Analysis of the early regulatory region of the human papovavirus BK

BKV is a human papovavirus which latently infects a majority of the world population and whose DNA has been found in human tumor tissue. Along with simian virus 40 (SV40) and JCV, it is one of several highly homologous polyomaviruses which display distinct host ranges, tissue tropisms, and transformation potentials. Determination of these properties is thought to reside, in part, in the noncoding regulatory region of these viruses. We have studied the regulation of gene expression by the early promoter and enhancer of BKV. Our results show that the early promoter of BKV consists of elements found both to the early side of and within the proximal 18 bp of the first enhancer element itself. At least one BKV regulatory element appears to be downstream of the mRNA start sites. The BKV enhancer consists of two different types of elements, three direct repeats, and an element (denoted "c") found in the 30 bp to the late side of the distal repeat. When used with the BKV promoter the enhancer repeat elements were found to be redundant, optimal promoter activity requiring only two of the three repeats plus the c element. Using the heterologous SV40 promoter the optimal BKV enhancer consisted of either three repeats or two repeats plus the c element. Subfragments of the enhancer region were capable of partial activation of homologous and heterologous promoters. We conclude that the regulation of BKV early gene expression involves novel elements arranged in ways not previously described in other papovaviruses.     

Immunoelectroosmophoresis for the human papovavirus BK

The reaction between BK virus and its antibodies may well be visualized with immunoelectroosmophoresis (IEOP). Serum antibody levels may be compared or graded by reacting them against a continuous antigen front. In seroepidemiologic surveys IEOP compares favourably with hemagglutination inhibition in terms of sensitivity, practicability and reproducibility. Technical conditions are described which are suitable for antigen detection, quantification and identification.     

Helper function for adenovirus replication in monkey cells by BK human papovavirus.

CV-1 monkey kidney cells are semipermissive for BK human papovavirus at 37°. Although infected cells synthesize T antigen at this temperature, only a small percentage of the cells (less than 5%) produce viral antigen. However, when infected cells were incubated at 40°, characteristic CPE was observed with high virus yields. The inhibition of BKV growth in CV-1 cells was thus shown to be a temperature-dependent phenomenon. Experiments were then conducted to determine whether BKV provided a helper function for adenovirus growth in CV-1 cells at temperatures which were either permissive (40°) or semipermissive (37°) for BKV replication. At 37°, there was a low level of adenovirus enhancement of 0.5 to 1.0 log increase. However, at 40°, there was a 1.5 to 2.5 log increase in adenovirus yields, comparable to those obtained by coinfection with SV40 and adenovirus. These data provide additional information on common viral functions shared by BKV and SV40.     

The late promoter of the human papovavirus BK is contained within the early promoter enhancer region

We have analyzed the sequences necessary for late promoter function and mapped the late mRNA start sites of the human papovavirus BK (prototype). Our results show that, under both replicating and nonreplicating conditions, the BKV late promoter is contained within the same region defined as the enhancer for the early promoter. This region consists of three 68-bp repeats (the middle one of which has an 18-bp deletion) and a 66-bp region containing an enhancer element denoted as c, located to the late side of the 68-bp repeats. Deletions within the early enhancer domain indicate that elements of the late promoter are found throughout the entire region.     

The persistence of papovavirus BK DNA sequences in normal human renal tissue

Evidence has accumulated indicating that BK virus, following an inapparent primary infection, persists in the renal organs of normal healthy individuals and reactivates upon immunosuppression. Data to support this hypothesis are presented and suggest that BK virus DNA sequences are present at very low levels in the kidneys of more than 50% of the population and that this persistence is localized in several foci within these organs.     

Transforming growth factor-beta-mediated regulation of BK virus gene expression

The increasing prevalence of BK virus (BKV)-associated diseases in immunosuppressed patients has prompted an investigation of the immune response to BKV, especially the role of cytokines in regulating viral replication. We examined the effect of TGF-beta, a cytokine that is stimulated by certain immunosuppressive therapies, on BKV gene expression during lytic infection of renal proximal tubule epithelial cells. Viral gene expression, and specifically the activity of the BKV early promoter, is regulated by TGF-beta in a strain-dependent manner. Promoter activity is upregulated in the presence of TGF-beta for the TU strain of BKV, and not for the Dik, Dunlop, or Proto-2 strains. Using site-directed mutagenesis, we have identified a small segment of the TU promoter that is required for stimulation in response to TGF-beta. These results demonstrate that BKV strains can respond differently to cytokine treatment and suggest that TGF-beta may play a role in the reactivation of BKV.     

Serological investigation of BK papovavirus infection in pregnant women and their offspring.

Paired sera from 150 pregnant women and 387 umbilical cord sera were tested for BK virus (BKV) antibodies. The hemagglutination inhibition, neutralization, and indirect immunofluorescence tests were employed for the detection of antibodies. Treatment of serum with anti-gamma Fc and tests of immunoglobulin M (IgM) fractions for antibodies were utilized as required to detect and validate the presence of virus-specific IgM. The BKV antibody prevalence in the sera collected at the time of the first prenatal visit was 75% by hemagglutination inhibition and 91% by neutralization tests. A total of 95% of the women had antibodies by at least one of the three serological tests. Five of 100 women with normal pregnancies exhibited BKV activity during pregnancy as evidenced by a greater than fourfold rise in BKV hemagglutination inhibition antibody titers and acquisition of BKV-specific IgM. The antibody rise occurred in the younger women and appeared to be a result of reactivation of the virus rather than of primary infection. Two instances of possible recent BKV infections were identified. BKV-specific IgM was not detected in any of the 387 umbilical cord sera which included three specimens from infants born to mothers with definite or probable BKV activity during pregnancy and 50 specimens with IgM levels of > 20 mg/100 ml. The results indicate that few women in the child-bearing age are nonimmune to BKV and that, although reactivation of infection occurs in pregnancy, congenital transmission of the virus either does not occur or is rare.     

Measurement of BK papovavirus IgG and IgM by radioimmunoassay (RIA)

Current techniques for the measurement of BK papovavirus (BKV) specific IgM include sucrose density gradient centrifugation followed by hemagglutination inhibition (HAI) or indirect immunofluorescent (IF) staining of BKV infected cells using a fluorescein conjugated anti-human IgM antibody. These techniques are cumbersome and labor intensive and do not lend themselves to testing large numbers of sera. A solid phase radioimmunoassay (RIA) was developed to facilitate the measurement of BKV IgG and IgM in large numbers of sera. Solid phase antigen was prepared by adsorbing CsCl purified BKV antigen to polyvinyl chloride microtiter plates. Following reaction with serum, bound immunoglobulin was detected with iodinated goat anti-human IgG or IgM. RIA for the measurement of BKV IgG was sensitive with titers approaching 10⁻⁶. Determination of IgG titers by RIA and HAI showed good agreement (P < 0.01, correlation coefficient = 0.74). Measurement of BKV IgM was not affected by the presence of BKV IgG as evidenced by sucrose density gradient fractionation of IgM positive sera, removal of IgG by treatment with S. aureus protein A, and addition of BKV IgG to BKV IgM. Rheumatoid factor (RF) gave false positive IgM titers in the presence of BKV IgG when RF titers were ≥ 1:640 by latex agglutination testing and BKV IgG levels exceed 1:256 by HAI. False positives due to RF could be eliminated by treatment of sera with sheep anti—human IgG antisera. RIA for BKV IgM was specific as sera containing JCV-, cytomegalovirus (CMV)-, rubella-, or hepatitis B core antibody (anti HBc)—IgM were negative by RIA. RIA detected BKV IgM in several sera from renal dialysis or allograft palienls with titers ranging from 1:400 to 1:128,000 and demonstrated that BKV IgM persisted in sera of renal allograft patients for as long as 343 days post transplantation.     

Induction of brain tumors in hamsters with BK virus, a human papovavirus.

The oncogenicity of BK virus for the central nervous system was studied in newborn hamsters. The virus was weakly oncogenic after intracerebral inoculation. Two of 45 hamsters treated with antithymocyte serum developed tumors whereas no untreated hamsters developed tumors. Both tumors were choroid plexus papillomas by histologic and electron microscopic examination. Cells cultured from one tumor had growth characteristics of transformed cells and had intranuclear T antigen; but infectious virus could not be rescued. Cultured tumor cells were weakly oncogenic for hamsters, but theoncogenicity of these cells was enhanced when the recipient animals were treated with antithymocyte serum. The possible role of host immune response as a basis for the weak oncogenicity of BK virus is discussed.     

Analysis of immediate-early and early proteins of murine cytomegalovirus in permissive and nonpermissive cells

Summary The immediate-early (IE) and early proteins induced by murine cytomegalovirus (Smith strain) in permissively infected 3T3-L1 murine fibroblasts were identified by SDS polyacrylamide gel electrophoresis. Ten proteins were classified as IE by their time of synthesis and by their synthesis in the presence of actinomycin D following reversal of a cycloheximide mediated protein synthesis block. By exclusion, seven proteins were classified as early class. Eleven of these proteins were precipitated by MCMV antiserum.The role of IE and early proteins in the replication of the virus was studied by infection of a murine macrophage cell line (J 774A.1) and human foreskin fibroblast (HFF) cells. The infections were characterized as nonpermissive by several criteria, including lack of production of infectious virus or viral DNA. However, the major IE and early MCMV proteins were detected in the nonpermissively infected cells. The block to virus replication in the macrophage and human fibroblast cells appeared to occur after the switch from IE to early protein synthesis, but before viral DNA replication.With 7 Figures     

Further characterization of the murine cytomegalovirus induced early proteins in permissive and nonpermissive cells

Summary Some of the properties of the immediate-early (IE) and early proteins induced by the murine cytomegalovirus (Smith strain) were examined in permissively infected 3 T 3-L 1 cells, and in non-permissively infected J 774 A. 1 (mouse macrophage) and human fibroblast cells, in order to determine differences that could account for the restriction in virus replication in the latter two cell lines. The different virus induced proteins had distinctive partitioning characteristics between nuclear and cytoplasmic fractions. The 96 K major IE protein had an exclusively nuclear association, as did the most abundant early proteins of 39 K and 36 K. The other viral proteins however were evenly distributed between nucleus and cytoplasm. In general these patterns were also seen in the infected non-permissive cells. Several proteins showed more than one charge isomer on two-dimension gels, and in addition five IE proteins and two early proteins were phosphorylated. Only two differences between the permissive and nonpermissive infections were observed; the IE proteins of 100 K and 89 K when synthesized in the human cells had a stronger affinity for the nuclear fraction; also a phosphorylated form of the 30 K IE protein was not detected in MCMV infected J 774 A.1 cells.     

Stable transformation of mouse, rabbit and monkey cells and abortive transformation of human cells by BK virus, a human papovavirus.

Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.     

Human monocytes kill M-CSF-expressing glioma cells by BK channel activation

In this study, human monocytes/macrophages were observed to kill human U251 glioma cells expressing membrane macrophage colony-stimulating factor (mM-CSF) via a swelling and vacuolization process called paraptosis. Human monocytes responded to the mM-CSF-transduced U251 glioma cells, but not to viral vector control U251 glioma cells (U251-VV), by producing a respiratory burst within 20 min. Using patch clamp techniques, functional big potassium (BK) channels were observed on the membrane of the U251 glioma cell. It has been previously reported that oxygen indirectly regulates BK channel function. In this study, it was demonstrated that prolonged BK channel activation in response to the respiratory burst induced by monocytes initiates paraptosis in selected glioma cells. Forced BK channel opening within the glioma cells by BK channel activators (phloretin or pimaric acid) induced U251 glioma cell swelling and vacuolization occurred within 30 min. U251 glioma cell cytotoxicity, induced by using BK channel activators, required between 8 and 12 h. Swelling and vacuolization induced by phloretin and pimaric acid was prevented by iberiotoxin, a specific BK channel inhibitor. Confocal fluorescence microscopy demonstrated BK channels co-localized with the endoplasmic reticulum and mitochondria, the two targeted organelles affected in paraptosis. Iberiotoxin prevented monocytes from producing death in mM-CSF-expressing U251glioma cells in a 24 h assay. This study demonstrates a novel mechanism whereby monocytes can induce paraptosis via the disruption of internal potassium ion homeostasis.     

Properties of influenzavirus nucleocapsids in nonpermissive cells.

The properties of fowl plague virus (Influenzavirus A) nucleocapsids isolated from the cytoplasm of infected Ehrlich ascites carcinoma cells and chick embryo cells were compared. Nucleocapsids isolated from both systems possessed similar polypeptides (P and NP) but differed in their biophysical characteristics. Nucleocapsids from ascites cells sedimented in velocity sucrose gradients slower (from 25 to 50 S) and the majority of them banded at higher density in CsCl gradients (rho 1.38 as compared to 1.34 g/ml) than nucleocapsids from chick embryo cells. In the electron microscope they appeared as thin threads 3--4 nm in diameter.     

Occurrence of free, defective viral DNA in a hamster tumor induced by human papovavirus BK

We characterized viral DNA present in an osteosarcoma cell line (Os-513), which had been originally induced in a hamster with human papovavirus BK (BKV), by hybridization of ³²P-labeled BKV DNA to DNA extracted from cells, undigested or digested with various restriction endonucleases, fractionated by electrophoresis, and transferred to a nitrocellulose filter. Os-513 was found to contain free viral DNA and high molecular weight DNA with viral sequences. The free viral DNA seemed to be defective, for its size was about two-thirds of the wild type. The deletion, one-third of the entire BKV DNA, extended over a segment between map positions 0.72 and 0 which includes the leader and intervening sequences and part of the body sequences for late mRNAs. The high molecular weight DNA with viral sequences appeared to be composed of tandem, head-to-tail repeats of the same defective BKV DNA as the free DNA.