the sequence unique for middle T, activated lymphocytes from polyoma-virus-immunized, but not from control mice, to release the lymphokine migration inhibitory factor (MIF). In contrast, purified, bacterially grown, full-length small T-antigen and a middle T-antigen mutant Py 1387T MT (lacking 37 C-terminal amino acids) did not induce lymphokine release, although they were capable of inducing an anti-polyoma TSTA response in vivo.

A polyomavirus tumor-specific transplantation antigen (TSTA) epitope is situated within the N-terminal amino acid sequence common to middle and small T-antigens.

An MT cDNA-transformed rat cell line (2.8), that contains only the polyoma middle T-antigen and expresses polyoma TSTA, can be rejected in rats immunized with 1837 cells that carry a host range (A185 hr-t), mutant which expresses a full-length large T-antigen, and nonfunctional N-terminal fragments of small and middle T. This shows that at least one polyoma TSTA epitope is situated in the 113 amino acid N-terminal region, which is common to middle and small T-antigens.

Immunization against the polyoma tumor-specific transplantation antigen (TSTA) with polyoma T-antigens.

The relationship between the polyoma virus tumor-specific transplantation antigen (TSTA) and 2 of the virus proteins coded from the early region of polyomavirus was investigated. Mice were immunized with small T antigen and a truncated mutant of middle T antigen, both purified from genetically engineered Escherichia coli. The 2 proteins induced protective immunity against polyomavirus-induced tumors, but not against non-polyoma tumors, indicating that one or more of the polyoma T antigens are directly involved in a TSTA function.

Studies on the polyoma tumor-specific transplantation antigen (TSTA): selection and characterization of TSTA-negative segregants from somatic hybrids.

Two polyoma-TSTA-negative variants were selected independently from a polyoma fibrosarcoma/Moloney lymphoma somatic hybrid, by repeated passages in polyoma-virus-preimmunized mice. One of the variants had lost all its polyoma DNA, while the other only retained a deleted piece of its integrated polyoma DNA. In contrast to the parental hybrid clone, none of the variants produced detectable amounts of T-antigens. This finding indicates that a detectable expression of the products of the polyoma virus early genome, the T-antigens, is important either directly or indirectly for the expression of TSTA.

Detection of a polyoma virus-induced tumor-associated membrane antigen in mouse cells by the macrophage migration inhibition test.

Soluble membrane fractions derived from polyoma tumor cells trigger lymphocytes, derived from polyoma-immunized animals, but not from nonimmunized controls, to release the lymphokine, macrophage migration-inhibitory factor. The reaction can be blocked by sera from polyoma-bearing animals. Absorption of these sera with polyoma cells, but not with nonpolyoma cell lines, abrogates this activity. These findings suggest that there is a polyoma virus-induced membrane component that can induce polyoma-specific macrophage migration inhibition.