Permissive Human Cells Research Articles

Characterization of germline porcine endogenous retroviruses from Large White pig.

Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses. Analysis of the envelope sequences revealed that PERV B demonstrated high similarity to the envelope sequences derived from replication-competent PERV, indicating that lack of replication competence does not appear to be attributable to this region of the provirus. These data complement recent findings that HTRC PERV are recombinants between the PERV A and PERV C subgroups, and that these recombinants are not present in the germline of miniature swine. Together, these results indicate that these individual PERV B proviruses are unlikely to give rise to HTRC PERV.

788. Imaging of Adenoviral Replication with a Fluorescent Reporter in the E3 Region

Top of pageAbstract Adenoviral vectors offer promise for gene therapy of various diseases. One recent development in this area is conditionally replicative adenoviruses (CRAds) which selectively kill cancer cells through tumor-specific replication. To maintain safety and practically apply these agents in a clinical context, a non-invasive detection system of viral replication is required. Current adenovirus detection methods used in virotherapy clinical trials include in situ hybridization, immuno-histochemistry, electron microscopy, and DNA amplification. Although these techniques are useful for examining the presence of adenovirus in a laboratory setting, they are not suitable for monitoring dynamic events and isolated such as viral replication in clinical context. These methods can only provide static data on replication since they rely on invasive biopsies. To address these issues, we hypothesized that expression of a reporter gene from a replicative adenovirus would yield valuable information on viral replication and localization. We chose a strategy to incorporate the EGFP fluorescent protein into the E3 region so that transgene expression under the adenovirus major late promoter would closely mimic the viral replication profile. In addition, the chosen fluorescent reporter protein has potential to be detected noninvasively. We constructed Ad5ΔE3ADPF by deleting proteins 6.7K, gp19K, RIDα (10.4K), RIDβ (14.5K), and a portion of 12.5K from the E3 region while retaining the adenovirus death protein (ADP) since this viral product is required for efficient cell lysis and viral release. This construct has been reported to overexpress ADP and cause enhanced cytopathic effect. Using this construct, we generated six different vectors with EGFP in six different configurations (forward / reverse directions and three reading frames). Vectors with the EGFP cassette in the forward direction (F0, F1, F2: left to right) expressed EGFP 12 hours after infection in A549 (adenovirus permissive human lung carcinoma cell line). On the other hand, no EGFP expression was seen in BNL1NGA.4 (mouse hepatoma cell line) in which human adenovirus can infect but not replicate productively. Of the three frames, F1 showed the clearest correlation of EGFP expression with viral replication. Vectors with reverse direction cassettes (R0, R1, R2: right to left) did not demonstrate any EGFP expression. In the context of cytocidal effect, viruses with ΔE3ADP structure showed same level of cell killing effect as Ad5 wild type in A549 cells. Our data suggest that our novel imaging system may provide a simple and convenient way to monitor viral replication in preclinical and clinical settings. Additionally, deletion of the E3 region while remaining ADP would yield more cloning capacity for transgenes in a replicative adenovirus without hampering cell killing effect. We are currently testing the functionality of our system in several CRAd configurations for noninvasive imaging purposes.

Binding to decay-accelerating factor is not required for infection of human leukocyte cell lines by enterovirus 70.

Enterovirus 70 (EV70) is one of several human enteroviruses that exhibit a propensity for infecting the central nervous system (CNS). The mechanisms by which neurotropic enteroviruses gain access to and invade the CNS are poorly understood. One possibility is that circulating leukocytes become infected and carry neurotropic enteroviruses to the CNS. We examined the ability of EV70 to infect cell lines derived from lymphoid, myeloid, and monocytic lineages. Most leukocyte cell lines tested bound radiolabeled EV70 and were permissive for EV70 replication, suggesting that EV70, in contrast to other enteroviruses, has an in vitro tropism that includes lymphoid, monocytic, and myeloid cell lines. For some of the cell lines, virus binding and infection correlated with surface expression of decay-accelerating factor (DAF), an attachment protein for EV70 on HeLa cells. However, EV70 also adsorbed to and infected cell lines that expressed little or no DAF. In contrast to what was observed for HeLa cells, neither DAF-specific monoclonal antibodies nor phosphatidylinositol-specific phospholipase C treatment inhibited EV70 binding to permissive leukocyte cell lines, and antibody blockade of DAF had little or no effect on EV70 replication. We also found that neither the human coxsackievirus-adenovirus receptor nor intercellular cell adhesion molecule 1, which mediate the entry of coxsackie B viruses and coxsackievirus A21, respectively, functions as a receptor for EV70. EV70 binding to all cell lines was sensitive to sialidase treatment and to inhibition of O glycosylation by benzyl N-acetyl-alpha-D-galactosaminide. Taken together, these results suggest that a sialylated molecule(s) other than DAF serves as a receptor for EV70 on permissive human leukocyte cell lines.

Differential display RT-PCR analysis of ECV304 endothelial-like cells infected with dengue virus type 2 reveals messenger RNA expression profiles of multiple human genes involved in known and novel roles.

Differential display (DD)-RT-PCR was employed to analyze mRNAs from ECV304 human endothelial-like cells undergoing apoptosis following infection with the virulent New Guinea C strain of dengue virus type 2 in order to elucidate the cellular gene responses to dengue viral infection at the transcriptional level. We isolated, sequenced, and identified 203 differentially expressed and overlapping cDNA fragments, all of which were of human origin except 1 that was of viral origin. Out of these, 78 were individual distinct clones comprising 46 and 32 expressed sequence tags (ESTs) that exhibited upregulated and downregulated trends, respectively. Of the 78 differentially expressed mRNAs, 16 did not match any characterized genes or ESTs. The remaining 62 mRNAs modified by dengue virus infection matched known genes, including those encoding components of the cell cycle (Anillin, CDC27), cytoskeleton (epsilon-tubulin), signal transduction (OPHN1, PPP2R2A, TIRAP), protein translation and modification (EIF3S10, IF2, TMEM1), transcriptional regulation (alpha-NAC, C20orf104, EGR1, ELP2), apoptotic cell death (RICK), membrane (BPAG1), and mitochondrial-related proteins. Semiquantitative RT-PCR and real-time RT-PCR authenticated further the altered expression patterns of selected genes of interest. These data demonstrate the feasibility of mRNA DD in providing insights into the complex responses of the transcriptional machinery of permissive and apoptotic human endothelial-like cells in the pathogenesis of dengue and/or its complications induced by the virulent dengue virus type 2.

The human cytomegalovirus major immediate-early enhancer determines the efficiency of immediate-early gene transcription and viral replication in permissive cells at low multiplicity of infection.

To determine the effect of the human cytomegalovirus (CMV) major immediate-early (MIE) enhancer or promoter on the efficiency of viral replication in permissive human cells, we constructed recombinant viruses with their human MIE promoter, enhancer, and promoter plus enhancer replaced with the murine CMV components. After a low multiplicity of infection (MOI) (0.01 PFU/cell), recombinant human CMV with the murine CMV promoter replicated like the wild type but recombinant virus with the murine enhancer replicated less efficiently. Immediate-early (IE) viral protein pIE72 (UL123), early viral protein (UL44), and viral DNA synthesis were significantly decreased. The effect of the human CMV enhancer substitution with the murine CMV enhancer was also demonstrated in different cell types by using recombinant virus with the UL127 promoter, driving the expression of green fluorescent protein (GFP). After an MOI of 1, GFP expression was high with the human CMV enhancer and significantly lower with the murine CMV enhancer. Even though at a high MOI (10 PFU/cell), the murine CMV enhancer was as efficient as the human CMV enhancer for the transcription of IE genes in human foreskin fibroblast cells, at lower MOIs, the murine CMV enhancer was less efficient. Proximal and distal chimeras of the human and murine enhancers also replicated less efficiently at a low MOI and expressed lower levels of GFP from the UL127 promoter. These experiments demonstrate that the entire human CMV enhancer has evolved for the efficient expression of the viral IE and early genes in human cells. Possible functions of the human CMV enhancer and promoter at a low MOI are discussed.

A virus-virus interaction circumvents the virus receptor requirement for infection by pathogenic retroviruses.

During ongoing C-type retrovirus infection, the probability of leukemia caused by insertional gene activation is markedly increased by the emergence of recombinant retroviruses that repeatedly infect host cells. The murine mink cell focus-inducing (MCF) viruses with this property have acquired characteristic changes in the N-terminal domain of their envelope glycoprotein that specify binding to a different receptor than the parental ecotropic virus. In this report, we show that MCF virus infection occurs through binding to this receptor (termed Syg1) and, remarkably, by a second mechanism that does not utilize the Syg1 receptor. By the latter route, the N-terminal domain of the ecotropic virus glycoprotein expressed on the cell surface in a complex with its receptor activates the fusion mechanism of the MCF virus in trans. The rate of MCF virus spread through a population of permissive human cells was increased by establishment of trans activation, indicating that Syg1 receptor-dependent and -independent pathways function in parallel. Also, trans activation shortened the interval between initial infection and onset of cell-cell fusion associated with repeated infection of the same cell. Our findings indicate that pathogenic retrovirus infection may be initiated by virus binding to cell receptors or to the virus envelope glycoprotein of other viruses expressed on the cell surface. Also, they support a broader principle: that cooperative virus-virus interactions, as well as virus-host interactions, shape the composition and properties of the retrovirus quasispecies.

The abortive infection of Syrian hamster cells with human adenovirus type 12.

Human adenovirus type 12 (Ad12) induces undifferentiated tumors in newborn Syrian hamsters, and this tumor model has been investigated in detail in our laboratory. One of the characteristics of the Ad12-hamster cell system is a strictly abortive infection cycle. In this chapter, we summarize previous and more recent results of studies on the interaction of Ad12 with the nonpermissive BHK21 hamster cell line. The block of Ad12 replication lies before viral DNA replication and late gene transcription which cannot be detected with the most sensitive techniques. Ad12 adsorption, cellular uptake and transport of the viral DNA to the nucleus are less efficient in the nonpermissive hamster cells than in permissive human cells. However, most of the early functions of the Ad12 genome are expressed in BHK21 cells, though at a low level. In the downstream region, the first exon, of the major late promoter (MLP) of Ad12 DNA, a mitigator element of 33 nucleotide pairs in length has been identified which contributes to the inactivity of the MLP in hamster cells and its markedly decreased activity in human cells. The E1 functions of Ad2 or Ad5 are capable of partly complementing the Ad12 deficiencies in hamster cells in that Ad12 viral DNA replication and late gene transcription can proceed, e.g. in a BHK hamster cell line, BHK297-C131,which carries in an integrated form and constitutively expresses the E1 region of Ad5 DNA. Nevertheless, the late Ad12 mRNAs, which are synthesized in this system with the authentic nucleotide sequence, fail to be translated to structural viral proteins. Hence, infectious virions are not produced in the partly complementing system. Probably there is also a translational block for late Ad12 mRNAs in hamster cells. We have recently shown that the overexpression of the Ad12 preterminal protein (pTP) gene or of the E1A gene facilitates the synthesis of full-length, authentic Ad12 DNA in BHK21 cells infected with Ad12. Apparently the pTP has a hitherto unknown function in eliciting full cycles of Ad12 DNA replication even in nonpermissive BHK21 cells when sufficient levels of Ad12 pTP are produced. We pursue the possibility that the completely abortive infection cycle of Ad12 in hamster cells ensures the survival of Ad12-induced hamster tumor cells which all carry, integrated in their genomes, multiple copies of Ad12 DNA. In this way, the viral genomes are immortalized and expanded in a huge number of tumor cells.

Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: a DNA microarray analysis.

We have previously reported that the immediate early (IE)-86 protein of human cytomegalovirus (HCMV) pushes the cell cycle toward S phase but inhibits cell division [Murphy, E. A., Streblow, D. N., Nelson, J. A. & Stinski, M. F. (2000) J. Virol. 74, 7108-7118]. We determined the cellular genes activated by the IE86 protein in permissive human fibroblast cells. A 4-fold or greater increase in the steady-state RNA from many cellular genes that regulate the cell cycle, the enzymes for DNA precursor synthesis, and the initiation of cellular DNA replication was detected by high-density DNA microarray analysis. Northern blot analysis confirmed the DNA microarray data. The viral IE86 protein induced a significant increase in the cellular steady-state RNA level from the B-myb, cyclin E, cdk-2, E2F-1, ribonucleotide reductase 1, ribonucleotide reductase 2, thymidylate synthetase, MCM3, and MCM7 genes, but actin RNA was not affected. Cellular genes regulated by the E2F transcription factors were strongly activated by the IE86 protein. In most cases, the cellular genes induced by the IE86 protein were also induced by HCMV infection. This study demonstrates the global array of cellular genes activated by the IE86 protein that pushes progression of the cell cycle from G0/G1 toward the G1/S transition point.

Adeno-associated virus type 2-mediated gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts.

Adeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, nonpathogenic human parvovirus that is currently in use as a vector for human gene therapy. However, the transduction efficiency of AAV vectors in different cell and tissue types varies widely. In addition to the lack of expression of the viral receptor and coreceptors and the rate-limiting viral second-strand DNA synthesis, which have been identified as obstacles to AAV-mediated transduction, we have recently demonstrated that impaired intracellular trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J. Hansen, K. Qing, H. J. Kwon, C. Mah, and A. Srivastava, J. Virol. 74:992-996, 2000). In this report, we document that escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293. While virions were found in both early and late endosomes or lysosomes of infected 293 cells, they were localized predominantly to the early endosomes in NIH 3T3 cells. Moreover, treatment of cells with bafilomycin A1 (Baf), an inhibitor of the vacuolar H(+)-ATPase and therefore of endosomal-lysosomal acidification, decreased the transduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated transduction in general, virions were detected in late endosomes and lysosomes, and these cells became sensitive to Baf-mediated inhibition of transduction. Thus, HU treatment overcomes defective endocytic processing of AAV in murine fibroblasts. These studies provide insights into the underlying mechanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy.

Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes.

Much evidence suggests that the major immediate-early (IE) transactivator of human cytomegalovirus (HCMV), IE-2, is likely to be critical for efficient viral replication; however, the lack of an IE-2 mutant HCMV has precluded an experimental test of this hypothesis. As an initial step toward characterizing an IE-2 mutant, we first cloned the HCMV Towne genome as a bacterial artificial chromosome (BAC) and analyzed the ability of transfected Towne-BAC DNA (T-BACwt) to produce plaques following introduction into permissive human fibroblasts. Like Towne viral DNA, transfected T-BACwt DNA was infectious in permissive cells, and the resulting virus stocks were indistinguishable from Towne virus. We then used homologous recombination in Escherichia coli to delete the majority of UL122, the open reading frame encoding the unique portion of IE-2, from T-BACwt. From this deleted BAC, a third BAC clone in which the deletion was repaired with wild-type UL122 was created. In numerous transfections of permissive human foreskin fibroblast cells with these three BAC DNA clones, the rescued BAC and T-BACwt consistently yielded plaques, while the UL122 mutant BAC never generated plaques, even after 4 weeks. Protein and mRNA of other IE genes were readily detected from transfected UL122 mutant BAC DNA; however, reverse transcription-PCR failed to detect mRNA expression from any of five early genes examined. The generalized failure of this mutant to express early genes is consistent with expectations from in vitro assays which have demonstrated that IE-2 transactivates most HCMV promoters. These experiments provide the first direct demonstration that IE-2 is required for successful HCMV infection and indicate that virus lacking IE-2 arrests early in the replication cycle.

A block to human immunodeficiency virus type 1 assembly in murine cells.

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24(gag) into the culture medium. A small amount of p24(gag) was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.

A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer.

The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18-25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

Cryptosporidium parvum apical complex glycoprotein CSL contains a sporozoite ligand for intestinal epithelial cells.

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, has become a well-recognized diarrheal disease of humans and other mammals throughout the world. No approved parasite-specific drugs, vaccines, or immunotherapies for control of the disease are currently available, although passive immunization with C. parvum-specific antibodies has some efficacy in immunocompromised and neonatal hosts. We previously reported that CSL, an approximately 1,300-kDa conserved apical glycoprotein of C. parvum sporozoites and merozoites, is the antigenic species mechanistically bound by neutralizing monoclonal antibody 3E2 which elicits the circumsporozoite precipitate (CSP)-like reaction and passively protects against C. parvum infection in vivo. These findings indicated that CSL has a functional role in sporozoite infectivity. Here we report that CSL has properties consistent with being a sporozoite ligand for intestinal epithelial cells. For these studies, native CSL was isolated from whole sporozoites by isoelectric focusing (IEF) following observations that the approximately 1,300-kDa region containing CSL as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was comprised of approximately 15 molecular species (pI 3 to 10) when examined by two-dimensional (2-D) electrophoresis and silver staining. A subset of six approximately 1,300-kDa species (pI 4.0 to 6.5) was specifically recognized by 3E2 in 2-D Western immunoblots of IEF-isolated CSL. Isolated native CSL bound specifically and with high affinity to permissive human intestinal epithelial Caco-2 cells in a dose-dependent, saturable, and self-displaceable manner. Further, CSL specifically bound to the surface of live Caco-2 cells inhibited sporozoite attachment and invasion. In addition, sporozoites having released CSL after incubation with 3E2 and occurrence of the CSP-like reaction did not attach to and invade Caco-2 cells. These findings indicate that CSL contains a sporozoite ligand which facilitates attachment to and invasion of Caco-2 cells and, further, that ligand function may be disrupted by CSL-reactive monoclonal antibody. We conclude that CSL is a rational target for passive or active immunization against cryptosporidiosis.

A cis repression sequence adjacent to the transcription start site of the human cytomegalovirus US3 gene is required to down regulate gene expression at early and late times after infection.

Human cytomegalovirus has two enhancer-containing immediate-early (IE) promoters with a cis repression sequence (CRS) positioned immediately upstream of the transcription start site, designated the major IE (MIE) promoter and the US3 promoter. The role of the CRS upstream of the US3 transcription start site in the context of the viral genome was determined by comparing the levels of transcription from these two enhancer-containing promoters in recombinant viruses with a wild-type or mutant CRS. Upstream of the CRS of the US3 promoter was either the endogenous enhancer (R2) or silencer (R1). The downstream US3 gene was replaced with the indicator gene chloramphenicol acetyltransferase (CAT). Infected permissive human fibroblast cells or nonpermissive, undifferentiated monocytic THP-1 cells were analyzed for expression from the US3 promoter containing either the wild-type or mutant CRS. With the wild-type CRS, the maximum level of transcription in permissive cells was detected within 4 to 6 h after infection and then declined. With the mutant CRS and the R2 enhancer upstream, expression from the US3 promoter continued to increase throughout the viral replication cycle to levels 20- to 40-fold higher than for the wild type. In nonpermissive or permissive monocytic THP-1 cells, expression from the US3 promoter was also significantly higher when the CRS was mutated. Less expression was obtained when only the R1 element was present, but expression was higher when the CRS was mutated. Thus, the CRS in the enhancer-containing US3 promoter appears to allow for a short burst of US3 gene expression followed by repression at early and late times after infection. Overexpression of US3 may be detrimental to viral replication, and its level of expression must be stringently controlled. The role of the CRS and the viral IE86 protein in controlling enhancer-containing promoters is discussed.

The Acidic Domain of pUL37x1 and gpUL37 Plays a Key Role in Transactivation of HCMV DNA Replication Gene Promoter Constructions

Transient complementation of human cytomegalovirus (HCMV)oriLyt DNA replication in permissive human diploid cells expressing replication genes under native promoters requires its UL36-38 gene products. Two of the immediate early (IE) proteins encoded by this locus, pUL37x1 and, to a lesser extent, gpUL37, activated expression of HCMV early gene promoter constructions. The other IE protein encoded by the UL36-38 locus, pUL36, and the early product, pUL38, did not transactivate the HCMV early promoter constructions under similar conditions. The acidic domain, common to both pUL37x1 and gpUL37, is required for activation of HCMV early promoter constructions. Conversely, gpUL37 sequences downstream of amino acid 199 are not required for transactivation of viral early promoters. Taken together, these results suggest that the requirement for UL36-38 products for HCMV DNA replication results, at least in part, from the requirement of the transactivation of HCMV early DNA replication promoters by pUL37x1 and, to a lesser extent, by gpUL37 and that the acidic domain is critical for this activity.

Propagation of the Human Polyomavirus, JCV, in Human Neuroblastoma Cell Lines

Susceptibility to infection by the human polyomavirus, JCV, is determined by intracellular mechanisms which control transcription and replication. Originally thought to propagate well only in human cells of oligodendroglial lineage, JCV has recently been shown to infect astrocytes, astrogliomas, and a neuroblastoma cell line. The data reported here describe two cell types that have been subcultured from a human neuroblastoma cell line, SK-N-SH. The SH-SY5Y subclone displays neuronal phenotypes and is not susceptible to JCV infection, while the SH-EP subclone displays glial cell phenotypes and is susceptible to infection. Binding of nuclear proteins from the permissive SH-EP cells to the nuclear factor-1 (NF-1) site in the JCV regulatory DNA sequences results in a gel shift pattern that is different from the nonpermissive SH-SY5Y cell proteins. Northern analysis of mRNA for the four classes of NF-1 proteins showed a predominance of the NF-1/X class in SH-EP cells similar to the highly permissive human fetal glial cells. Very low levels of mRNA for NF-1/X were seen in the nonpermissive SH-SY5Y cells, similar to that seen for the nonpermissive HeLa cells. Several other cell lines tested that were permissive for JCV infection also showed synthesis of the NF-1/X class of proteins. SH-EP cells represent a cell line in a glial cell lineage which is susceptible to JCV multiplication. These cells may be a useful cell culture system for the investigation of DNA binding factors which correlates with viral susceptibility.

Insufficient levels of NFIII and its low affinity for the origin of adenovirus type 12 (Ad12) DNA replication contribute to the abortive infection of BHK21 hamster cells by Ad12.

Human adenovirus type 12 (Ad12) induces undifferentiated sarcomas in neonate Syrian hamsters and hence presents a suitable model for studies of the molecular mechanism of viral oncogenesis. Since we submit that an understanding of the early steps in the interaction between Ad12 and hamster cells might shed light on the initiation of malignant transformation, the abortive infection of BHK21 hamster cells with Ad12 has been investigated in detail. Ad12 replication in these cells is blocked in early stages, while Ad2 can replicate to moderate titers. Early Ad12 genes are expressed in BHK21 hamster cells, but there is a total block in Ad12 DNA replication and late gene transcription. The Ad5-transformed hamster cell line BHK297-C131, with the left terminus of Ad5 DNA chromosomally integrated and constitutively expressed, allows limited levels of Ad12 DNA replication and late transcription, probably through Ad5 E1 functions, but not the translation of late Ad12 gene products. We have now investigated the capacities of binding of nuclear proteins NFI and NFIII from permissive human KB cells, nonpermissive hamster BHK21 cells, and complementing BHK297-C131 cells to the origin of replication (ori) of Ad2 or Ad12 DNA. The electrophoretic mobility shift assay has been used to assess these binding reactions. The data support the notions that NFIII of BHK21 cells has a lower affinity for the ori of Ad12 DNA than for the ori of Ad2 DNA and that the levels of NFIII in BHK21 cells are markedly reduced compared with the levels in the permissive human KB cells or the complementing BHK297-C131 hamster cells. These deficiencies are contributing factors for the abortive infection of BHK21 hamster cells with Ad12. The lack of sufficient levels of NFIII in BHK21 cells is also consistent with the decreased replication capacity of Ad2 in hamster compared with human cell lines.

Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter

We have previously shown that the human cytomegalovirus early UL4 promoter has upstream negative and positive cis-acting regulatory elements. In the absence of the upstream negative regulatory region, the positive element confers strong transcriptional activity. The positive element contains a CCAAT box dyad symmetry and binds the cellular transcription factor NF-Y. The effect of the negative regulatory element is negated by the viral IE2 protein (L. Huang, C.L. Malone, and M.F. Stinski, J. Virol. 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis-acting negative regulatory element was located between -168 and -134 bp relative to the transcription start site. This element could be transferred to a heterologous promoter, and it functioned in either orientation. Mutational analysis demonstrated that a core DNA sequence in the cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3', was required for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in both nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to the upstream cis-acting negative regulatory element correlates with repression of transcription from the early UL4 promoter. Binding of the viral IE2 protein correlates with negation of the repressive effect.

Characterization of a 100-kilodalton binding protein for the six serotypes of coxsackie B viruses.

Viral infection of host cells primarily depends on binding of the virus to a specific cell surface protein. In order to characterize the binding protein for group B coxsackieviruses (CVB), detergent-solubilized membrane proteins of different cell lines were tested in virus overlay protein-binding assays. A prominent virus-binding protein with a molecular mass of 100 kDa was detected in various CVB-permissive human and monkey cell lines but was not detected in nonpermissive cell lines. The specificity of CVB binding to the 100-kDa protein on permissive human cells was substantiated by binding of all six serotypes of CVB and by competition experiments. In contrast, poliovirus and Sendai virus did not bind to the 100-kDa CVB-specific protein. A fraction of HeLa membrane proteins enriched in the range of 100 kDa showed functional activity by transforming infectious CVB (160S) into A-particles (135S). In order to purify this CVB-binding protein, solubilized membrane proteins from HeLa cells were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by elution of the 100-kDa protein. Amino acid sequence analysis of tryptic fragments of the CVB-binding protein indicated that this 100-kDa CVB-specific protein is a cell surface protein related to nucleolin. These results were confirmed by immunoprecipitations of the CVB-binding protein with nucleolin-specific antibodies, suggesting that a nucleolin-related membrane protein acts as a specific binding protein for the six serotypes of CVB.

Identification of three new JC virus proteins generated by alternative splicing of the early viral mRNA.

The genome of the human polyomavirus JC Virus (JCV) encodes two regulatory proteins, large and small T antigen which are expressed early in a lytic infection, and three structural proteins, VP1, VP2, VP3, which are produced late in an infection. A fourth late protein, agnoprotein, may contribute to the assembly of the virion. In this study, we demonstrate the presence of three additional early proteins, T'135, T'136, and T'165, which are expressed in lytically-infected cells; T'135 is also readily detected in JCV transformants. The three species of T' are phosphoproteins generated via an alternative splicing mechanism. This mechanism involves the excision of a second intron from the large T mRNA using a common donor splice site at JCV nucleotide 4274 and three unique acceptor splice sites at nucleotides 2918, 2777 and 2704 for T'135, T'136 and T'165, respectively. The mutant JCV delta T' was created by converting the G at nucleotide 4274 to an A, thereby disrupting the consensus sequence of the shared donor splice site without altering the amino acid sequence of any early JCV protein. Upon transfection of permissive human brain cells, JCV delta T' replicated its DNA 10-fold less efficiently than did wild type JCV. Passage of extracts of the infected cells on to fresh human brain cells revealed that the expression of T antigen was greatly reduced and the presence of T' proteins was undetectable in the mutant versus wild type JCV-infected cells.