Human endogenous retroviruses (HERVs)are very likely footprints of ancient germ-cell infections.

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by ϳ75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by ϳ78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.The long interspersed nuclear element 1 L1) 4 is the only autonomous non-LTR retrotransposon in the human genome. Approximately 520,000 L1 copies compose ϳ17% of the chromosomal DNA (1), but only ϳ80 -100 L1s are retrotransposition-competent in the average genome (2). As functional L1 proteins are also mobilizing nonautonomous non-LTR retrotransposons (e.g. Alus) in trans (3), they are overall responsible for the generation of at least 30% of the human genome. A functional full-length L1 element is ϳ6 kb long and includes a 5Ј-untranslated region (UTR) consisting of an internal promoter, two open reading frames (ORFs) separated by a 63-nucleotide intergenic region, and a 3Ј-UTR terminating in a poly(A) tail (4). ORF1 encodes an RNA-binding protein (ORF1p) that has nucleic acid chaperone activity in vitro (5-7). ORF2 is coding for a protein (ORF2p) with endonuclease (8) and reverse transcriptase activities (9, 10) and a 3Ј-terminal Zn 2ϩ finger-like domain (11). ORF1p and ORF2p demonstrate a profound cis preference for their encoding transcript resulting in the formation of a cytoplasmic ribonucleoprotein particle (RNP) (12-15). After RNP formation, the L1 RNA gains access to the nucleus where it is reverse-transcribed into a cDNA copy most likely via target site primed reverse transcription (8,16,17). Integration of the resultant L1 cDNA and the completion of retrotransposition were suggested to occur by alternative mechanisms (18). L1s have altered the genome structure in a multitude of ways (3, 19 -22). Host ge...

Retroviruses comprise strains with considerable disease potential in animals and humans. In addition to exogenous strains transmitted horizontally, endogenous proviruses are transmitted through the germ line. Some of these endogenous retroviruses can be pathogenic in mice and possibly in other animal species. They may also be considered as mobile genetic elements with the potential to produce mutations. In humans, genomic DNA contains numerous endogenous retroviral sequences detected by their partial relatedness to animal retroviruses. However, all proviruses sequenced so far have been found to be defective. In this communication, we describe the expression of a family of human endogenous retrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell line producing the human teratocarcinoma-derived retrovirus (HTDV) particles previously described by us. Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic mRNAs are generated by single or double splicing events. This expression pattern is reminiscent of the more complex control of virus gene regulation observed, for example, with lenti-or spumavirus strains, although HERV-K shows no sequence homology to human T-lymphotropic virus or human immunodeficiency virus. Sequence analysis of expressed HERV-K genomes revealed nondefective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family.

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)n repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.