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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 6, 2004 as doi:10.1096/fj.04-2467fje. |
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INSERM U526, IFR 50, Laboratoire de Virologie, Faculté de Médecine, Nice, France
2Correspondence: Laboratoire de Virologie, Faculté de Médecine, avenue de Valombrose, 06107 Nice cedex 2, France. E-mail: lefebvre{at}unice.fr
SPECIFIC AIM
Our aim was to determine the mechanism of the overall hyposialylation status of cells infected with HIV-1. Given that, in our system, both N- and O-glycans were hyposialylated, with fully preserved activity of specific sialyltransferases, a general defect in the various sialylation pathways could be speculated. Hence, we focused on the activity of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme implicated in the synthesis pathway of CMP-N-acetylneuraminic acid (i.e., CMP-sialic acid), the donor substrate of all sialyltransferases.
PRINCIPAL FINDINGS
1. Normal sialylation of HIV-infected CEM T cells can be completely restored by metabolic complementation with N-acetylmannosamine (ManNac)
On the basis of the sialic acid biosynthesis pathway, hyposialylated cells were cultured in the presence of several sialic acid precursors. Addition of 20 µM of ManNAc in the culture medium of hyposialylated CEM cells latently infected by the HIV-1 strain LAI (CEMLAI/NP) for 5 days allowed to obtain a quasi-complete restoration of sialylation to the level of parental CEM cells. Addition of N-acetylglucosamine (GlcNAc) or mannose (Man) did not bring any change. Indeed, GlcNAC and Man are sialic acid precursors synthesized upstream of the intervention of GNE, whereas the synthesis of ManNac is directly monitored by GNE. The restoration of sialylation was certified by increased binding of the plant lectins Maackia amurentis agglutinin II (MAL II) and Sambucus nigra agglutinin (SNA), which are sialic acid-specific lectins. In accordance with these results, we observed a decreased binding of Peanut agglutinin (PNA) that recognizes unsialylated galactoseß1,3N-acetylgalactosamine. We have previously shown oligosaccharides elongations by core 2 O-glycans and poly-N-acetyllactosamine chains (poly-LacNAc) in hyposialylated CEMLAI/NP (NP) cells. After metabolic complementation with ManNAc, a quasi-total correction of the reactivity of Lycopersicon esculentum agglutinin, specific for poly-LacNAc, was obtained in NP cells. It was thus indirectly demonstrated that elongation of poly-LacNAc sequences was a consequence of a defect in engrafting of sialic acids, known as carbohydrate chain terminators. More convincing results were obtained using visualization of purified CD43 molecules that are particularly convenient for the study of glycosylation changes because of their 70-85 O-glycans side chains. The important reduction in electrophoretic mobility of CD43 molecules from NP cells, due to a dramatic loss in electronegative charges and elongation with poly-LacNAc chains was completely corrected by nutrient complementation with ManNac (Fig. 1
). This was a direct demonstration of the role played by sialic acid residues as terminators of oligosaccharide chains.
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2. Transcription of GNE gene is down-regulated in HIV-infected CEM cells
The defect in sialic acid biosynthesis observed in HIV-infected cells led us to evaluate the expression of GNE, the key enzyme in the biosynthesis pathway of sialic acid. The transcriptional activity of the GNE gene appeared significantly decreased in latently infected NP cells and several virus-producing CEMRP cell lines which are CEM subclones infected with the strain BCL. This was demonstrated using a semiquantitative RT-PCR technique and Northern blot analysis of poly(A)-rich RNAs.
3. Identification of the GNE gene promoter
To characterize the promoter of GNE gene, a 5'RACE method was used to identify the Transcription start site (TpSst) of the gene. It was found that the 5'UTR of GNE primary mRNA was interrupted by an unusually long intron of 9 kb. The sequence surrounding the TpSst complied with the most strict definition of a CpG island with 67% G+C and a ratio of 0.80 between observed and expected CpGs. Several motifs CREB, SP1 and E2F were detected. These sequences are known to be implicated in transcription regulation by methylation. There was a putative CCAAT box located at –98 relative to the TpSst but no TATA box. A typical GC-box was found closely upstream of the TpSst. Within the +182/+254 nt sequence there was a striking cluster containing one canonical CCCCAGGC and five putative AP-2 motifs, three of which had no CpG, and one had overlapping an SP1 motif.
To evaluate the expected activity of the putative promoter of GNE (GNE-Prom), a 1.9 kb-long fragment surrounding the TpSst plus 3 truncated forms of GNE-Prom were inserted upstream of the luciferase reporter gene. The promoter activity of the long form appeared significant by comparison with a plasmid control. The truncated GNE-Prom forms, devoid of most of the transcription motifs and of the TpSst, displayed a near-complete loss of activity. These results identify GNE-Prom as an authentic promoter of the GNE gene.
4. GNE gene promoter is de novo hypermethylated in HIV-1-infected CEM cells
Epigenetic regulation of GNE has been proposed to explain hyposialylation of human B cell lines. The transcriptional level of GNE was entirely restored in NP cells, when they were incubated with 5 µM of the methylation inhibitor 5-azaC. This indirectly indicated that the transcription of GNE was very likely down-regulated by methylation in HIV-infected cells. Six useful HpaII/MspI endonuclease sites were found in the GNE-Prom. MspI, an isoschizomer of HpaII (C/CGG), is the only one able to cleave the methylated CCmGG site. After 18 h of cutting, HpaII did not prevent amplification of an expected 1.5 kb-long fragment from genomic DNA of latently HIV-infected virus nonproducing (NP) cells and chronically virus-producing HIV-infected (CEM-RP BCL) cells, in contrast to parental cells, whereas MspI cutting prevented all amplifications. These results were in favor of an overall methylation of GNE-Prom of HIV-infected cells.
A bisulfite-modified DNA fragment of 186 bp-long, located just upstream of the TpSst, was PCR amplified and sequenced. The defined sequence contains 19 CpGs and a total of 49 cytosines. Subcloned PCR fragments obtained from treated DNA were randomly selected for sequencing. A striking de novo hypermethylation pattern was detected in subclones from NP vs. parental CEM cells. All 30 cytosines outside CpGs were found to be unmethylated. The relative diversity of sequences warranted that the results provided by the semi-nested PCR technique were not biased. Conversely, only one methylated CpG was detected in sequences from parental CEM cells. Hence, hypermethylation of GNE-Prom associated with HIV-1-infection was clearly confirmed (Fig. 2
).
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CONCLUSIONS AND SIGNIFICANCE
Glycolipid and glycoprotein biosynthesis can be modified to varying degrees by addition of sialic acids. GNE is the key enzyme in the biosynthetic pathway of CMP-NAN, the donor substrate of all sialyltransferases. We show that the transcription level of the GNE gene is decreased in HIV-infected cell lines, and can be corrected by the methylation inhibitor 5-azaC. In accordance with this result, sialylation on the surface of HIV-infected cell can be entirely restored by nutrient complementation with ManNAc, a sialic acid precursor, the synthesis of which is monitored by GNE. It was noted that the glycosylation changes associated with hyposialylation, consisting of core 2 O-glycans and poly-LacNAc elongations, were also corrected by ManNAC complementation. Hence, the crucial role played by sialic engraftments in the control of extension of carbohydrate chains, shown by other, was directly confirmed.
It can be deduced that all glycoproteins in cells showing GNE down-regulation yield a modified level of sialylation. Hyposialylation of oligosaccharides such as Lewis-x, present on heavily glycosylated molecules such as CD43, CD7, CD45, or PSGL-1, which participate in activation, survival/cell death, and trafficking of lymphocytes, can greatly modify their functions.
Gene silencing by DNA methylation associated with retroviral infections is a well-known general phenomenon. Several results indicate that HIV-1 is responsible for an increased overall genomic methylation. Promoters of IFN-
, tumor suppressor p16INK4.A gene, O6-methylguanine-DNA methyltransferase, and the LTR promoter of HIV-1 itself can be down-regulated by methylation. Our findings are in keeping with this general context. The promoter that we characterized around the TpSst of GNE-Prom was embedded in a CpG-rich region strictly complying with the most stringent definition of a CpG island, and containing several regulatory motifs (CREB, E2F and SP1) favorable to reprogramming by methylation. Almost all 19 CpG dinucleotides, located upstream of the TpSst, including those within the GC-Box and CREB motifs, were found to be methylated. Consequently, the entire CpG island containing the GNE-Prom was likely to be hypermethylated in NP cells. We had previously shown that NP cells were only hyposialylated and could be over-desialylated. The partial decrease in GNE gene expression, in spite of an extended hypermethylation of its promoter, might be explained by a bi-modal system of regulation controlled by methylation-dependent and –independent transcriptional motifs.
Highly active antiretroviral therapy has brought considerable benefits to the control of HIV replication and the increase in CD4 cell count. However, the sustained control of virus replication, that is commonly observed, is not correlated with a complete immune restoration in the T cell count and functions, even when HAART has been initiated right at the beginning of clinical disorders. This misunderstood paradox is likely to be clarified by in vivo approaches on the basis of epigenetic T cell reprogramming. Moreover, given several studies reporting HIV-1 infection of hematopoietic progenitors, long-term epigenetically inherited disorders associated with infection might be transmitted through this pathway.
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FOOTNOTES
1 Both authors contributed equally to this work 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2467fje;
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