Reconstruction of the cell entry pathway of an extinct virus
Fig 3
(A) Purified VSV-HERVK or VSV were incubated with heparin (H) or protein A (A) beads, with (+) or without (-) soluble heparin added as a competitor. Bound virions were analyzed by Western blot against HERV-K Env, VSV G, and VSV M. Input: total input virus. (B) Schematic of HERV-K Env and HERV-K SU used in this study. SP: signal peptide. RBD: receptor binding domain. TM: transmembrane subunit. CT: cytoplasmic tail. 3C: 3C protease cleavage site. (C) HERV-K SU or Influenza A HA receptor binding domain (HA) were pre-incubated with or without soluble heparin prior to incubation with either cobalt (C, maximum pull-down control), heparin (H), or protein A (A) agarose beads. Bound protein was eluted from the beads and subjected to SDS-PAGE followed by Western blot against the HA tag. Input: 10% of total input protein. (D) Top: Structure of glycosaminoglycans. The repeating disaccharides of heparan sulfate/heparin (left) and chondroitin/dermatan sulfate (right) are shown. Sulfates are highlighted in red. Positions of O-sulfations are indicated with circled numbers. Disaccharides are shown as fully sulfated, however individual sugars will not always be sulfated at each position. Bottom: HERV-K SU was pre-incubated with soluble competitor compounds (heparin, heparan sulfate, 2-O-desulfated heparin, 6-O-desulfated heparin, chondroitin sulfate A, and dermatan sulfate) prior to incubation with either cobalt, heparin, or protein A agarose beads. Bound protein was eluted from the beads and subjected to SDS-PAGE followed by Western blot against the HA tag. Input: 10% of total input.