FIG 2.

Generation and characterization of VSV-HERVK and VSV-HERVK⁺. (A) Genomic organization of VSV-eGFP, VSV-HERVK, and VSV-HERVK⁺. The negative-sense RNA genomes are shown in the 3′-to-5′ orientation. The five viral genes are shown for VSV-eGFP: N, nucleocapsid; P, phosphoprotein; M, matrix; G, glycoprotein; L, large polymerase. The noncoding leader (le) and trailer (tr) genomic regions are also indicated. Each virus contains the eGFP gene, which serves as a marker for infection. To generate VSV-HERVK and VSV-HERVK⁺, the glycoprotein gene of VSV-eGFP was replaced with the gene encoding the HERVK or HERVK⁺ENV, respectively. Viruses generated from these genomic plasmids were replication competent. Representative plaque assays in BSRT7 cells are shown for each virus, as well as the endpoint titers. (B) SDS-PAGE analysis of purified virions. (Left) Sypro Ruby-stained gel showing all the viral proteins. Molecular mass markers are indicated on the left, and the positions of viral proteins are indicated on the right. For both VSV-HERVK and VSV-HERVK⁺, the SU subunit is indicated (approximately 50 kDa). TM runs in the same position as VSV N (approximately 42 kDa). (Right) Western blots (WB) probed with antibodies against HERV-K TM, VSV-M, and VSV-G cytoplasmic tail. The lower band in the bottom right gel corresponds to the TM of HERVK⁺, which reacts with the VSV-G cytoplasmic-tail antibody. (C) Electron micrographs of purified particles stained with 1% PTA. Glycoprotein spikes are visible coating the surfaces of the virions. (Left) Magnified portions of the virions corresponding to the boxed areas on the right.