Analysis of transcribed human endogenous retrovirus W env loci clarifies the origin of multiple sclerosis-associated retrovirus env sequences - PubMed (original) (raw)

Analysis of transcribed human endogenous retrovirus W env loci clarifies the origin of multiple sclerosis-associated retrovirus env sequences

Georg Laufer et al. Retrovirology. 2009.

Abstract

Background: Multiple sclerosis-associated retrovirus (MSRV) RNA sequences have been detected in patients with multiple sclerosis (MS) and are related to the multi-copy human endogenous retrovirus family type W (HERV-W). Only one HERV-W locus (ERVWE1) codes for a complete HERV-W Env protein (Syncytin-1). Syncytin-1 and the putative MSRV Env protein have been involved in the pathogenesis of MS. The origin of MSRV and its precise relation to HERV-W were hitherto unknown.

Results: By mapping HERV-W env cDNA sequences (n = 332) from peripheral blood mononuclear cells of patients with MS and healthy controls onto individual genomic HERV-W env elements, we identified seven transcribed HERV-W env loci in these cells, including ERVWE1. Transcriptional activity of individual HERV-W env elements did not significantly differ between patients with MS and controls. Remarkably, almost 30% of HERV-W env cDNAs were recombined sequences that most likely arose in vitro between transcripts from different HERV-W env elements. Re-analysis of published MSRV env sequences revealed that all of them can be explained as originating from genomic HERV-W env loci or recombinations among them. In particular, a MSRV env clone previously used for the generation of monoclonal antibody 6A2B2, detecting an antigen in MS brain lesions, appears to be derived from a HERV-W env locus on chromosome Xq22.3. This locus harbors a long open reading frame for an N-terminally truncated HERV-W Env protein.

Conclusion: Our data clarify the origin of MSRV env sequences, have important implications for the status of MSRV, and open the possibility that a protein encoded by a HERV-W env element on chromosome Xq22.3 may be expressed in MS brain lesions.

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Figures

Figure 1

Expression of HERV-W env in human PBMC. RT-PCR using HERV-W _env_-specific primers was carried out on total RNA isolated from human PBMC which was subjected (+) or not (-) to reverse transcription. The expected size of the amplified HERV-W env fragment is ~640 bp. M, DNA size marker; H2O, PCR negative control.

Figure 2

Examples of recombined HERV-W env cDNA sequences. A multiple alignment of the genomic DNA sequences (March 2006 human genome assembly) of the seven HERV-W env loci identified as transcriptionally active in human PBMC in this study is shown. HERV-W env loci are designated according to their chromosomal location. The 7q21.2 HERV-W env locus (ERVWE1) serves as reference sequence. Note that the 7 HERV-W env loci can be distiguished by unique nucleotides and/or indels. Two of the cloned HERV-W env cDNA sequences, MS-III-K11 (from a patient with MS) and KO-IV-K6 (from a healthy control) are shown as examples of recombined cDNAs. The proviral origin of cDNA sequence portions is indicated by a color code. Gray shaded areas represent regions in which recombination events have taken place. Sequences of the primers used in this study are underlined.

Figure 3

Nucleotide mismatches between HERV-W env cDNAs and best matching genomic HERV-W env loci. White bars represent the number of nucleotide mismatches between HERV-W env cDNAs (n = 332) and their best matching genomic HERV-W env locus without assuming the presence of recombined HERV-W env sequences among those cDNAs. Black bars indicate the number of nucleotide mismatches between HERV-W env cDNAs and their best matching genomic HERV-W env loci when the presence of recombination events in 99 out of 332 HERV-W env cDNAs (see Table 2) was taken into account.

Figure 4

Relative cloning frequencies of transcriptionally active HERV-W env loci in human PBMC. The relative cloning frequencies are given as the number of cDNA clones from a particular HERV-W env locus relative to the number of all cDNA clones analyzed. Frequencies were calculated separately for all non-recombined clones (n = 233 sequences; white bars), all recombined clones (n = 99 sequences, originating from 238 transcripts [see text and Table 2]; black bars), and for non-recombined and recombined clones together (n = 332; originating from 471 transcripts [see text and Table 2]; gray bars).

Figure 5

Relative cloning frequencies of transcriptionally active HERV-W env loci in human PBMC from patients with MS and healthy controls. Relative cloning frequencies were calculated for recombined and non-recombined clones together (n = 332 sequences, originating from 471 transcripts [see text and Table 2]). The box represents the mean, and the whiskers represent the minimum and maximum of the relative cloning frequencies of cDNAs from individual HERV-W env elements for the groups of patients with MS (n = 4) and healthy controls (n = 4). There were no statistically significant differences between patients and controls (p > 0.05; two-tailed Fisher's exact test).

Figure 6

Relationship between Xq22.3 HERV-W Env, MSRV Env, and Syncytin-1. An amino acid sequence alignment of Xq22.3 HERV-W Env, MSRV Env (clones AF127228 and AF331500), and Syncytin-1 is shown. The sequence of a HERV-W element on chromosome 5p12 from which the C-terminal region of the MSRV env clone AF331500 is derived (see also Table 3 and Additional file 2) is also shown. For the sake of simplicity, only the C-terminal region of the 5p12 element is included. The region of MSRV Env (AF331500) originating from HERV-W 5p12 is highlighted in yellow. Predicted signal peptides (according to SignalP 3.0,

) are shaded in gray. The stop codon at position 39 of Xq22.3 HERV-W Env and AF127228 is indicated by a dot (·). The consensus C-X-X-C motif conserved among C-type and D-type retroviral Env proteins [42] is shown in boldface. The border between the SU and TM regions is indicated by arrows. The proteolytic cleavage site (consensus R/K-X-R/K-R) between SU and TM is highlighted in red letters. The sequences of the MSRV Env SU protein (generated using the MSRV env clone AF331500) studied by Rolland et al. [24] is marked in red. The fragment of the MSRV env clone AF127228 used for generation of the anti HERV-W Env monoclonal antibody 6A2B2 [16] is shown in green.

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Analysis of transcribed human endogenous retrovirus W env loci clarifies the origin of multiple sclerosis-associated retrovirus env sequences - PubMed (original) (raw)