Fig 1.
Haploid genetic screen identifies host factors required for infection by VSV-HERVK.
(A) Viral genome structures. Sequences from VSV are shown in grey: N, nucleocapsid; P, phosphoprotein; M, matrix; G, glycoprotein; L, large polymerase; le: leader; tr: trailer. Viruses also encode an eGFP reporter gene. VSV-HERVK encodes the HERV-K glycoprotein, which contains the signal peptide (SP), surface (SU) subunit, transmembrane subunit (TM), and membrane-spanning domain of HERV-K env, and cytoplasmic tail of VSV G. (B) Schematic of haploid genetic screen. HAP1 cells were subjected to insertional mutagenesis, followed by selection with VSV-HERVK. Surviving cells were deep sequenced to identify the position of insertion sites. The number of insertions per gene in the selected set was compared to that of an unselected set to identify genes that were associated with survival of infection. (C) Screen results. The y-axis indicates the significance of enrichment of gene-trap insertions compared with unselected control cells. Circles represent individual genes and their size corresponds to the number of unique insertion sites in the selected population. Genes with significance scores above 10 are colored according to function and grouped horizontally. Genes with significance score above 25 are labeled. The number of unique insertions is shown in parentheses. (D) HAP1 cells were gene edited to lack the indicated genes and infected with VSV or VSV-HERVK. The fold difference in percent infected cells (top) and mean fluorescence intensity (MFI, bottom) of VSV-HERVK infected cells normalized to that of VSV is shown. Error bars represent standard error of the mean (SEM) for at least three independent experiments.
Fig 2.
Heparan sulfate facilitates HERV-K Env-mediated entry and attachment.
(A) WT HAP1, EXT1KO Neor Ctrl (transduced with a control retrovirus), EXT1KO EXT1-HA (+ addback), SLC35B2KO Neor Ctrl and SLC35B2KO SLC25B2-HA (+ addback) cells were infected with VSV-HERVK or VSV and infectivity analyzed by flow cytometry. Fold difference in percent infected cells compared to WT for VSV-HERVK was normalized to that of VSV for each condition. Error bars represent SEM for four independent experiments. (B) Relative MFI of cells from (A). Data were normalized as in (A). (C) BSRT7 cells were treated with 50mM sodium chlorate and infected with either VSV or VSV-HERVK. Fold difference in both percent infected cells (left) and MFI (right) compared to untreated cells for VSV-HERVK was normalized to that of VSV. Error bars represent SEM for three independent experiments. (D) VSV or VSV-HERVK was incubated with the indicated soluble glycosaminoglycans prior to infecting BSRT7 cells. Percent infected cells was normalized to untreated virus controls. Error bars represent SEM for three independent experiments. (E) Schematic of virus attachment experiment. Cells were blocked with BSA then incubated with both fluorescently labeled VSV-HERVK and VSV at either 37°C or 4°C. (F) Representative images from 4°C attachment experiment. Red: VSV. Green: VSV-HERVK. Blue: DAPI. Grey: calcein. (G) Results of attachment experiment. Numbers of particles/μm2 are plotted. Grey circles indicate outliers. Total number of particles counted per condition is indicated above each box.
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.
Fig 4.
Acidic pH is sufficient to trigger HERV-K Env.
(A) HERV-K Env is inactivated by exposure to acidic pH. VSV-HERVK and VSV were incubated in buffer at the indicated pH for 30 minutes at 37°C. Samples were returned to neutral pH before infecting BSRT7 cells. Cells were collected 5 hours post infection and percent GFP-expressing cells quantified by flow cytometry. Values are normalized to the pH 7 condition. Error bars represent SEM from three independent experiments. (B) VSV-HERVK fuses at the plasma membrane when treated with acidic pH. BSRT7 cells were pre-treated with bafilomycinA1 prior to binding virus at 4°C. Cells were treated with buffer at pH 7 or 5. Unbound virus was washed off and cells were collected 6 hours post-infection. Percent infected cells was normalized to cells not treated with bafilomycinA1. Error bars represent SEM for three independent experiments. (C) Endogenous HERV-K Envs are fusogenic at acidic pH. BSRT7 cells were transfected with envs from Phoenix, Xq21.33, and HERV-K 108 and subsequently exposed to buffer at the indicated pH. Syncytia are highlighted with arrows. Data are from a single representative experiment. (D) Endogenous HERV-K Envs bind heparin. 293T cells were transfected with the envs from Phoenix, Xq21.33, HERV-K 108, or VSV G. Cell lysates were incubated with either heparin (H) or protein A (A) beads and bound protein analyzed by Western blot against HERV-K Env and VSV G. T: 10% of total input. Data are from a single representative experiment.
Fig 5.
Proposed model of HERV-K entry.
We propose a 3-step model for HERV-K entry: 1. HERV-K binds heparan sulfate on the cell surface to attach to the cell. 2. The virus is taken up by dynamin-dependent, clathrin-independent endocytosis. 3. Exposure to low pH following endosomal acidification triggers Env to fuse the viral and cellular membranes, releasing the viral core into the cytoplasm.