Oligomerization of Uukuniemi virus nucleocapsid protein.

BACKGROUND: Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail. RESULTS: A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact alpha-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s). This structure is formed by two alpha-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31) or long aliphatic (I14, I24) side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A) affected the N protein functionality both in mammalian two-hybrid and minigenome assays. CONCLUSIONS: UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N-terminal region of the UUKV-N protein is essential for the N-N interaction.

Entry of bunyaviruses into mammalian cells.

The Bunyaviridae constitute a large family of enveloped animal viruses, many members of which cause serious diseases. However, early bunyavirus-host cell interactions and entry mechanisms remain largely uncharacterized. Investigating Uukuniemi virus, a bunyavirus of the genus Phlebovirus, we found that virus attachment to the cell surface was specific but inefficient, with 25% of bound viruses being endocytosed within 10 min, mainly via noncoated vesicles. The viruses entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomes. Acid-activated penetration, occurring 20-40 min after internalization, required maturation of early to late endosomes. The pH threshold for viral membrane fusion was 5.4, and entry was sensitive to temperatures below 25 degrees C. Together, our results indicate that Uukuniemi virus penetrates host cells by acid-activated membrane fusion from late endosomal compartments. This study also highlights the importance of the degradative branch of the endocytic pathway in facilitating entry of late-penetrating viruses.

A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-beta mediated epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-beta (TGF-beta) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-beta-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.

The coxsackie and adenovirus receptor (CAR) is required for renal epithelial differentiation within the zebrafish pronephros.

The coxsackie and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily and a component of vertebrate tight junctions. CAR protein is widely expressed in fish and mammals in organs of epithelial origin suggesting possible functions in epithelial biology. In order to gain insight into its function, we knocked the CAR gene down in zebrafish using antisense morpholinos. We identified a requirement for CAR in the terminal differentiation of glomerular podocytes and pronephric tubular epithelia. Podocytes differentiate in CAR morphants but are not able to elaborate a regularly patterned architecture of foot processes. In the tubules, CAR was required for the apposition of plasma membranes from adjacent epithelial cells but did not appear to be necessary for the formation of tight junctions. Additionally, tubular epithelia lacking CAR were not able to elaborate apical brush border microvilli. These results establish a requirement for CAR in the terminal differentiation of renal glomerular and tubular cell types.

The cytoplasmic tails of Uukuniemi Virus (Bunyaviridae) G(N) and G(C) glycoproteins are important for intracellular targeting and the budding of virus-like particles.

Functional motifs within the cytoplasmic tails of the two glycoproteins G(N) and G(C) of Uukuniemi virus (UUK) (Bunyaviridae family) were identified with the help of our recently developed virus-like particle (VLP) system for UUK virus (A. K. Overby, V. Popov, E. P. Neve, and R. F. Pettersson, J. Virol. 80:10428-10435, 2006). We previously reported that information necessary for the packaging of ribonucleoproteins into VLPs is located within the G(N) cytoplasmic tail (A. K. Overby, R. F. Pettersson, and E. P. Neve, J. Virol. 81:3198-3205, 2007). The G(N) glycoprotein cytoplasmic tail specifically interacts with the ribonucleoproteins and is critical for genome packaging. In addition, two other regions in the G(N) cytoplasmic tail, encompassing residues 21 to 25 and 46 to 50, were shown to be important for particle generation and release. By the introduction of point mutations within these two regions, we demonstrate that leucines at positions 23 and 24 are crucial for the initiation of VLP budding, while leucine 46, glutamate 47, and leucine 50 are important for efficient exit from the endoplasmic reticulum and subsequent transport to the Golgi complex. We found that budding and particle generation are highly dependent on the intracellular localization of both glycoproteins. The short cytoplasmic tail of UUK G(C) contains a lysine at position -3 from the C terminus that is highly conserved among members of the Phlebovirus, Hantavirus, and Orthobunyavirus genera. Mutating this single amino acid residue in G(C) resulted in the mislocalization of not only G(C) but also G(N) to the plasma membrane, and VLP generation was compromised in cells expressing this mutant. Together, these results demonstrate that the cytoplasmic tails of both G(N) and G(C) contain specific information necessary for efficient virus particle generation.

The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging.

We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein G(N) for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the G(N)/G(C) glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein G(N) cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.

Generation and analysis of infectious virus-like particles of uukuniemi virus (bunyaviridae): a useful system for studying bunyaviral packaging and budding.

In the present report we describe an infectious virus-like particle (VLP) system for the Uukuniemi (UUK) virus, a member of the Bunyaviridae family. It utilizes our recently developed reverse genetic system based on the RNA polymerase I minigenome system for UUK virus used to study replication, encapsidation, and transcription by monitoring reporter gene expression. Here, we have added the glycoprotein precursor expression plasmid together with the minigenome, nucleoprotein, and polymerase to generate VLPs, which incorporate the minigenome and are released into the supernatant. The particles are able to infect new cells, and reporter gene expression can be monitored if the trans-acting viral proteins (RNA polymerase and nucleoprotein) are also expressed in these cells. No minigenome transfer occurred in the absence of glycoproteins, demonstrating that the glycoproteins are absolutely required for the generation of infectious particles. Moreover, expression of glycoproteins alone was sufficient to produce and release VLPs. We show that the ribonucleoproteins (RNPs) are incorporated into VLPs but are not required for the generation of particles. Morphological analysis of the particles by electron microscopy revealed that VLPs, either with or without minigenomes, display a surface morphology indistinguishable from that of the authentic UUK virus and that they bud into Golgi vesicles in the same way as UUK virus does. This infectious VLP system will be very useful for studying the bunyaviral structural components required for budding and packaging of RNPs and receptor binding and may also be useful for the development of new vaccines for the human pathogens from this family.

The coxsackie- and adenovirus receptor (CAR) is an in vivo marker for epithelial tight junctions, with a potential role in regulating permeability and tissue homeostasis.

The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.

Coxsackievirus and adenovirus receptor (CAR) is expressed in male germ cells and forms a complex with the differentiation factor JAM-C in mouse testis.

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.

Oligomerization and interacellular localization of the glycoprotein receptor ERGIC-53 is independent of disulfide bonds.

ERGIC-53 is a type I transmembrane lectin facilitating the efficient export of a subset of secretory glycoproteins from the endoplasmic reticulum. Previous results have shown that ERGIC-53 is present as reduction-sensitive homo-oligomers, i.e. as a balanced mixture of disulfide-linked hexamers and dimers, with the two cysteine residues located close to the transmembrane domain playing a crucial role in oligomerization. Here, we demonstrate, using sucrose gradient sedimentation, cross-linking analyses, and non-denaturing gel electrophoresis, that ERGIC-53 is present exclusively as a hexameric complex in cells. However, the hexamers exist in two forms, one as a disulfide-linked, Triton X-100, perfluoro-octanic acid, and SDS-resistant complex, and the other as a non-covalent, Triton X-100, perfluoro-octanoic acid-resistant, but SDS-sensitive, complex made up of three disulfide-linked dimers that are likely to interact through the coiled-coil domains present in the luminal part of the protein. In contrast to what was previously believed, neither of the membrane-proximal cysteine residues plays an essential role in the formation, or maintenance, of the latter form of hexamers. Subcellular fractionation revealed that the double-cysteine mutant was present in the endoplasmic reticulum-Golgi-intermediate compartment, indicating that the two cysteine residues are not essential for the intracellular distribution of ERGIC-53. Based on these results, we present a model for the formation of the two hexameric forms.

The cell surface protein coxsackie- and adenovirus receptor (CAR) directly associates with the Ligand-of-Numb Protein-X2 (LNX2).

The coxsackievirus and adenovirus receptor (CAR) is a cell surface protein that is proposed to be involved in cell-cell adhesion. Based on a yeast two-hybrid screen, co-immunoprecipitation and binding experiments, the intracellular tail of CAR was found to interact both in vivo and in vitro with the Ligand-of-Numb Protein-X2 (LNX2). The interacting domains between the two proteins were identified by truncation analyses and affinity chromatography. CAR and LNX2 protein expression in embryonic mouse tissues was analyzed by immunohistochemistry. The results suggest that CAR is a partner in a protein complex organized at specific subcellular sites by LNX2.

Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments.

The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5' and 3' ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.

Cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells.

Endothelial cells have the ability to change their complement of cell surface proteins in response to inflammatory cytokines. We hypothesized that the expression of the coxsackievirus-adenovirus receptor (CAR), a viral receptor and putative cell-cell adhesion molecule, may be altered during the response of endothelial cells to inflammation. To test this hypothesis, we evaluated CAR protein and mRNA levels in human umbilical vein endothelial cells after they were exposed to tumor necrosis factor alpha, gamma interferon, or a combination of the two cytokines. Flow cytometric and Western blot analyses indicated that cytokine treatment led to a synergistic decrease in CAR protein expression. A Western blot analysis showed that CAR levels decreased to 16% +/- 4% or 1% +/- 4% of the CAR protein levels in untreated cells with either 24 or 48 h of cytokine treatment, respectively. Quantitative reverse transcription-PCR demonstrated that the combination treatment caused CAR mRNA levels to decrease to 21% +/- 12% or 5% +/- 3% of the levels in untreated cells after a 24- or 48-h cytokine treatment, respectively. Reduced CAR expression led to a decrease in adenovirus (Ad) binding of 80% +/- 3% (compared with untreated endothelial cells), with a subsequent decrease in Ad-mediated gene transfer that was dependent on the dose and duration of cytokine treatment but not on the dose of Ad. A similar decrease in CAR protein level and susceptibility to Ad infection was observed in human microvascular endothelial cells, while CAR expression on normal human bronchial epithelial cells or A549 lung epithelial cells was less affected by cytokine treatments. Taken together, the data demonstrate that inflammatory cytokines decrease CAR mRNA and protein expression with a concomitant decrease in Ad binding, reflecting the impact of cell physiology on the function of CAR and the potential effect of inflammation on the ability of Ad to transfer genes to endothelial cells.

CLMP, a novel member of the CTX family and a new component of epithelial tight junctions.

The CTX family is a growing group of type I transmembrane proteins within the immunoglobulin superfamily (IgSF). They localize to junctional complexes between endothelial and epithelial cells and seem to participate in cell-cell adhesion and transmigration of leukocytes. Here, we report the identification of a new member of the CTX family. This protein, which was designated CLMP (coxsackie- and adenovirus receptor-like membrane protein), is composed of 373 amino acids including an extracellular part containing a V- and a C2-type domain, a transmembrane region and a cytoplasmic tail. CLMP mRNA was detected in a variety of both human and mouse tissues and cell lines. The protein migrated with an Mr of around 48 on SDS-PAGE and was predominantly expressed in epithelial cells within different tissues. In cultured epithelial cells, CLMP was detected in areas of cell-cell contacts. When exogenously expressed in polarized MDCK cells, CLMP was restricted to the subapical area of the lateral cell surface, where it co-localized with the tight junction markers ZO-1 and occludin. Also endogenous CLMP showed association with tight junctions, as analyzed in polarized human CACO-2 cells. This suggested a role for CLMP in cell-cell adhesion and indeed, overexpressed CLMP induced aggregation of non-polarized CHO cells. Furthermore, CLMP-expressing MDCK cells showed significantly increased transepithelial resistance, indicating a role for CLMP in junctional barrier function. Thus, we conclude that CLMP is a novel cell-cell adhesion molecule and a new component of epithelial tight junctions. We also suggest, based on phylogenetic studies, that CLMP, CAR, ESAM, and BT-IgSF form a new group of proteins within the CTX family.

The crystal structure of the carbohydrate-recognition domain of the glycoprotein sorting receptor p58/ERGIC-53 reveals an unpredicted metal-binding site and conformational changes associated with calcium ion binding.

p58/ERGIC-53 is a calcium-dependent animal lectin that acts as a cargo receptor, binding to a set of glycoproteins in the endoplasmic reticulum (ER) and transporting them to the Golgi complex. It is similar in structure to calcium-dependent leguminous lectins. We have determined the structure of the carbohydrate-recognition domain of p58/ERGIC-53 in its calcium-bound form. The structure reveals localized but large conformational changes in relation to the previously determined metal ion-free structure, mapping mostly to the ligand-binding site. It reveals the presence of two calcium ion-binding sites located 6A apart, one of which has no equivalent in the plant lectins. The second metal ion-binding site present in that class of lectins, binding Mn(2+), is absent from p58/ERGIC-53. The absence of a short loop in the ligand-binding site in this protein suggests that it has adapted to optimally bind the high-mannose Man(8)(GlcNAc)(2) glycan common to glycoproteins at the ER exit stage.

VIPL, a VIP36-like membrane protein with a putative function in the export of glycoproteins from the endoplasmic reticulum.

Subsets of glycoproteins are thought to require lectin-like membrane receptors for efficient export out of the endoplasmic reticulum (ER). To identify new members related to two previously characterized intracellular lectins ERGIC-53/p58 and VIP36, we carried out an extensive database search using the conserved carbohydrate recognition domain (CRD) as a search string. A gene, more closely related to VIP36 than to ERGIC-53/p58, and hence called VIPL (VIP36-Like), was identified. VIPL has been conserved through evolution from zebra fish to man. The 2.4-kb VIPL mRNA was widely expressed to varying levels in different tissues. Using an antiserum prepared against the CRD, the 32-kDa VIPL protein was detected in various cell lines. The single N-linked glycan of VIPL remained endoglycosidase H-sensitive during a 2-h pulse-chase, even when the protein was overexpressed or mutated to allow export to the plasma membrane. VIPL localized primarily to the ER and partly to the Golgi complex. Like VIP36, the cytoplasmic tail of VIPL terminates in the sequence KRFY, a motif characteristic for proteins recycling between the ER and ERGIC/cis-Golgi. Mutating the retrograde transport signal KR to AA resulted in transport of VIPL to the cell surface. Finally, knock-down of VIPL mRNA using siRNA significantly slowed down the secretion of two glycoproteins (M(R) 35 and 250 kDa) to the medium, suggesting that VIPL may also function as an ER export receptor.

Rescue of Hantaan virus minigenomes.

Hantavirus infections are a major public health concern worldwide. Their widespread geographical distribution and their ability to produce serious, often fatal, human disease underline the need for a system that allows manipulation of these viruses. We describe here the first successful establishment of a reverse genetics technology for Hantaan virus, the prototype of the genus Hantavirus. The system offers a unique opportunity to study the biology of hantaviruses, the pathogenesis of the diseases, and the efficacy of antiviral and prophylactic measures against hantavirus infections.

Apoptosis-related fragmentation, translocation, and properties of human prothymosin alpha.

Human prothymosin alpha is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. We show here that caspase-3 is the principal executor of prothymosin alpha fragmentation in vivo. In apoptotic HeLa cells as well as in vitro, caspase-3 cleaves prothymosin alpha at one major carboxy terminal (DDVD(99)) and several suboptimal sites. Prothymosin alpha cleavage at two amino-terminal sites (AAVD(6) and NGRD(31)) contributes significantly to the final pattern of prothymosin alpha fragmentation in vitro and could be detected to occur in apoptotic cells. The major caspase cleavage at D(99) disrupts the nuclear localization signal of prothymosin alpha, which leads to a profound alteration in subcellular localization of the truncated protein. By using a set of anti-prothymosin alpha monoclonal antibodies, we were able to observe nuclear escape and cell surface exposure of endogenous prothymosin alpha in apoptotic, but not in normal, cells. We demonstrate also that ectopic production of human prothymosin alpha and its mutants with nuclear or nuclear-cytoplasmic localization confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis.

Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4A/pRb.

Recent studies show that stable expression of the human telomerase catalytic subunit, hTERT, alone can lead several types of normal human somatic cells to bypass replicative senescence and become immortal. The molecular mechanisms by which telomerase immortalizes cells are not fully understood, although a key function of telomerase is to maintain a critical length of telomeres in order to preserve the stability and integrity of the genome. Here we report that stable transfection of hTERT alone was sufficient to allow bovine capillary endothelial (BCE) cells to bypass senescence and acquire immortality. Surprisingly, telomere lengths in these stable transfectants were progressively shortened during an increasing number of population doublings (PDLs), despite high telomerase activity. The expression of the cyclin-dependent kinase inhibitors (CDKIs) p16INK4A and p21CIP1/WAF1 was concomitantly repressed, and the retinoblastoma protein (pRb) was maintained in a hyperphosphorylated state in the telomerase-expressing cells. Re-expression of p16INK4A in these cells by either treatment with a demethylating agent or by adenovirus-mediated expression reinduced a senescence-like phenotype, suggesting that the inactivation of p16INK4A was due to DNA methylation and was crucial for the immortalization process. In agreement with this finding, the expression levels of the prototypic DNA methyltransferase DNMT1 were elevated in the hTERT-positive cells.

Expression of the coxsackie and adenovirus receptor in human astrocytic tumors and xenografts.

The sensitivity of human tissues and tumors to infection with type C adenoviruses correlates with the expression of the human coxsackie B- and adenovirus receptor, hCAR. HCAR is heterogeneously expressed in various tissues and types of human cancer cells, which has implications for the use of adenoviruses as vectors in cancer gene therapy. Using immunoblotting, real-time PCR, FACS-analysis and sensitivity to infection with adenovirus-lacZ, we analyzed the expression level of hCAR in glioma Grade IV cell lines. With real-time PCR, we also analyzed hCAR expression in primary human astrocytomas of different malignancy grades, as well as in their xenograft derivatives. Analysis of a set of 10 cell lines showed great variation in hCAR expression. Susceptibility to Ad5lacZ correlated well with hCAR expression, whereas no correlation was observed with the expression of alphavbeta3/alphavbeta5 integrins, proposed to function as co-receptors for adenoviruses. A great variation of CAR expression was also observed in primary astrocytomas of different malignancy grades. The mean value of CAR expression was significantly lower in 22 Grade IV tumors as compared to the values for 6 Grade II (p = 0.01) and 6 Grade III (p = 0.01) tumors. When the hCAR expression in 11 xenografts derived from Grade IV gliomas were compared to the levels detected in the original parental tumors, a mean 12-fold higher expression was seen in the xenografts (P = 0.01). Two xenografts with low hCAR expression grew considerably faster than the hCAR-expressing cells. Our results have relevance for the use of adenoviruses in gene therapy against astrocytomas.