Functional effects of coxsackievirus and adenovirus receptor glycosylation on homophilic adhesion and adenoviral infection

The coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and homophilic adhesion protein. The extracellular portion of CAR consists of two immunoglobulin (Ig)-like domains, each with a consensus sequence for N-glycosylation. We used chemical, genetic, and biochemical studies to show that both sites are glycosylated and contribute to the function of CAR. Although the glycosylation of CAR does not alter cell surface levels or junctional localization, it affects both adhesion and adenovirus infection in unique ways. CAR-mediated adhesion appears to require at least one site of glycosylation since cells expressing CAR without glycosylation do not cluster with each other. In contrast, glycosylation of the Ig-like domain proximal to the membrane is key to the cooperative behavior of adenovirus binding and infection. Contrary to the hypothesis that cooperativity improves viral infection, our data show that although glycosylation of the D2 domain is required for adenovirus cooperative binding, it has a negative consequence upon infection. This is the first report dissecting the adhesion and receptor activities of CAR, revealing that factors other than the binding interface play a significant role in the function of CAR. These data have important implications for both cancers with altered glycosylation states and cancer treatments using oncolytic adenovirus.     

Differential expression of the coxsackievirus and adenovirus receptor regulates adenovirus infection of the placenta

The molecular mechanisms and pathologic significance of placental viral infections are poorly understood. We investigated factors that regulate placental infection by adenovirus, which is the most common viral pathogen identified in fetal samples from abnormal pregnancies (i.e., fetal growth restriction, oligohydramnios, and nonimmune fetal hydrops). We also determined the pathologic significance of placental adenovirus infection. Northern hybridization, flow cytometry, and immunostaining revealed that placental expression of the coxsackievirus and adenovirus receptor (CAR) varied with gestational age and trophoblast phenotype. The CAR was continuously expressed in invasive or extravillous trophoblast cells but not in villous trophoblast cells. We postulate that the villous syncytiotrophoblast, which does not express CAR and is resistant to adenovirus infection, limits the transplacental transmission of viral pathogens, including adenovirus. Conversely, extravillous trophoblast cells underwent apoptosis when infected by adenovirus in the presence of decidual lymphocytes (which simulated the maternal immune response to viral infection). Thus, adenovirus infection and/or the maternal immune response to adenovirus infection induced the death of placental cell types that expressed CAR. Consequently, we speculate that adenovirus infection of extra-villous trophoblast cells may negatively impact the process of placental invasion and predispose the mother and fetus to adverse reproductive outcomes that result from placental dysfunction.     

A human cell line selected for resistance to adenovirus infection has reduced levels of the virus receptor.

To investigate determinants of host cell susceptibility to infection, cells partially resistant to infection were selected from the rare cells which remained adherent after infection of a culture of A549 cells with Ad2RAE, a mutant of adenovirus type 2 whose vertex capsomers lack an Arg-Gly-Asp (RGD) sequence which mediates binding of wild-type virus to integrins. Integrins promote the internalization of attached virions, whereas adsorption itself results from binding of the viral fibers to an unidentified cellular receptor. Following three rounds of selection, a persistently infected culture was established in which virus replication was detected in approximately 5% of the cells. Uninfected cells were readily cloned from the culture, indicating that at any particular time the majority of cells in the culture were uninfected. The resistance of one clone of uninfected cells to infection was correlated with a 10-fold reduction in the concentration of fiber receptors on these cells compared with the parental A549 cell line, indicating that efficiency of virus adsorption depends on the receptor concentration. Surprisingly, the rate at which host cells internalized RGD-negative virus also was strongly dependent on the fiber receptor concentration. While internalization of wild-type virus is promoted by the binding of integrins to the penton base RGD sequence, these results suggest that virus also can enter cells by an alternate pathway which requires binding of virions to multiple fiber receptors.     

Differentiation of an adult neuron cell line increases susceptibility to rabies infection

A wide variety of in vitro models have been used for studying rabies infection, however, currently, no central nervous system (CNS) adult neuron cultures are available. The current study determined the susceptibility to rabies infection in an adult CNS neuron cell line (CAD-R1). Cultures of CAD-R1 cells were held for 5 days in medium containing serum (undifferentiated CAD-R1 cells) or in serum-free medium (differentiated CAD-R1 cells). They were then infected with highly neurotropic rabies virus (RV) strain (CVS), obtained from fibroblastic cells (CVS-BHK) or from adult mouse brain (CVS-MB). Undifferentiated and differentiated cells were infected with the two RV strains, but the percentage of infected cells in differentiated cultures was significantly greater (83% and 79%, respectively) than in undifferentiated cells (51% and 60%) (Student's t test<0.05). Susceptibility to infection apparently depended on cellular differentiation state, possibly due to acquisition of additional morphological and biochemical characteristics during the differentiation process that made them more susceptible to RV infection. Therefore, CAD R1 cells may represent a good model for RV infection, making them a useful tool for studying RV neurotropism, infection pathogeny, isolation of street virus or producing safer and most potent vaccines.     

Proteasome inhibitor MG-132 modifies coxsackie and adenovirus receptor expression in colon cancer cell line lovo

The efficacy of adenovirus vector-based cancer gene therapy is controversial. Its uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Low transduction is believed to be one of the main barriers as the expression of CAR on tumor cells is frequently reduced. Increasing CAR expression on tumor cells thus offers a promising opportunity for more effective adenovirus based treatment. Expression of CAR in 62 cases of colon tumor specimens were examined with immunohistochemistry. To modify the CAR expression, the effects of proteasome inhibitor MG132 on CAR expression of colon cancer cell lines were determined by flow cytometry, RT-PCR, and western blot. To evaluate adenovirus transfer, we further used rAd.EGFP, rAd.p53, and oncolytic adenovirus to infect target cells. The CAR expression was significantly decreased in colon carcinomas, both in primary tumors and lymphonode metastasis. Though the deregulation of CAR occurred in early disease and showed no relationship with TNM stage, when primary tumors are more than 5 cm in diameter, this deregulation becomes more frequent. More importantly, proteasome inhibitor MG-132 could enhance CAR expression in colon carcinoma cell line lovo, accompanied with enhanced adenovirus transfer, target gene expression, and oncolysis. These data provide a rational basis for evaluation of CAR expression in tumors and pretreatment with CAR conditioner prior to adenovirus vector-based gene therapy.     

Fatty acid modification of the coxsackievirus and adenovirus receptor

Membrane-proximal cysteines 259 and 260 in the cytoplasmic tail of the coxsackievirus and adenovirus receptor (CAR) are known to be essential for the tumor suppression activity of CAR. We demonstrate that these residues provide an S-acylation motif for modification of CAR with the fatty acid palmitate. Substitution of alanine for cysteines 259 and 260 results in the additional localization of CAR in perinuclear compartments with no effect on the efficiency of adenovirus infection. The results indicate that palmitylation is important for stable plasma membrane expression and biological activity of CAR but is not critical for adenovirus receptor performance.     

Genetic susceptibility to infection with human papillomavirus and development of cervical cancer in women in Brazil

Human papillomavirus (HPV) is considered to be a necessary but not sufficient cause for cervical cancer and, therefore, other factors contribute to the carcinogenic process. A hereditary component for this neoplasia has been reported and several studies indicate that genetic background of the host is important for cervical cancer susceptibility. Among genetic factors that could participate in the susceptibility to this tumor and disease outcome, polymorphic genes of the major histocompatibility complex (MHC), as well as a particular polymorphism in the p53 gene have been intensely investigated. From our analysis of 613 samples in Brazil, we found evidence to indicate that different polymorphic human leukocyte antigen (HLA) genes are involved in the clearance and maintenance of HPV infection. In addition, the homozygous codon 72 p53-Arg gene allele is associated with susceptibility to HPV-associated cervical carcinogenesis. However, supportive and opposing data have been reported in different populations. Therefore, international collaborative studies need to be conducted to define the consistency of the associations described.     

Solution structure of the coxsackievirus and adenovirus receptor domain 2

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR-D1 and CAR-D2. In the present work, the solution structure of CAR-D2, consisting of residues 142-235 of human CAR, has been determined by NMR spectroscopy. CAR-D2 is shown to be a beta-sandwich motif comprised of two beta-sheets, which are stabilized by two disulfide bonds. The first beta-sheet is comprised of beta-strands A, B, and E, and the second beta-sheet is comprised of beta-strands C, F, and G. A relatively hydrophobic helix is found between beta-strands C and E, which replaces beta-strand D of the classical c-type immunoglobulin fold.     

Solution structure of the coxsackievirus and adenovirus receptor domain 1

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus B (CVB) and adenovirus (Ad). The normal cellular function of CAR, which is expressed in a wide variety of tissue types, is thought to involve homophilic cell adhesion in the developing brain. The extracellular domain of CAR consists of two immunoglobulin (Ig) domains termed CAR-D1 and CAR-D2. CAR-D1 is shown by sedimentation velocity to be monomeric at pH 3.0. The solution structure and the dynamic properties of monomeric CAR-D1 have been determined by NMR spectroscopy at pH 3.0. The determinants of the CAR-D1 monomer-dimer equilibrium, as well as the binding site of CVB and Ad on CAR, are discussed in light of the monomer structure.     

Structural basis for variation in adenovirus affinity for the cellular coxsackievirus and adenovirus receptor

The majority of adenovirus serotypes can bind to the coxsackievirus and adenovirus receptor (CAR) on human cells despite only limited conservation of the amino acid residues that comprise the receptor-binding sites of these viruses. Using a fluorescence anisotropy-based assay, we determined that the recombinant knob domain of the fiber protein from adenovirus serotype (Ad) 2 binds the soluble, N-terminal domain (domain 1 (D1)) of CAR with 8-fold greater affinity than does the recombinant knob domain from Ad12. Homology modeling predicted that the increased affinity of Ad2 knob for CAR D1 could result from additional contacts within the binding interface contributed by two residues, Ser408 and Tyr477, which are not conserved in the Ad12 knob. Consistent with this structural model, substitution of serine and tyrosine for the corresponding residues in the Ad12 knob (P417S and S489Y) increased the binding affinity by 4- and 8-fold, respectively, whereas the double mutation increased binding affinity 10-fold. X-ray structure analysis of Ad12 knob mutants P417S and S489Y indicated that both substituted residues potentially could form additional hydrogen bonds across the knob-CAR interface. Structural changes resulting from these mutations were highly localized, implying that the high tolerance for surface variation conferred by the stable knob scaffold can minimize the impact of antigenic drift on binding specificity and affinity during evolution of virus serotypes. Our results suggest that the interaction of knob domains from different adenovirus serotypes with CAR D1 can be accurately modeled using the Ad12 knob-CAR D1 crystal structure as a template.     

A comprehensive review on host genetic susceptibility to human papillomavirus infection and progression to cervical cancer

Cervical cancer is the second most common cancer in women worldwide. This is caused by oncogenic types of human papillomavirus (HPV) infection. Although large numbers of young sexually active women get HPV-infected, only a small fraction develop cervical cancer. This points to different co-factors for regression of HPV infection or progression to cervical cancer. Host genetic factors play an important role in the outcome of such complex or multifactor diseases such as cervical cancer and are also known to regulate the rate of disease progression. The aim of this review is to compile the advances in the field of host genetics of cervical cancer. MEDLINE database was searched using the terms, 'HPV', 'cervical', 'CIN', 'polymorphism(s)', 'cervical'+ *the name of the gene* and 'HPV'+ *the name of the gene*. This review focuses on the major host genes reported to affect the progression to cervical cancer in HPV infected individuals.     

Curcumin causes promoter hypomethylation and increased expression of FANCF gene in SiHa cell line

Curcumin and resveratrol were evaluated for their potential to cause reversal of promoter hypermethylation and associated gene expression of FANCF in SiHa cell line. Methylation specific PCR along with bisulphite sequencing revealed the demethylation of 12 CpG sites out of 15 CpG sites spanning ?280 to ?432 region of FANCF promoter after treatment with curcumin and fivefold up regulation of FANCF gene expression as shown by qRT-PCR. In vitro methylation assay also showed that M.SssI an analogue of DNMT1 was effectively inhibited at 50 lM concentration of curcumin. Resveratrol was not found to be effective in causing reversal of promoter hypermethylation of FANCF gene when used at 20 lM for 4 days in SiHa cell line.     

Susceptibility of human bone marrow cells and hematopoietic cell lines to coxsackievirus B3 infection.

Viremia is commonly observed in association with enterovirus infections, and during this phase viruses can be transmitted to secondary target organs in the body. It is not known, however, whether blood cells play a role in the pathogenesis of enterovirus infection supporting virus replication. Our earlier work (T. Vuorinen, R. Vainionpää, H. Kettinen, and T. Hyypiä, Blood 84:823-829, 1994) demonstrated that coxsackievirus B3 is able to replicate in representatives of B- and T-cell lines but not in a monocytic cell line or peripheral blood mononuclear cells, indicating that virus replication may depend on the differentiation and maturation stages of the cells. Therefore, we have broaden our studies and analyzed the susceptibility of granulocyte-macrophage CFU and hematopoietic cell lines with various differentiation and maturation stages to coxsackievirus B3 infection. Virus replication was detected in B- and T-cell lines with no direct correlation to the maturation stage. Granulocyte-macrophage CFU were also able to support virus multiplication.     

Activation of human neutrophils increases thrombospondin receptor expression.

Thrombospondin (TSP), a 450-kDa extracellular matrix protein secreted by platelets may be instrumental in triggering polymorphonuclear leukocyte (PMN) activation and mediating PMN-endothelial cell interactions. TSP alone had no effect on O-2 generation but caused a significant increase in the chemoattractant FMLP-mediated O-2 generation. Purified HBD, but not the 140-kDa COOH-terminal fragment of TSP, retained the priming activity indicating that the priming effect was mediated through the HBD of TSP. The priming of FMLP-mediated O-2 generation by TSP, and our recent studies demonstrating that TSP stimulates PMN adhesion and motility suggested the presence of specific receptors for TSP on PMN. Binding studies on unactivated PMN, using 125I-TSP and competition with excess unlabeled TSP, demonstrated 2.4 x 10(3) binding sites/cell with an apparent Kd of 7 nM. Heparin did not compete for binding as effectively as unlabeled TSP. There were 1.5 x 10(3) heparin-inhibitable binding sites/cell with an apparent Kd of 8 nM that represented approximately 60% of the TSP-specific sites. Therefore, two distinct TSP receptors appeared to exist on unactivated PMN; one interacting with the heparin-binding domain of TSP and one interacting with a different site. Treating PMN with cytochalasin B followed by FMLP caused a 30-fold increase in TSP receptor expression. Binding studies on activated PMNs revealed 7.6 x 10(4) sites/cell; 60% of which were heparin inhibitable. The majority (5.3 x 10(4) sites/cell) of receptors expressed had an affinity of approximately 20 nM. About 50% of these sites were heparin inhibitable. In addition, there were 2.3 x 10(4) higher affinity sites/cell with an apparent Kd of 6 nM. Heparin-inhibitable sites comprised 70% of the higher affinity sites. The existence of a subset of TSP receptors that were heparin-inhibitable on PMN suggests that binding of TSP may trigger functionally independent responses. Increased receptor expression and expression of two high affinity binding sites following PMN activation may modulate PMN-endothelium or PMN-basement membrane interactions localized at the blood vessel wall.     

Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.     

The expression and biological activity of IL-2 receptor on a human pancreas cancer cell line

To ascertain whether the tumor cells can regulate the host immune systems through the production of the cytokines or their receptors, we examined the expressions of tumor necrosis factor (TNF), tumor necrosis factor (TNF), interleukin 2 (IL-2) and interleukin 2 receptor alpha chain (IL-2R) on the human cancer cell lines by Northern blot analysis. We used K562 (leukemia cell line), MCF-7 (breast cancer cell line), LS180, HT29 (colon cancer cell lines), SH101 (gastric cancer cell line) and PH101 (pancreas cancer cell line). Expressions of TNF, TNF and IL-2 mRNA were not detected in any of the tumor cell lines. However, 1.4 and 3.5 kilobases of the IL-2R mRNA were expressed in the PH101 cells, but not in the other five cell lines. Furthermore, IL-2R was detected on the cell surface of the PH101 cells by the flow-cytometric analysis with an anti-IL-2R monoclonal antibody. Interestingly, the soluble IL-2R (sIL-2R) was found in the conditioned media obtained from the PH101 cell culture with a sandwich enzyme immunoassay. Moreover, the sIL-2R secreted from the PH101 cells blocked the IL-2 dependent lymphocyte proliferation. These results indicate that the expression of IL-2R on PH101 might suppress the IL-2 induced lymphocyte proliferation.