Figure 4

HERV-K(HML-2) SP localizes to nucleoli. (A) Schematic representation of proteins encoded by Env and EnvΔRec expression vectors. Env-expressing cells produce Env and Rec. Both contain the epitope recognized by the anti-SP antibody (black bars). The EnvΔRec construct harbors silent point mutations (asterisks) in splice donor (SD) and splice acceptor (SA) sites, eliminating rec mRNA splicing and Rec protein production. (B) Western blot analysis of HeLa cells transiently expressing Env, EnvΔRec and SP_96/94/75-RFP. The Western blot was stained with anti-SP antibody. Env-expressing cells produce the 15 kDa Rec protein and a lower amount of SP (asterisk) with an approximate molecular weight of 13 kDa. In EnvΔRec-expressing cells only SP is detected. SP_96/94/75-RFP constructs produce SP-RFP fusion proteins and (RFP) degradation products. SP₉₆-RFP releases SP while SP₇₅-RFP and SP₉₄-RFP do not due to engineered deletions (see text). An SP-like band produced by SP₇₅-RFP is very likely an unspecific mRFP degradation product (see text and Figure 3B). (C) Western blot analysis of fractionated Hela cells transiently expressing Env and EnvΔRec. Cell lysates were probed with anti-SP and anti-GAPDH antibodies, the latter verifying proper separation of fractions. L: full lysate; Cy: cytoplasm; Nu: nucleus; Np: nucleoplasm; Ni: nucleoli. (D and E) Confocal sections of HeLa cells fixed 24 hours post-transfection and co-immunostained with anti-SP for detection of SP or Rec (in red), and with antibodies detecting B23/nucleophosmin or C23/nucleolin (in green). White bar = 10 μm. (D) SP distribution in EnvΔRec-expressing cells. (E) Rec distribution in Env-expressing cells. The merge panels show, in yellow, co-localization of both SP and Rec with B23/nucleophosmin in the granular component of nucleoli.