(A) Genomic organization and transcripts of HTDV/HERV-K type 2. SA, splice acceptor; SD, splice donor. (B) Comparison of arginine-rich NLSs. (C) Comparison of leucine-rich NESs. Amino acids in boldface are essential for function.

Mapping of the HTDV/HERV-K cORF-responsive element. (A) Mapping of RcRE: genomic localization of 3′-LTR subfragments tested for RRE-like activity in combination with cORF. Fragments depicted as solid lines were active; that depicted by a hatched line was inactive. (B) Mammalian expression vector encoding HTDV/HERV-K cORF (effector plasmid). BGH, bovine growth hormone; CMV, cytomegalovirus; pA, polyadenylation sequence. (C) Mammalian HIV Gag expression vectors containing the HIV RRE or different sections of the HTDV/HERV-K 3′ LTR (U3RIII, nt 8720 to 9149; U3RIX, nt 8720 to 9093; RU5, nt 9065 to 9492) and the negative-control vector lacking an RRE element (reporter plasmid). (D) Cotransfection of reporter and effector plasmids into HLtat cells. Panel 1, pHIVgagU3RIII and pcORF; panel 2, pHIVgagU3RIII and p(−); panel 3, pHIVgagU3RIX and pcORF; panel 4, pHIVgagU3RIX and p(−); panel 5, pHIVgagRU5 and pcORF; panel 6, pHIVgagRU5 and p(−). Staining was performed with a mouse monoclonal α-HIV p24 antibody (panels 1 to 6) (orange) and with a rabbit α-cORF antibody (panels 1, 3, and 5) (green). A filter suitable for both fluorescence markers was used. (E) Determination of the HIV Gag precursor levels in particle preparations by an HIV p24 capture assay (Abbott). HLtat cells were transfected with the vector combinations listed in the table below. The cutoff value was determined by the extinction of the lysis buffer. (F) Detection of the HIV Gag precursor by Western blot analysis. The p55 Gag precursor was present in combination 1 (Rev and RRE), in 5 and 8 (cORF and RcRE), and faintly in 4 and 7 (Rev and RcRE). The positions of molecular mass markers (M) are indicated on the left.

Analyses of HTDV/HERV-K nuclear export. (A) Northern blot of cytoplasmic (C) and nuclear (N) mRNAs of transfected HeLa cells. Cells were transfected with pHKRcRE (lanes 1 and 2) or pHK (lanes 3 and 4) or were not transfected (lanes 5 and 6). The probe was the first exon of cORF. The two cORF bands detected with the vector pHKRcRE are a consequence of the two polyadenylation signals present in pHKRcRE, one in the HERV-K LTR and the other in the plasmid backbone. The membrane was rehybridized with an actin mRNA probe to show that there were equal amounts of mRNA in the different cytoplasmic and nuclear fractions. The positions of molecular size markers (M) are indicated on the left. (B) Quantification of the radioactive signals by phosphorimaging. The signals were normalized to those of the actin mRNA.

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) were compared by using the algorithm contained in the DNASIS software (Hitachi). Similar to the RxRE binding sites, the putative HTDV/HERV-K binding site is located on a stem-loop structure displaying a characteristic bulge.

Expression of HTDV/HERV-K Gag in the presence of the viral regulatory elements Rev and RRE. (A) Mammalian expression vectors encoding HIV Rev and the respective control plasmid (effector plasmids). SV40-pA, simian virus 40 polyadenylation sequence (for plasmid construction, see Materials and Methods). (B) Mammalian expression vectors encoding HIV or HERV-K Gag proteins as well as the HIV RRE (reporter plasmids). (C) Cotransfection of reporter and effector plasmids into HLtat cells. Panel 1, pHIVgagRRE and pREV; panel 2, pHIVgagRRE and p(−); panel 3, pHKgagRRE and pREV; panel 4, pHKgagRRE and p(−). Staining was achieved with a mouse monoclonal α-HIV p24 antibody (panels 1 and 2) or with a rabbit α-HERV-K Gag antibody (panels 3 and 4). Bar, 25 μm.

Posttranscriptional regulation of HTDV/HERV-K protein expression in full-length clones. (A) Expression vectors containing the complete HTDV/HERV-K genome with the exception of the 5′ LTR (starting with nt 1012): a cytomegalovirus (CMV) promoter was used to overcome the tissue specificity of HTDV/HERV-K LTRs. In the negative-control vector pHK, the RcRE and the HTDV/HERV-K termination signals were deleted. SA, splice acceptor; SD, splice donor; BGH, bovine growth hormone. (B) Transfections into HeLa cells. Panel 1, pHKRcRE: expression of HERV-K Gag (orange) and cORF (green); panel 2, pHK: expression of cORF (green). Staining was achieved with goat α-HERV-K Gag (panels 1 and 2) (orange) and with rabbit α-cORF (panels 1 and 2) (green). (C) Transfections of pHKRcRE into HeLa cells. Panel 1, expression of HERV-K Gag (green); panel 2, expression of Env (orange). Staining was performed with rabbit α-HERV-K Gag (panel 1) (green) and with goat α-HERV-K Env (panel 2) (orange). The same cell was photographed with an FITC filter to demonstrate Gag expression and with a Cy3 filter to show Env expression. (D) Quantification of Gag- and cORF- double-positive cells after transfection of pHKRcRE (hatched bars) or pHK (white bars) in HeLa (right) and COS7 (left) cells. cORF-positive cells were set as 100%. (E) Western blot analysis of Gag proteins in GH virus pellets (lane 1), in lysates of HeLa cells transfected with pHKRcRE (lane 2), or pHK (lane 3), and in lysates of untransfected HeLa cells (lane 4). PC, precursor; IP, intermediate proteins; CA, capsid; MA, matrix; NC, nucleocapsid. The positions of molecular mass markers (M) are indicated on the left.

Blocking of cORF-mediated nuclear export by LMB, a CRM1 inhibitor. (A) Cotransfection of reporter and effector plasmids into HLtat cells without (−) or with (+) 4 nM LMB. Panels 1 and 2, pHIVgagRRE and pREV; panels 3 and 4, pHIVgagU3RIII and pcORF. Staining was achieved with a mouse monoclonal α-HIV p24 antibody (all panels) and with a rabbit α-cORF antibody (panels 3 and 4). (B) Determination of the levels of HIV Gag precursor in lysates of cells that were or were not treated with LMB, using the HIV p24 capture assay. HLtat cells were transfected with the vector combinations listed in the table below. Each variant was analyzed without (−) and with (+) 5 nM LMB. The cutoff value was determined by the extinction of the lysis buffer. (C) Detection of the HIV Gag precursor by Western blot analysis. The p55 Gag precursor was present only in the absence of LMB in Rev-RRE combinations (lane 1), cORF-RcRE (lane 9), and (very faintly) Rev-RcRE (lane 7). LMB completely blocked HIV Gag expression at a 5 nM concentration. The positions of molecular mass markers are indicated on the left.