The HTDV/HERV-K family is a unique group of human endogenous retroviruses in so far as they contain open reading frames for all viral proteins necessary for the formation of retroviral particles. Such particles have been detected in teratocarcinoma cell lines (HTDV particles [5
]). In these cell lines, HTDV/HERV-K are expressed with an RNA pattern reminiscent of that seen for complex retroviruses (26
). One of the predominant proteins (cORF), encoded by a small, doubly spliced mRNA, resembles the viral regulatory proteins Rev and Rex in having an arginine-rich NLS and a leucine-rich NES (27
). The presented study demonstrates that the cORF protein, interacting with a responsive element in the HTDV/HERV-K sequence, exerts a Rev-like function.
This conclusion is supported by several lines of evidence. (i) HTDV/HERV-K Gag proteins, like HIV Gag proteins, need a Rev-RRE-like export system for proper expression. (ii) For HIV gag
-containing constructs, Rev and RRE can be replaced by cORF and a responsive element in the 3′ LTR of the HTDV/HERV-K mRNA, as demonstrated by immunofluorescence, determination of p24 levels, and Western blotting. This element, designated RcRE, maps to nt 8720 to 9148 in U3R. (iii) With full-length HTDV/HERV-K constructs, the expression of structural proteins is significantly enhanced by the presence of RcRE. (iv) As shown by Northern blot analysis, the amount of cytoplasmic viral mRNA specific for the structural proteins is also significantly increased when the constructs contain RcRE and when cORF is expressed, demonstrating the stabilization and enhanced nuclear export of these mRNAs by cORF and RcRE. (v) The expression of structural proteins is inhibited by LMB, a potent inhibitor of CRM1-mediated nuclear export. This export pathway has been shown to be used by Rev and Rex (4
Since RRE and RxRE are complex mRNA structures, the possible existence of similar features in RcRE has been investigated by using the DNASIS RNA secondary structure prediction software. Two adjacent energetically favored stem-loop structures are consistently identified (nt 8839 to 8945). The exact binding sites for Rev and Rex on their responsive elements have been described in detail (see, e.g., references 2
, and 22
). Rev binds to the sequence 5′UGGGCG/5′CGGUACA, forming a short stem with an internal loop located in stem-loop 2 of the RRE (17
). The Rex binding core is mapped to the sequence 5′CUCAGGUCGA(G)/5′(C)UCCCUUGGAG in HTLV-1 (1
) and to the sequence 5′GAGCUCG/5′CGCUC in HTLV-2. A putative binding site [5′GGGUCGA(G)/5′CUCCCC] has been identified in simian T-cell leukemia virus (STLV) PH969, a primate T-cell lymphotropic virus isolated from an African baboon (47
). In the stem of the first stem-loop structure of RcRE, the sequence 5′CUCCC/5′GGAAGGG shows a striking similarity to the Rex binding domains of the HTLV/STLV group (Fig. ). Further investigations will aim at high-resolution mapping of the biologically relevant nucleotides to define the core binding sites in RcRE of cORF and of Rev, which seems to be able to replace cORF to some extent. In addition, whether Rex can replace cORF on RcRE or cORF can substitute for Rex on the RxRE will be examined.
FIG. 7 Comparison of primate RRE sequences. Secondary structures of the experimentally determined (HTLV-1 [HTLV-I] and HTLV-2 [HTLV-II]) or predicted (STLV and HTDV/HERV-K) Rex or cORF binding sites (relevant nucleotides are underlined) (more ...)
HTDV/HERV-K genomes exist in two subgroups which differ by a deletion of 292 nt at the boundary between the pol
reading frames. This deletion affects the splice acceptor site for the formation of the subgenomic env
transcript and, most importantly, removes the first coding exon of cORF (27
). The subclass of proviruses harboring the deletion has been designated type 1 in order to credit the first sequencing of a provirus of this family, the HERV-K 10 provirus (38
); the subclass of the prototypic complete genomes has been designated type 2 (Fig. A) (27
). Type 1 proviruses do not code for the cORF protein but contain RcRE. Therefore, their proper expression may still depend on a cORF-RcRE-based nuclear export system. A survey of whole-cell RNA prepared from different human tissues showed widespread expression of full-length type 1 sequences. However, spliced viral mRNAs or viral proteins could not be detected in those tissues (40
). One possible explanation is that the nuclear export of viral RNA is disturbed due to the lack of cORF.
Expression of type 2 proviruses, which encode cORF, is mainly restricted to testicular cells. The precise mechanism leading to the differential expression in testicular cells is not known but may involve a cell type-specific regulation of transcription of putative type 2 specific LTRs (unpublished data). The presence of cORF, translated from type 2 specific mRNA, allows the high-level export of spliced and unspliced viral RNAs into the cytoplasm and their subsequent translation into proteins. Type 1 sequences may contribute to the pool of viral cytoplasmic mRNAs and viral proteins expressed in cases in which cORF is provided in trans
. Especially in testicular tumor cell lines, both type 1 and 2 proviruses may be a source for upregulation of viral proteins to a level which allows the formation of particles (24
). Surprisingly, HTDV/HERV-K proteins, predominantly cORF, can be detected in normal human testis tissue (40
). This finding raises the question of whether cORF has taken on some role in the development of testes in the primate lineage. In this case, the persistence of open reading frames in this class of HERVs would be obvious. It will be interesting to study whether dysregulation of cORF expression contributes to the onset or progression of germ cell tumors.
The particles observed in teratocarcinoma cell lines may be solely Gag particles, because Env is present in only limited amounts in the cells as well as in particle preparations (unpublished data). A similar, albeit less dramatic, imbalance can be seen in transient-transfection assays with a full-length HTDV/HERV-K clone: only 50 to 80% of Gag-producing transfectants express Env. These observations do not conform with the pattern expected for a functional cORF-RcRE interaction and may be explained by as-yet-unidentified deficiencies in HTDV/HERV-K proviruses.
In sequence composition, gag and pol genes of HTDV/HERV-K proviruses resemble those of type D viruses, but their Rev-RRE-like posttranscriptional regulation resembles that of the bovine leukemia virus-HTLV and lentivirus genera. Therefore, these endogenous proviruses could be seen as intermediates in the evolution to contemporary primate retroviruses. Bearing this example in mind, ERVs in general can be regarded as remnants of the evolution buried in the genomes of living cells. Uncovering and analyzing these sequences as fossils may extend our understanding of the different steps taken during the evolution of retroviruses. Our findings, for instance, indicate that the strategy of regulation of gene expression by a viral protein which binds to a responsive element may have evolved 40 million years ago and was not first achieved during the recent evolution of contemporary retroviruses. The observation that exogenous retroviruses frequently recombine with endogenous counterparts may indicate that ERVs provide a useful reservoir of genes by which exogenous retroviruses can evolve new functions. One might even cautiously speculate that an HIV predecessor could have picked up the Rev gene by swapping sequences with an HTDV/HERV-K provirus in the course of evolution. However, other hypotheses, like derivation of HTDV/HERV-K from an even more complex ancestor or the independent evolution of strategies for nuclear export, are still possible.
Having shown the Rev-like function of the cORF protein, the abbreviation of the corresponding gene needs a new interpretation: cORF is convergent to Rev in function.