Development of an antigen-capture ELISA for the detection of the p27-CA protein of HERV-K(HML-2).

The detection or quantification of retroviruses is often achieved using an antigen-capture ELISA (AC-ELISA) that targets the Gag capsid (CA) protein. We report here the development of an AC-ELISA specific for the p27-CA protein of HERV-K(HML-2). A monoclonal p27-specific antibody is used for capture and a polyclonal anti-p27-CA immune serum generated in rabbits serves for detection. The assay was shown to be specific for HERV-K(HML-2), showing no evidence of cross reactivity with the human retroviruses HIV-1, HIV-2 and HTLV-1 or with XMRV (as a model non-human gammaretrovirus). Using purified recombinant antigen, the limit of detection was shown to be 130pg/ml. The AC-ELISA can be used to quantify HERV-K(HML-2) expression in teratocarcinoma cell lines and to normalize HERV particles generated by transfecting HEK 293T cells with full-length molecular clones. This novel AC-ELISA also proved useful in studies of virus regulation, for example in demonstrating that HERV-K(HML-2) expression is dramatically enhanced by overexpression of Staufen-1, a binding partner of the HERV-K(HML-2) Rec protein. This specific and sensitive HERV-K(HML-2) AC-ELISA will be a useful tool for investigating many aspects of endogenous retroviruses, from basic research to the role they may play in human diseases or as a surrogate marker for particular diseases.

CMV-promoter driven codon-optimized expression alters the assembly type and morphology of a reconstituted HERV-K(HML-2).

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.