Organization and integration sites in the human genome of endogenous retroviral sequences belonging to HERV-E family

The present paper reports the characterization of HERV-E endogenous retroviral sequences in the human genome by using three complementary approaches. Firstly, we identified genomic clones containing HERV-E by BLAST screening of human DNA databases by using the entire sequence of a characterized HERV-E clone (M10976) as a query. The genomic structure and integration sites of the HERV-E elements were characterized. Secondly, new integration sites of HERV-E elements were revealed by a retroviral LTR-arbitrary primer-PCR (RELAP-PCR) technique. BLAST analysis of the PCR products identified a subgroup that shows low identity (75%) to the original clone M10976 and slightly higher identity (80%) to a closely related HERV-E (Ac. n. K02166). Finally, we performed FISH mapping, which revealed sites of integration of HERV-E not previously identified at the cytogenetic level. A preliminary analysis of genes mapping in the same bands as HERV-E integration sites was performed: several loci relevant to physiological and/or pathological processes were detected. Our findings may provide clues to identify HERV-E integration sites adjacent to genes with important biological roles.     

Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases.     

Presence of a retroviral encapsidation sequence in nonretroviral RNA increases the efficiency of formation of cDNA genes.

We showed previously that retrovirus vector particles can encapsidate RNAs without retroviral cis-acting sequences, that such RNAs are reverse transcribed in infected target cells, and that the cDNA copies are inserted into the host genome resulting in cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To provide further evidence that this retrovirus-mediated gene transfer occurred through an RNA intermediate, we constructed retroviral vectors containing an intron from a cellular gene. This intron was lost in a cDNA gene formed after infection with retroviral particles, establishing that an RNA intermediate had existed. Retroviral vectors with additional encapsidation sequences were constructed. The presence of a murine leukemia virus encapsidation sequence in an mRNA transcribed from the hygromycin B phosphotransferase gene increased the efficiency of encapsidation into spleen necrosis virus vector particles and the formation of cDNA genes by approximately 2 orders of magnitude.     

Structure and genomic organization of a new family of murine retrovirus-related DNA sequences (MuRRS).

A new class of murine retrovirus-related sequences (MuRRS) is described. These 5.7 kb long transposon-like DNA-elements start and end with approximately 600 bp long repeats identical to previously identified solitary LTR-like elements (LTR-IS). There are about 50 - 100 5.7 kb elements and about 500 - 1000 solo LTR-IS elements per mouse haploid genome. Sequence analysis of one cloned MuRRS element revealed several possible open reading frames with partial sequence homologies to retroviral gag, pol and env genes.     

Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene.

Human immunodeficiency virus (HIV) regulates the expression of its genes temporally at the mRNA processing step. A subset of the mRNA species which encode the structural and some accessory genes contains inhibitory sequences (INS or CRS elements) which prevent nuclear export of the RNA or its utilization in the cytoplasm. Such inhibition is overridden by the interaction of a viral protein, Rev, with its RNA target sequence, RRE. The vif gene product, which is essential for virus replication in vivo, is encoded by a singly spliced mRNA, and its expression is dependent on rev in infected cells. However, INS elements have not been found in the HIV-1 vif gene itself, although such elements have been observed in Gag, Pol, and Env coding sequences. We have now identified an INS within the 5' half of HIV-2 vif which does not show any homology with cellular mRNAs or other previously identified INS and CRS elements of HIV. These results suggest that retroviral mRNAs have novel labile sequences different from those of cellular mRNAs.     

The transformation booster sequence from Petunia hybrida is a retrotransposon derivative that binds to the nuclear scaffold

The transformation booster sequence (TBS) from Petunia hybrida enhances transformation frequencies in P. hybrida, Nicotiana tabacum and Zea mays. TBS also stimulates homologous inter- and intramolecular recombination in P. hybrida, the molecular basis for this stimulation is not known. We investigated whether TBS contains sequence elements that might contribute to the stimulation of recombination and whether its recombinogenic potential reflects a biological function of TBS. We identified a scaffold attachment region (SAR) within TBS and analysed its distribution in the genome and its homologies to other genomic sequences. A 516 by subfragment of TBS binds to the nuclear scaffold. The sequence of the TBSSAR fragment shows strong homologies to retroviral elements from plants, suggesting that TBS is an inactive derivative of a retrovirus that still promotes DNA recombination.     

The transformation booster sequence from Petunia hybrida is a retrotransposon derivative that binds to the nuclear scaffold

The transformation booster sequence (TBS) from Petunia hybrida enhances transformation frequencies in P. hybrida, Nicotiana tabacum and Zea mays. TBS also stimulates homologous inter- and intramolecular recombination in P. hybrida, the molecular basis for this stimulation is not known. We investigated whether TBS contains sequence elements that might contribute to the stimulation of recombination and whether its recombinogenic potential reflects a biological function of TBS. We identified a scaffold attachment region (SAR) within TBS and analysed its distribution in the genome and its homologies to other genomic sequences. A 516 by subfragment of TBS binds to the nuclear scaffold. The sequence of the TBSSAR fragment shows strong homologies to retroviral elements from plants, suggesting that TBS is an inactive derivative of a retrovirus that still promotes DNA recombination.     

Identification and phylogenetic analysis of novel human endogenous retroviral sequences belonging to the HERV-H family on human X and Y chromosomes

HERV-H, a family of endogenous retroviral elements that has undergone successive expansions in the human genome, includes sequences that are expressed in placenta and T cells. With a PCR approach to the HERV-H using human monochromosomal somatic cell hybrid DNA, we identified 8 new HERV-H sequences on the X chromosome, and one novel HERV-H element, HY-1, the first reported such element on the Y chromosome, and compared these with sequences in the nucleotide sequence database. Phylogenetic analysis indicated that clone HX-1 and BAC clone 523A23 on the X chromosome were found to be in close relationship to the sequences of DJ088A21 on the human chromosome 7q31. This finding allows us to speculate that HERV-H elements may have evolved by intra-chromosomal spread. Our data may relevant to an understanding of human genomic plasticity.     

Human transposon-like elements insert at a preferred target site: evidence for a retrovirally mediated process.

Members of the human transposon-like family of repetitive sequences (called THE 1 repeats) like many other repetitive DNA sequences are flanked by short direct repeats. Comparison of the base sequences of twelve examples of these flanking direct repeats indicates that THE 1 repeats insert into a preferred genomic target site. In one case, we have identified the sequence of an empty site into which a THE 1 element inserted. The sequence of this empty site and sequences of truncated THE 1 LTRs are consistent with a retroviral mechanism for the insertion of THE 1 elements. Truncated transposon structures illustrate for the first time that intermediate structures of retrotransposition may also be integrated into the genome.     

Sequence heterogeneity of theenvelope-like domain in cultivated allotetraploidGossypium species and their diploid progenitors

Retroviralenvelope (env)-like sequences in 2 cultivated allotetraploid cottons and their diploid progenitors have been identified and characterized in this study. DNA sequence analysis reveals that these sequences are heterogeneous. The observed sequence diversity, however, seems to preserve coding information. This is evidenced by the detection of the transmembrane domain (TM), which is the most conserved feature of the divergent retroviral env genes. The high ratio of synonymous to nonsynonymous changes suggests that these sequences are evolving under purifying selection. Phylogenetic analysis shows thatGossypium sequences closely cluster with a lineage of plant endogenous retroviruses that have anenv-like gene. These results provide evidence for the antiquity and the wide diversity ofenv-like sequences in theGossypium genome.