Glycobiology Advance Access originally published online on January 3, 2007
Glycobiology 2007 17(4):355-366; doi:10.1093/glycob/cwl083
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Virus-induced transcriptional activation of host FUT genes associated with neo-expression of Ley in cytomegalovirus-infected and sialyl-Lex in varicella-zoster virus-infected diploid human cells
2 Department of Virology
3 Department of Clinical Chemistry and Transfusion Medicine, University of Göteborg, Göteborg, Sweden
4 Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden
5 Dental School, University of Copenhagen, Copenhagen, Denmark
1 To whom correspondence should be addressed; Tel: +46-31-342 4659; Fax: +46-31-82 7032; e-mail: sigvard.olofsson{at}microbio.gu.se
Received on March 21, 2006; revised on December 19, 2006; accepted on December 21, 2006
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Cell surface carbohydrate structures including sialyl-Lewis X (sLex) and Lewis Y (Ley) are important ligands in normal and malignant tissues. The aim here was to determine the possible influence on the expression of such antigens by two viruses varicella-zoster virus (VZV) and cytomegalovirus (CMV) involved in persistent infections of humans. We found that infection of human diploid fibroblasts with both viruses resulted in transcriptional activation of several fucosyltransferase (FUT) genes that were either dormant or expressed at low levels in uninfected cells. Both viruses induced FUT3, FUT5, and FUT6, encoding
1,3- and/or 1,4-specific fucosyltransferases. CMV, but not VZV, induced transcription of FUT1 (encoding an 1,2-specific fucosyltransferase), FUT7, and FUT9. The changes in transcription of FUT genes were expectedly associated with expression of Ley in CMV-infected cells and sLex in the VZV-infected fibroblasts although no expression of these antigens was observed in uninfected cells. One major explanation for this difference between CMV- and VZV-infected cells was that CMV, but not VZV, induced expression of FUT1, necessary for Ley expression. The induced carbohydrate antigens in CMV- and VZV-infected cells could be of significance for virus spread and possible escape from immune responses. Key words: cytomegalovirus / fucosyltransferase / Lewis Y / sialyl-Lewis X / varicella-zoster virus
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Several fucosylated histo-blood group antigens, including sialyl-Lewis X (sLex) and Lewis Y (Ley, Figure 1) are deeply involved in cancer dissemination and metastasis (Irimura et al. 1993
; Thurin and Kieber-Emmons 2002; Klinger et al. 2004; Magnani 2004), most likely by decoying the functions of these structures in normal cells. Thus, sLex or its sulfated derivatives participate in specific binding to E- and P-selectins, thereby promoting activation and homing of various white blood cells active in innate as well as acquired immune responses (Muramatsu 2000; Kannagi and Hakomori 2001; Hiraiwa et al. 2003; Uchimura et al. 2005). Ley is found at the surface of activated granulocytes, possibly engaged in appropriate adhesion of circulating granulocytes although this process is less characterized (Dettke et al. 2000). Owing to biological significance of sLex and Ley, the critical genes for their synthesis are only active on the appropriate occasions. The changes in expression of carbohydrate antigens in the tumor cells are in most cases achieved by tumor-induced changes in the transcription rates of one or more quiescent glycosyltransferase (FUT) genes, whose gene products synthesize sLex or Ley (Taniguchi et al. 2000). For human T-cell leukemia virus type 1 (HTLV-1)-dependent leukemia, induction of sLex is accomplished by transactivation via HTLV-1 encoded Tax of the human fucosyltransferase VII (Fuc-T VII) gene (FUT7) (Kannagi and Hakomori 2001; Hiraiwa et al. 2003). This finding raised the question whether viral control of host cell fucosylation as a pathogenic factor could also be used by other viruses, where latency and/or persistent infection are characteristic for the virus–host relationship.
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The genetics behind the synthesis of fucosylated carbohydrate antigens is however complex, involving a family of eight active human fucosyltransferase human genes, designated FUT1–FUT7 and FUT9, encoding key enzymes in the formation of sLex and Ley and related histo-blood group antigens of the ABH and the Lewis systems [Reviewed in de Vries et al. (2001)
and Becker and Lowe (2003)]. The eight FUT genes mentioned belong to two groups with respect to catalytic activity, where the gene products of FUT1 and FUT2 add fucose in an 1,2 linkage, whereas the others may add fucose in an 1,3 and/or 1,4 linkage (de Vries et al. 2001). In spite of these similarities between the members of each of the two groups, there are important differences between all enzymes with respect to a number of parameters including acceptor specificity and tissue localization (Table I).
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There are reasons to believe that also nontransforming viruses may significantly influence the structures of the carbohydrate antigens of viruses and the infected cell surface by controlling the regulation of the fine-tuned transcriptional program of these glycosyltransferase genes (Cebulla et al. 2000
; Nystrom et al. 2004). The aim of the present study was therefore to explore the interactions of varicella-zoster virus (VZV) and human cytomegalovirus (CMV) with the host cell glycosylation machinery, focusing on the eight fucosyltransferases (FUT1–FUT7 and FUT9). VZV, a representative for -herpesviruses, causes chickenpox, and thereafter life-long latency in sensory neurons, from which reactivations may present as herpes zoster or, rarely, as encephalitis. This virus was chosen because of its tight interactions with T memory cells during viremia (Ku et al. 2005). CMV, a representative for ß-herpesviruses, may cause fevrile illness including mononucleosis and is thought to remain life-long in monocytes and lymphocytes. Understanding the mechanisms of CMV infection has become increasingly important, because it may cause life-threatening infections in transplanted and other immunocompromised subjects. CMV was chosen because of its close interactions with the endothelium and various white blood cells (Mocarski and Tan Courcelle 2001). As a target cell an embryonic human diploid fibroblast cell line was chosen, maintained in our laboratory at low passages and characterized in previous studies (Lundström et al. 1987; Nystrom et al. 2004), being permissive for both CMV and VZV. We report that infection with both CMV and VZV viruses resulted in increased transcription rates by several orders of magnitudes for at least three of the FUT genes, but there were also differences between CMV and VZV, the most important being that only CMV induced FUT1, encoding a Fuc 1,2-transferase. This resulted in prominent differences between VZV and CMV with respect to the neo-carbohydrate antigens in the infected cells. |
Results |
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Cell histo-blood group genotyping
As target cells for the present studies, we chose a diploid cell line of human embryonic fibroblasts (HEL), available at low passage numbers and equally permissive for both CMV and VZV. This enabled studies on the influence of the two viruses on expression of glycosyltransferase genes in one and the same cell type. Moreover, as several of the FUT genes studied are not vital for growth in cell culture, it was deemed advantageous to avoid transformed, aneuploid cells with possible asymmetric distribution of the actual FUT genes among individual cells. The cells were genotyped, using polymerase chain reaction-sequence specific primers (PCR-SSP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFPL) (Larson et al. 1996
; Procter et al. 1997; Elmgren et al. 2000; Bengtson et al. 2001), with respect to those glycosyltransferases previously shown to be subject to allovariation (de Vries et al. 2001) and which are synthesized histo-blood group antigens of relevance for the present study. The results are presented in Table I. The most important finding was that the cells were homozygously defective in FUT2 (stop codon), implying that they are neither able to express Leb, nor type 1 ABO antigens, despite the existence of one functional B allele. Furthermore, the cells contained at least one functional FUT3, FUT6, and FUT7 allele of each gene.
Time course of CMV and VZV infection and FUT transcription in virus-infected cells
Replicate cell cultures were infected with CMV or VZV (see Materials and methods), and virus-infected cells were extracted for DNA and assayed via real-time PCR for viral progeny DNA (Figure 2). Typical cytopathic effects (CPE) were observed for both CMV and VZV at 24 h postinfection (p.i). Production of progeny VZV DNA was detected at 48 h p.i. and a plateau phase was reached at 72 h p.i., which is in accordance with the previously published data (Arvin 2001; Pass 2001). Production of progeny CMV DNA was detected at 24 h p.i. and maximal levels were reached at 72 h p.i.
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Total RNA from VZV- and CMV-infected cells was extracted and analyzed by Taqman real-time PCR using the PCR systems described in Table II. Linear levels of different FUT transcripts were calculated from
CT values (see Materials and methods). The arbitrary RNA concentration values were normalized against 18S RNA enabling direct comparisons of differences in the transcript abundances of each and one of the analyzed FUT genes. The FUT genes studied could be divided into three different groups with respect to their transcriptional patterns after infection with CMV or VZV: (i) FUT genes induced by CMV and VZV (Figure 3), (ii) FUT genes only induced by CMV (Figure 4), and (iii) FUT genes whose transcription remained high and relatively constant in virus-infected cells as well as uninfected cells during the time interval studied (Figure 5). The transcription rates in mock-infected cells of the FUT genes of groups (i) and (ii) were low or undetectable (Figures 3 and 4).
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–) and mock-infected cells (–
–) was analyzed using real-time PCR. FUT gene assignations are indicated in the figure. The RNA concentrations indicate units are comparable between all graphs in Figures
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Three FUT genes encoding
1,3/1,4- (FUT3 and FUT5), or 1,3- (FUT6) fucosyltransferases belonged to the first group i.e. their transcription rates were strongly induced in VZV- as well as in CMV-infected cells (Figure 3). The maximal transcription rates of these FUT genes in virus-infected cells were 100–1600 times higher than the baseline levels obtained for mock-infected cells.
The second group of FUT genes represents exclusively CMV-induced genes, containing one 1,2-fucosyltransferase (FUT1) whose transcription was induced to 20 000 times over the baseline level by CMV, but not altered by VZV (Figure 4). The maximal levels during the observation interval were reached about 70 h p.i. Also, FUT7 and FUT9 transcription was induced at lower levels and different kinetics (FUT7) in CMV-infected cells, but not altered in VZV-infected cells.
The third group comprised FUT genes that remained relatively unaffected by virus infection compared with the other two groups (Figure 5). The FUT4 RNA concentration was high and in the same range (approximately 20 000 arbitrary units) as the maximal value observed for FUT1 through CMV induction. In contrast to all other differences between virus-infected and mock-infected cells, the extraordinary high value recorded for CMV-infected cells at 24 h p.i. was not consistently observed from one set of infected cells to another. The transcription of the inactive (see Table I) FUT2 gene was relatively constant in the CMV-, VZV-, and mock-infected cells.
Expression of carbohydrate neo-antigens in CMV- and VZV- infected cells
Next, we explored whether the induced changes in FUT transformation influenced the expression pattern of fucosylated carbohydrate antigens in the virus-infected cells. Confluent HEL cell cultures were cultured in teflon-coated glass wells where 30–60% of the cells were infected with either CMV or VZV during conditions in which cell-to-cell spread of virus was permitted as described in Materials and methods, Immunofluorescence. The cells were examined in immunofluorescence using antibodies to viral antigens and/or specific monoclonal antibodies to defined histo-blood group antigens. CMV-infected cells were identified by a typical granular intranuclear stain with a CMV-specific antibody, whereas the VZV antibody resulted in a cytoplasmic granular staining (Figure 6). Uninfected cells, used for control purposes, expressed little if any detectable fluorescence with any of the antibodies used (K. Nyström unpublished data). In contrast, 30–40% of the CMV-infected cells demonstrated a prominent cytoplasmic and surface staining for Ley. In addition, 5% of the cells were positive for sLex, preferentially in the cytoplasm, although the intensity of the fluorescence was less pronounced than that observed for the Ley antibody. In contrast, the VZV-infected cells demonstrated no Ley signal, but a clear, mainly Golgi-like sLex fluorescence, considerably more intense than in the CMV-infected cells. A weak but yet consistent surface-associated sLex surface staining, that was dimmed by the VZV-specific Tetramethyl Rhodamine Iso-Thiocyanate (TRITC)-fluorescence in the double-fluorescence setting, became readily visible in Fluorescein Iso-Thiocyanate (FITC) single fluorescence (Figure 6, inset in the right middle panel). About 50% of the VZV-infected cells were positive for sLex. In some experiments, a weak Lex fluorescence was detected in the CMV- or VZV-infected cells, but this phenomenon was occasional and not reproducible. No fluorescence was detected with antibodies towards Lea, Leb, or sLea (K. Nyström, unpublished data).
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To further explore the role of sialyltransferase gene expression, we analyzed the levels of
(2,3)-sialyltransferase III (ST3Gal-III) and (2,3)sialyltransferase IV (ST3Gal-IV) transcription in VZV- and CMV-infected cells (Figure 7). VZV- as well as CMV-infected cells expressed a high constitutive transcription of these human sialyltransferase genes with levels of 2000 arbitrary units for ST3Gal-III and 5000 arbitrary units for ST3Gal-IV using an identical arbitrary unit as in Figures 3, 4, and 5. This is in line with previous results regarding the sialylation capacity of the HEL cells (Lundström et al. 1987).
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The relationship among the FUT genes studied and the possible carbohydrate structures generated by the gene products are depicted in Figure 8. The presence of Ley in CMV-infected cells and absence in VZV-infected cells is well in line with the absence of FUT1 transcription in the infected cells, as formation of H Type 2 is a rate limiting step in Ley synthesis. The observation that sLex is detectable in VZV- as well as CMV-infected in the absence of Lex expression is most likely explained by a high ratio between sialylated Type 2 structure and Type 2 structure, which is in accordance with the data above and previous reports regarding expression of highly sialylated glycoproteins in HEL cells (Lundström et al. 1987
). The absence of detectable sLex in spite of high levels of FUT4 expression in mock-infected cells may be explained by a reported restricted capacity of Fuc-T IV to fucosylated sialylated Type 2 structure (Satoh et al. 2005). The absence of Lea and sLea is most likely reflected low levels of Type 1 structures in the virus- and mock-infected HEL cells.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Here, we report that infection of diploid human fibroblasts with VZV and CMV resulted in expression of novel carbohydrate epitopes in a manner that is specific for each of the viruses. Moreover, we showed that this was correlated with virus-induced changes in the transcription rates of fucosyltransferases engaged in the synthesis of histo-blood group antigens (Figure 8). The particular carbohydrate antigens induced sLex and Ley are involved in processes by which tumor cells spread and colonize distal tissues. One intriguing question is whether this principle is also used by viruses for spread of infected cells.
Regarding the molecular background to the differences in carbohydrate antigen expression between Ley expressing CMV-infected cells and sLex expressing VZV-infected cells, the most prominent factor is the dramatic induction of FUT1 in CMV-infected cells compared with no induction in VZV-infected cells. A fucosyltransferase I(Fuc-T I) enzyme driven switch from sLex to Ley has been analyzed in other experimental systems (Mathieu et al. 2004). Thus, transgenic expression of FUT1 in a number of cell types initiates an efficient formation of the H Type 2 structure that efficiently competes with the synthesis of the sialylated Type 2 structure, i.e. the precursor to sLex (see Figure 8). Accordingly, in CMV-infected cells, the possibilities for Fuc-T III, IV, V, VII, and IX to form Lex or sLex must be restricted at high Fuc-T I activities. On the other hand, in VZV-infected cells, the absence of Fuc-T I is favorable for sLex synthesis, but not for Lex synthesis, owing to scarcity of nonsialylated structures in HEL cells (Lundström et al. 1987). The potential of the FUT1 gene product to intercept the sLex synthesis is further underscored by the fact that this enzyme is localized to the medial Golgi region in contrast to its competitor for the Type 2 structure, the trans-Golgi-localized 2,3-sialyltransferase (Zerfaoui et al. 2002). Our results regarding CMV-infected cells is somewhat at variance with those published by Cebulla et al. (2000), reporting induction of sLex and Lex, but not Ley, in human endothelial cells. The reasons for this discrepancy could be that other cells were used in that study, no assays for Ley were reported, and the cells were examined only after long-term culture.
One interesting feature is that only 30–40% of the CMV-infected cells expressed Ley and about 50% of the VZV-infected cells expressed sLex. These seemingly low figures probably reflected that the cells in the object glass cultures for immunofluorescence were infected at low multiplicity of infection during longer periods of time to allow cell-to-cell spread of virus to ensure the existence of cells at different stages of infection. Carbohydrate signaling is usually transient in nature (Tsuboi and Fukuda 2001) and this is probably also true for the present sLex and Ley appearance in the virus-infected cells. The FUT transcription data depicted in Figures 3–5 represent the sum of FUT transcription in all the cells, and it is reasonable that some of the cells are more active in FUT transcription and others are less active or even inactive, resulting in the heterogeneous Ley and sLex expression pattern observed in immunofluorescence. As expected we found surface-associated sLex in the VZV-infected cells, but much of the sLex fluorescence was associated with Golgi-like structures in VZV-infected cells. This could reflect the fact that VZV glycoproteins generally remain Golgi-associated for a relatively long time resulting in a comparatively low surface exposition, compared with its Golgi appearance (Wang et al. 1998).
One key function of sLex is to act as a receptor for primarily E- and P-selectins during leukocyte trafficking and homing via the high-endothelial venules (Kannagi 2002; Lowe 2002; Schottelius et al. 2003). In this context, it is tempting to compare the early appearance of sLex in VZV-infected cells with the parallel phenomenon in adult T leukemia cells, where sLex is strongly upregulated due to transcriptional induction of the human FUT7 gene by the human T-cell leukemia virus type 1 (HTLV-1) Tax protein (Hiraiwa et al. 2003). The degree of skin infiltration of leukemia in patients is directly proportional to the fraction of cells displaying HTLV-1-induced sLex (Kannagi and Hakomori 2001). It is now established that VZV viremia in primary virus infection involves T memory cells, infected by VZV in the tonsils for subsequent cell-bound transport to the skin [Reviewed in Ku et al. (2005)]. It is easy to imagine that the efficiency of such a viremic colonization should be further potentiated, given that the virus transiently could induce sLex as a tag for addressing the appropriate target skin cells. One VZV advantage in expressing sLex on the T memory cells used in viremia is that sLex does not only permit specific adhesion to the target tissue, but also offers the intricate selectin-mediated mechanism for endothelial passage and tissue extravasation. It should, however, be born in mind that the present study was performed in HEL cells and further proofs for a possible VZV use of sLex in viremia must await studies with VZV-infected T memory cells.
So far, very little is known about the biological functions of Ley, in spite of the fact that it is expressed very selectively in biologically active cells. Thus, Ley is expressed during short intervals in embryogenesis, in activated granulocytes, and in several types of neoplasia (Dettke et al. 2000; Kannagi 2002), and possible functions suggested include engagement in the apoptosis process, in activated granulocyte behavior and not least specific cellular adhesion (Hiraiwa et al. 2003; Azuma et al. 2004). Thus, polymorphonuclear leukocytes play a central role in CMV dissemination and it is therefore possible that the Ley induction is engaged in specific adhesion of infected cells in analogy with previous observations for Ley-expressing tumor cells (Kannagi and Hakomori 2001; Kim et al. 2005).
Interference with sLex and Ley expression in tumor cells including oligosaccharide mimics and humanized anti-carbohydrate antibodies have been considered for anti-tumor therapy, and a few of these antibodies have been approved by Food and Drug Administration for use in humans [Reviewed by Saleh et al. (2000), Ramsland et al. (2004), and Brodzik et al. (2006)]. Therefore, should viral expression of sLex and Ley or related epitopes prove to be of biological relevance for development of viral disease, as described for the HTLV-1 induced leukemia, it seems reasonable to assume that the therapeutical modalities, described above, could be of interest also for treatment of e.g. CMV or VZV infections in immunocompromised patients.
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Materials and methods |
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Cells and viruses
HEL cells (Lundström et al. 1987
) that were kept at a low passage level were used throughout the study. The fibroblasts were cultivated in Eagle's MEM with Penicillin, Streptomycin, and 1% L-glutamine. 10% fetal calf serum (FCS) was used during cell growth. The CMV laboratory strain Towne (ATCC-977) was used throughout the study. The virus titers for Towne were determined by plaque titration on HEL cells (Lundström et al. 1987) as previously described (Chiba et al. 1972). A patient VZV isolate C822 that was typed by PCR (Bergstrom 1996) was used for the VZV studies.
Viral infection of cells
One plaque forming unit per cell of CMV strain Towne was added to HEL cells in six-well culture plates (700 000 cells/well) and one well was used for each infectious sample or mock-infection. Cell-associated VZV (180 000 particles/cell; see Real-time PCR), where dimethylsulfoxide was removed prior to infection, was added to HEL cells at 500 µL/well. The viruses were allowed to attach to the cells for 3 h at 37 °C and 5% CO2 before the inoculum was removed. The cells were washed with phosphate-buffered saline (PBS) and fresh Eagle MEM with penicillin, streptomycin, and 1% L-glutamine were added. Virus- and mock-infected cells were harvested by removing the medium from the well and washing the cells in PBS. The cells were lysed in 2 x Nucleic Acid Purification Lysis Solution (Applied Biosystems, Foster City, CA).
Immunofluorescence
Mock-, CMV-, or VZV-infected HEL cells were trypsinated and resuspended in Eagle's MEM, penicillin, streptomycin, 1% L-glutamine, and 10% FCS. Mock-infected and CMV- or VZV-infected cells (trypsinated at 48 h p.i.) were mixed and added to teflon-coated object slides and allowed to further infect for at least 2 days to ensure infected cells at different stages. The slides were fixated in cold acetone and stored at –80 °C.
Immunofluorescence was performed on the slides by incubating the cells with human sera containing antibodies directed against CMV or VZV diluted 1:400 and 1:200, respectively. Lex, Ley, and sLex were detected using immunoglobulin (Ig)M antibodies P12, (abcam, Cambridge, UK), F3 (abcam, Cambridge, UK), and KM93 (Chemicon International, Temecula, CA), respectively. Possible cross-reactions between sLea and sLex were ruled out by the use of MCA2031 (Serotec, Oxford, UK). Lea and Leb were detected using IgG1 antibodies 7LE and 2-25LE (abcam, Cambridge, UK). The primary antibodies were incubated for 1 h at 37 °C. TRITC-conjugated anti-human antibody (Jackson ImmunoResearch, West Grove, PA) and FITC-conjugated anti-mouse antibody (abcam) were used at dilutions of 1:200 and 1:50 and incubated on the cells for 45 min at 37 °C. The slides were washed thoroughly in PBS between each of the antibody incubations. Prolong Antifade Kit (Molecular Probes, Eugene, OR) was used as a mounting media on the slides. The slides were analyzed in a Zeiss LSM 510 Meta confocal microscope using a Plan-Apochromat 36x objective in oil immersion, and the possibility of cross-reactivity between TRITC and FITC was checked for in each well.
RNA and DNA extraction
The 6100 Prep Station (Applied Biosystems) was used for the isolation of RNA (Isolation of Total RNA from Cultured Cells, Applied Biosystems) and DNA (BloodPrep Chemistry, Applied Biosystems) from cells and supernatant according to the manufacturer's instructions. Briefly, 400 µL of lysed virus-infected cells or mock-infected cells were added to 96-well RNA or DNA isolations trays used as described by the manufacturer. Purified total RNA and DNA were eluded in a final volume of 100 µL. The concentration and purity of the RNA was checked using spectrophotometry, measuring the samples at 260 and 280 nm. The 260/280 nm ratio was 2.0 ± 0.2.
Real-time PCR
Real-time PCR was performed as previously described (Nystrom et al. 2004), and the primers and probes are listed in Table II. Briefly, RNA analysis was carried out using TaqMan One-Step RT-PCR Mastermix (Applied Biosystems) and 40 ng of total RNA. A volume of 2 µL of DNA extraction was added to the real-time PCR to control the infection. The reverse transcription reaction was performed at 48 °C for 30 min and the following PCR according to the manufacturer's instructions, for 40 cycles (95 °C 15 s, 60 °C 60 s) using 2 x AmpliTaq Gold DNA Polymerase mix and 40 x RT enzyme mix, forward and reverse primers (0.5 µM and probe (0.3 µM). The DNA real-time PCR was performed in the same manner, with omission of the reverse transcription step and the reverse transcription enzyme mix. All real-time PCR reactions were performed using 30 µL reaction volumes and carried out in 96-well plates, which were centrifuged for 1 min at 1000g in a swing-out rotor (Rotina 48R; Hettich, Tuttlingen, Germany) before the PCR. The amplification and detection was carried out with an ABI prism 7000 sequence detection system (Applied Biosystems). All RNA samples were subject to a ß-actin PCR without added reverse transcriptase to check for DNA contamination. Samples where DNA was detected were DNase treated with Turbo DNAfree (Ambion, Austin, TX) according to the manufacturer's instructions.
The CT values were normalized to 18S, which is a housekeeping gene previously shown to be constantly expressed in herpesvirus-infected cells (Nystrom et al. 2004). Linearized results were calculated using the CT method [(Livak and Schmittgen 2001); Applied Biosystem's User Bulletin #2: Relative Quantitation of Gene Expression 2001] relative 18S expression as follows. The linearized CT expression of a sample (Arbitrary unit, A) was obtained from the formula: A = 2(–CTsample+CTreference), where the reference was the one sample giving the highest FUTCT value selected among all the FUT assays depicted in Figures 3, 4, and 5. This enables direct comparison of the arbitrary unit values given in these three figures. As the CT values obtained rather than the linearized values are normally distributed, it is not possible to use the standard deviations directly in the graphs. In order to transform the standard deviation values, derived from CT measurements, to surrogate error estimates applicable in graphs presenting the linearized results, the following formula was used for each value: S = 2(–CT sample+CT reference+std 18S+std sample)–A, where S represents the length of the error bar, standard deviation values (std) obtained for 18S PCR and the sample (FUT PCR).
Genotyping
PCR-SSP genotyping of ABO, FUT2, FUT3, and FUT6 mutations
The PCR-SSP assay conditions and the primers for the ABO mutations, the FUT2 428G > A, 385A > T, and 571C > T, FUT3 59T > G, 202T > C, 314C > T, 508G > A, 1067T > A mutations have been described previously (Procter et al. 1997; Grahn et al. 2001). The FUT6 PCR-SSP assay was optimized using DNA samples from individuals with known FUT6 genotypes (Larson et al. 1996; Elmgren et al. 2000) and was designed to use the same PCR-program as described in Real-time PCR. The primers (A. Grahn, unpublished data) were selected to identify the 370C > T, 730C > G, 738C > T, 739G > A, 907C > G, 945C > A, and 977G > A mutations and to give products of 819, 462, 455, 453, 282, 245, and 212 bp not interfering in size with each other or with the human growth factor or developmentally regulated RNA-binding protein internal controls of 428 and 796 bp, respectively. All products were analyzed on UltraPURE 1% agarose gel (Gibco BRL, Paisley, Scotland) in 0.5 mg/mL ethidium bromide at 125 V and visualized under UV-light.
PCR-RFLP genotyping of FUT7 329G > A mutation
The genotyping for the FUT7 329G > A mutation was performed as described in Bengtson et al. (2001) using the primer pair VII-3s/VII-4as to generate a 1404 bp product further cleaved by NotI into 791 and 613 bp products.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The authors wish to thank Dr Julia Fernandez-Rodriguez and the Center for Cellular Imaging at the Sahlgrenska Academy, University of Göteborg for assistance during confocal microscopy. This work was supported by the Swedish Research Council (grants 8266 and 15283) and by grants from the LUA-ALF foundation, Sahlgrenska University Hospital, Göteborg.
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Abbreviations |
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CMV, human cytomegalovirus; CPE, cytopathic effects; FCS, fetal calf serum; FITC, Fluorescein Iso-Thiocyanate; Fuc, fucose; FUT, fucosyltransferase gene; Fuc-T, fucosyltransferase enzyme; HEL, human embryonic diploid fibroblats; HTLV, human T-cell leukemia virus type 1; Lex, Lewis X; Ley, Lewis Y; p.i., postinfection; PBS, phosphate-buffered saline; PCR-RFLP, PCR-restriction fragment length polymorphism; PCR-SSP, PCR-sequence specific primers; PFU, plaque forming unit; ST3Gal III,
(2, 3)-sialyltransferase III; ST3Gal IV, (2, 3)-sialyltransferase IV; TRITC, Tetramethyl Rhodamine Iso-Thiocyanate; VZV, varicella-zoster virus |
References |
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