Human endogenous retroviruses and pathogenicity: genomic considerations.
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Animals↗
Biomedical subjects
D L Mager
Publications and source records attributed to D L Mager.
Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2.
The ability to sense orientation relative to gravity requires dense particles, called otoconia, which are localized in the vestibular macular organs. In mammals, otoconia are composed of proteins (otoconins) and calcium carbonate crystals in a calcite lattice. Little is known about the mechanisms that regulate otoconial biosynthesis. To begin to elucidate these mechanisms, we have partially sequenced and cloned the major protein component of murine otoconia, otoconin-90 (OC90). The amino acid sequence identified an orphan chimeric human cDNA. Because of its similarity to secretory phospholipase A2 (sPLA2), this gene was referred to as PLA2-like (PLA2L) and enabled the identification of human Oc90. Partial murine cDNA and genomic clones were isolated and shown to be specifically expressed in the developing mouse otocyst. The mature mouse OC90 is composed of 453 residues and contains two domains homologous to sPLA2. The cloning of Oc90 will allow an examination of the role of this protein in otoconial biosynthesis and in diseases that affect the vestibular system.
Amino Acid Sequence↗
Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor.
The recognition of class I MHC molecules on target cells by the Ly-49 family of receptors regulates NK cytotoxicity. Previous studies have suggested that carbohydrates are involved in the recognition of class I MHC by Ly-49, although their precise role remains unclear. Here, we examined the role of asparagine-linked carbohydrates of the murine class I MHC in the binding to Ly-49A and Ly-49C. We have generated H-2Dd mutants that lack the highly conserved glycosylation sites at amino acid residues 86 in the alpha1 domain and 176 in the alpha2 domain, respectively. These mutant Dd cDNAs were transfected into leukemic cell lines, and the binding of the transfected cells to COS cells expressing Ly-49A or Ly-49C, as well as their susceptibility to lysis by Ly-49A+ NK cells, was examined. Only the mutation of the alpha2 domain glycosylation site significantly reduced the binding of Dd to Ly-49A and Ly-49C. Cells expressing Dd with the mutation at this site were partially resistant to killing by Ly-49A+ NK cells. These results suggest that, while carbohydrates linked to residue 176 seem to function as a part of the ligand structure for the Ly-49 family of NK receptors, there are additional structural features involved in this recognition. This glycosylation site is highly conserved among murine class I MHC but is not found among those of other species, suggesting that its role is unique to the murine immune system. It further suggests that murine class I MHC and Ly-49 gene families may have evolved in concert.
Amino Acid Sequence↗
Localization of five new Ly49 genes, including three closely related to Ly49c.
Nine genes belonging to the mouse Ly49 multigene family of natural killer cell receptors have been identified to date. Two of these genes, Ly49h and i, are very closely related to the well characterized Ly49c gene in the carbohydrate recognition domain. Here we show by Southern blotting that at least two additional new sequences exist in C57BL/6 mice that are also closely related to Ly49c in the carbohydrate recognition domain. Furthermore, in contrast to Ly49a, extensive variation in the arrangement and number of Ly49c-related genes in different mouse strains was observed. To characterize and localize the new Ly49c-related genes in C57BL/6 mice, we isolated and mapped genomic P1 clones hybridizing to an Ly49C exon 7 probe. Locations and the relative order of all Ly49 genes found within the clones was determined. We also used polymerase chain reaction to sequence exons 2, 4, and 7 from all genes. In this manner, we identified five new potential Ly49 genes which have been tentatively termed Ly49j-n. Ly49j, k, and n belong to the Ly49c-related subfamily, whereas Ly49l and Ly49m are most similar to Ly49d and g, respectively. Interestingly, the members of the Ly49c-related subfamily are not clustered as a unit but are interspersed among other Ly49 genes. These results illustrate the complex nature of the Ly49 gene family and should aid in the understanding of functions, such as the mediation of hybrid resistance, in which Ly49c-related genes play a role.
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Human-specific integrations of the HERV-K endogenous retrovirus family.
Several distinct families of endogenous retrovirus-like sequences (HERVs) exist in the genomes of humans and other primates. One of these families, the HERV-K group, contains members that encode functional proteins and that have been implicated in the etiology of insulin-dependent diabetes mellitus (IDDM). Because of potential functional and disease relevance, it is important to determine if there are HERV-K-associated genetic differences between individuals. In this study, we have investigated the divergence and evolutionary age of HERV-K long terminal repeats (LTRs). Thirty-seven LTRs, taken primarily from random human clones in GenBank, were aligned and grouped into nine clusters with decreasing sequence divergence. Cluster 1 sequences are 8.6% divergent, on average, whereas cluster 9 LTRs, represented by the LTRs of the fully sequenced HERV-K10 clone, show an average of only 1.1% divergence from each other. The evolutionary age of 18 LTRs from different clusters was then investigated by genomic PCR to determine presence or absence of the retroviral element in different primate species. LTRs from clusters of higher divergence were detected in monkeys and apes, whereas LTRs in clusters with lower divergence were acquired later in evolution. Notably, LTRs of cluster 9 were found only in humans at all nine loci examined. Genomic Southern analysis with an oligonucleotide probe specific for cluster 9 LTRs suggests that HERV-K elements with this type of LTR expanded independently in the genomes of humans and the great apes. This is the first report of endogenous retroviral integrations that are specific to humans and indicates that some HERVs have amplified much later than previously thought. These elements may still be actively transposing and may therefore represent a source of genetic variation linked to disease development.
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A human endogenous retrovirus suppresses translation of an associated fusion transcript, PLA2L.
Human endogenous retroviruses (HERVs) are repetitive, noninfectious chromosomal elements degenerated from exogenous retroviruses. The HERV-H family is composed of approximately 1,000 elements which are dispersed throughout the human genome. We have shown previously that an HERV-H element splices into a downstream locus, termed PLA2L, which has a large open reading frame (ORF) containing two domains with phospholipase A2 homology. Over half of the putative 5' untranslated region (5' UTR) of the resulting fusion transcript is derived from HERV-H long-terminal-repeat and internal sequences. As 5' UTRs are known to modulate translation initiation, we tested for possible effects upon gene expression at the translation level due to the 5' fusion with HERV-H sequences. No PLA2L protein was detected in teratocarcinoma cell lines in which PLA2L mRNA is abundantly expressed. In addition, despite a high level of transcription, no protein synthesis was detected when the full-length PLA2L cDNA was expressed in COS cells. Upon removal of the 5'-terminal HERV-H sequences, PLA2L protein was seen in transfectants. The 5' UTR contains both small ORFs and a strong predicted RNA secondary structure, both of which have been shown to contribute to translation suppression. The HERV-H sequences, combined with a unique PLA2L 5' UTR sequence, form a predicted RNA stem-loop that has a stability greater than that proposed to negatively affect translation. Interestingly, this stem-loop is abolished when the HERV-H sequences are removed. We hypothesize that the PLA2L 5' HERV-H sequences function as an abnormally long and complex 5' UTR, resulting in suppression of translation in both teratocarcinoma cell lines and full-length cDNA transfectants. This is the first known example of a endogenous retrovirus integration affecting expression of a heterologous human gene at the translational level.
Amino Acid Sequence↗
Genomic structure and evolution of a novel gene (PLA2L) with duplicated phospholipase A2-like domains.
In a previous study, we isolated a novel human cDNA with two domains of homology to secreted phospholipase A2 (sPLA2) embedded within a much larger open reading frame. The corresponding gene, termed PLA2L, is also unusual in that it is transcribed from an endogenous retroviral long terminal repeat promoter in teratocarcinoma cell lines. The associated retroviral element, a member of the HERV-H family of sequences, is found within an intron of the human PLA2L gene and has apparently assumed transcriptional regulatory functions at this locus. In this study we have isolated genomic clones spanning the human PLA2L locus and have determined the intron/exon structure of the PLA2-like domains. This intron/exon structure is very similar to that of known sPLA2s despite the fact that the PLA2L gene is highly diverged and has a novel duplicated structure. We also mapped PLA2L to chromosome 8q24, a location that differs from the known locations of human sPLA2s. Genomic PCR across primate species was performed to determine the approximate time of integration of the HERV-H element. Results indicate that the element integrated 15-20 million years ago since it is present in chimpanzee and gorilla but absent in orangutan and lower primates. Although the function of the PLA2L gene is not known, genomic Southern analyses suggest evolutionary conservation in mammals. These results contribute to our understanding of the unique and complex evolutionary history of the PLA2L gene.
Amino Acid Sequence↗
Structure and genomic organization of a novel human endogenous retrovirus family: HERV-K (HML-6).
Prototypic elements of a novel human endogenous retrovirus (HERV) family were identified and cloned from a human genomic library by the use of a pol fragment, HML-6, related to type A and type B retroviruses and class II HERVs. Out of 39 polhybridizing clones, five contained structures of full-length retroviral proviruses, with regions showing similarity to gag, pol and env, flanked by long terminal repeats (LTRs). Restriction mapping and partial sequence analysis of each full-length clone revealed few conserved restriction sites among HML-6 genomes, and about 20% sequence divergence over the reverse transcriptase region sequenced, suggesting that HML-6 constitutes a heterogeneous, but distinct family of elements belonging to the HERV-K superfamily. Sequence analysis of two clones, HML-6p and HML-6.17, revealed a lysine (K) tRNA UUU primer-binding site, and 40-68% nucleotide sequence similarity to LTR, gag, pro, pol and env regions of type B retroviruses and class II HERVs. HERV-K (HML-6) elements are present at about 30-40 copies per haploid genome. The HML-6 LTRs contain putative progesterone-responsive elements, which may be involved in the regulation of HML-6 expression. Furthermore, there are about 50 additional solitary HML-6 LTRs per haploid genome. Such LTRs were integrated within the pol region of two clones belonging to the same HML-6 family, indicating that some site preference may be involved in HERV integration.
Amino Acid Sequence↗
The Ly-49 family: genes, proteins and recognition of class I MHC.
The Ly-49 family consists of at least nine members, of which Ly-49A and C have been found to be NK-cell inhibitory receptors specific for class I MHC. The functions of other Ly-49 molecules are still unclear. Further analysis of Ly-49 is complicated by the cross-reactivities of some anti-Ly-49 antibodies initially thought to be specific for individual Ly-49 molecules. Studies on the role of Ly-49 in hybrid resistance as well as on allelic exclusion are also complicated by our recent finding that a novel Ly-49CB6 gene is the likely allelic form of Ly-49CBALB as opposed to a previously reported highly related but distinct gene in B6 mice. In cell-cell binding assays, only Ly-49A and C show significant binding to class I MHC. Ly-49A and C also bind some polysaccharides, and carbohydrates on class I MHC seem to be important for its binding to Ly-49. However, this interaction involves not only the carbohydrate recognition domain of Ly-49 but also a part of the stalk region, suggesting that both carbohydrates and peptide backbone of class I MHC may be recognized by Ly-49. It is likely that additional Ly-49 molecules yet to be identified function as NK-inhibitory receptors specific for class I MHC.
Animals↗
Heterogeneity among Ly-49C natural killer (NK) cells: characterization of highly related receptors with differing functions and expression patterns.
Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2d bone marrow by F1 mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49CB6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49CBALB at only 4 residues, whereas the previously reported Ly-49CB6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49CB6. Two-color flow cytometric analysis detected 5E6+4LO3311- as well as 5E6+4LO3311+ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49CB6 was indistinguishable from that of Ly-49CBALB, whereas no binding was detectable with previously reported Ly-49CB6. These results demonstrate that the newly described Ly-49CB6, not the previously reported Ly-49CB6, is the probable B6 allelic form of Ly-49C. The previously reported Ly-49CB6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.
Amino Acid Sequence↗
Gain of Sp1 sites and loss of repressor sequences associated with a young, transcriptionally active subset of HERV-H endogenous long terminal repeats.
HERV-H sequences comprise a large family of human endogenous retrovirus-like elements. Previous DNA sequence comparisons of HERV-H long terminal repeats (LTRs) have led to their classification into three subtypes, Types I, Ia, and II. Type Ia appears to have been generated by recombination between Type I and Type II LTRs. These subtypes differ in evolutionary age and transcriptional activity with Type Ia LTRs being younger in evolutionary terms and possessing stronger promoter function than the other two subtypes. In this study, possible mechanisms responsible for the functional difference between LTRs have been explored. Types I and II LTRs each contain different sets of repeated segments in their U3 regions which are disrupted in Type Ia LTRs. Using reporter gene assays, we have shown that both types of repeated segments can suppress activity of the human beta-globin gene promoter when cloned at a distant site. Both sets of repeats also repress promoter activity of a Type Ia LTR when directly inserted within its U3 region. In addition, using deletion constructs, we have localized two positive regulatory segments within the Type Ia LTR, both of which contain a potential binding site for the transcription factor Sp1. Gel mobility shift assays demonstrated that fragments containing these sites do bind Sp1. Although Type I LTRs are generally similar to Type Ia LTRs in the regions surrounding the Sp1 sites, there are sequence differences within the sites. Gel-shift analysis revealed no or much reduced Sp1 binding of Type I LTR fragments containing these sites. Thus, it appears that the loss of repeated suppresser elements and the acquisition of Sp1-binding sites have both contributed to the relatively strong transcriptional activity of the Type Ia LTRs.
Base Sequence↗
Transcripts from a novel human KRAB zinc finger gene contain spliced Alu and endogenous retroviral segments.
During the course of an investigation into the potential effects of endogenous retroviruses on adjacent gene expression, we isolated two cDNA clones containing a small sequence segment belonging to the human endogenous retrovirus family, HERV-H. Characterization of the clones revealed that they represent transcripts from a novel KRAB zinc finger gene termed ZNF177. The two cDNA clones differ at their 5' termini and in the presence of a 559-bp internal exon. The clone containing this internal exon has six imperfect zinc finger motifs followed by seven perfect copies of the C2H2 type but has a frame shift between the KRAB domain and the downstream zinc finger region. The smaller clone lacks the six imperfect motifs and has an intact ORF. The 5' putative untranslated regions of both cDNAs contain an 86-bp HERV-H env segment and a segment of an Alu repeat, both in the antisense orientation, that have been incorporated by splicing. RT-PCR experiments show evidence of alternative splicing but the majority of transcripts appear to contain the Alu and env segments. Genomic PCR and hybridization experiments suggest that a partial HERV-H element is integrated within the ZNF177 locus, which Southern analysis has shown to be a single-copy gene. Northern and RT-PCR analyses suggest that ZNF177 is transcribed at a low level in a variety of cell types.
Amino Acid Sequence↗
Recognition of class I major histocompatibility complex molecules by Ly-49: specificities and domain interactions.
Ly-49 is a family type II transmembrane proteins encoded by a gene cluster on murine chromosome 6. One member of this family, Ly-49A, is expressed by a natural killer (NK) cell subset, binds to class I major histocompatibility complex (MHC) molecules, and blocks the killing of target cells bearing the appropriate H-2 antigens. Here we show that another member of this family which is expressed by an NK cell subset, Ly-49C, recognizes H-2b and H-2d structures which are distinct from and overlapping with those recognized by Ly-49A. Interactions between Ly-49A and C and their class I ligands are entirely blocked by the antibodies 5E6, YE1/48, YE1/32, and A1, all of which were found to recognize epitopes contained within the carbohydrate recognition domain (CRD). However, cell-cell binding assays revealed that class I binding specificity is conferred by a combination of sequences within both the CRD and a 19-amino acid adjacent region. We also investigated the question of whether Ly-49A and C form dimers on cells which express both receptors. When coexpressed on COS cells, sequential immunoprecipitation demonstrated that these receptors pair exclusively as homodimers, with no evidence for heterodimeric structures. These observations provide insight into both the biochemical nature of the Ly-49 family as well as the receptor functions of Ly-49C on NK cells.
Amino Acid Sequence↗
Expression of novel-transposon-containing mRNAs in human T cells.
The HERV-H family of endogenous retrovirus like elements is the largest such human family known. Using an HERV-H LTR probe, 6 and 4.5 kb transcripts were detected by Northern blot analysis which were induced in normal peripheral T cells after treatment with phytohaemaglutinin (PHA). Expression was not evident 30 min after treatment with phorbol ester, was increased within 3-4 h after treatment, reached a maximum after 8 h and then declined to low levels 24 h after treatment. Expression was inhibited totally by cycloheximide (10 micromolar) and by the immunosuppressant cyclosporin A (1 microgram/ml). Using probes specific for the U3 and U5 regions of the HERV-H LTR, in combination with internal HERV-H probes, evidence was obtained that the 6 and 4.5 kb transcripts are polyadenylated from an HERV-H LTR. A cDNA library was constructed from T cells which had been treated with PHA for 8 h and a 1.7 kb clone was isolated using the HERV-H LTR probe. The insert contained a novel tandem array of an Alu, a LINE-1 element, two endogenous retroviral LTRs and an A-T-rich sequence. The A-T-rich sequence contained multiple copies of AUUUA mRNA regulatory motifs. Because of its high expression level, defined transcription kinetics, novel cassette-like composition and the presence of conserved mRNA stabilization sequences, we hypothesize that the transcript may play a biological role during T cell activation.
Base Sequence↗
HERV-H endogenous retroviruses: presence in the New World branch but amplification in the Old World primate lineage.
The evolutionary origin and age of the HERV-H family of human endogenous retrovirus-like sequences was investigated in this study. HERV-H elements exist in approximately 900 partially deleted copies and 50-100 more intact forms in humans and Old World monkeys. However, their possible presence in more divergent species is unknown. We have isolated a 1.6-kb genomic DNA segment from the New World monkey marmoset that had been PCR amplified using human HERV-H primers. DNA and protein comparisons and database searches indicate that this marmoset clone is more closely related to human HERV-H elements than to any other sequence, indicating that HERV-H-related sequences do exist in New World monkeys. In contrast to the high copy numbers of deleted elements in Old World primates. Southern blot analysis shows that such elements are present in less than 50 copies in two different species of New World monkey. To estimate evolutionary ages of the common deleted form of the element, a selected DNA segment from the pol region was compared from multiple human HERV-H elements. This comparison suggests that many HERV-H elements of the abundant deleted subfamily integrated approximately 30-35 million years ago. Very similar percentage divergence values between 5' and 3' long terminal repeats of individual elements of the deleted subfamily also suggest that these elements are close in age. These results indicate that HERV-H elements first appeared in the germline prior to the New World/Old World divergence over 40 million years ago. Interestingly, they remained in low numbers in the New World branch while a subfamily underwent a major amplification in Old World primates before the time of divergence of hominoids from Old World monkeys.
Animals↗
Carbohydrate recognition by a natural killer cell receptor, Ly-49C.
Ly-49 represents a family of type II transmembrane proteins containing C-type lectin domains. At least two members of the Ly-49 family, namely Ly-49A and Ly-49C, are expressed by distinct subsets of natural killer cells and bind to class I major histocompatibility complex antigens on the surface of target cells. In this report we have established that Ly-49C mediates carbohydrate recognition. The sulfated glycans fucoidan, lambda-carrageenan, and dextran sulfate were found to be potent inhibitors of Ly-49C-mediated cell adhesion, whereas other polysaccharides of similar size, charge, or sulfate content were noninhibitory. All of the polysaccharides which inhibited Ly-49C adhesion also blocked the binding of the antibody 5E6 to Ly-49C-expressing COS cells, confirming the direct protein-carbohydrate interaction. The enzymatic removal of specific carbohydrates from the target cell surface has shown that Ly-49C-mediated adhesion is not sialic acid-dependent, but is significantly decreased following fucosidase treatment. These results suggest an important role for carbohydrate recognition by natural killer cell receptors.
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Spliced HERV-H endogenous retroviral sequences in human genomic DNA: evidence for amplification via retrotransposition.
HERV-H elements are a large family of endogenous retrovirus-like sequences found in approximately 1000 dispersed copies in the genomes of humans and other primates. The most abundant subclass of these elements is a partially deleted form of 5.8 kb which is transcribed primarily as a 5.6-kb unit length RNA and a 3.7-kb spliced derivative. The provirus-like structure of these elements suggests that their numbers have increased in the genome through retrotransposition. However, this has not been demonstrated for HERV-H. To determine if genomic expansion of HERV-H elements involved an RNA intermediate, primate DNAs were screened by PCR for elements that were transcribed, spliced, reverse transcribed, and integrated back into the genome. This PCR screen detected several genomic HERV-H fragments that appear to be derived from spliced transcripts. Interestingly, the presence of one of these fragments is polymorphic in humans, suggesting that its integration was a relatively recent event. Another PCR strategy was used to determine that at least one of the spliced elements has an intact 5' LTR, indicating that it is not simply a "processed pseudogene" or cDNA copy of a HERV-H transcript. Genomic cloning and sequencing of a human locus harboring a spliced element revealed the expected structure, e.g., intact LTRs and flanking 5-bp direct repeats, for a virally retrotransposed element. A genomic library screening method also indicated that very few HERV-H elements (less than 1%) have the structure of processed pseudogenes. These results suggest that most HERV-H elements amplified in the genome as viral retrotransposons.
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A modified indicator gene for selection of retrotransposition events in mammalian cells.
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