Autophagy protein ATG5 interacts transiently with the hepatitis C virus RNA polymerase (NS5B) early during infection

Abstract

Autophagy is an important cellular process by which ATG5 initiates the formation of double membrane vesicles (DMVs). Upon infection, DMVs have been shown to harbor the replicase complex of positive-strand RNA viruses such as MHV, poliovirus, and equine arteritis virus. Recently, it has been shown that autophagy proteins are proviral factors that favor initiation of hepatitis C virus (HCV) infection. Here, we identified ATG5 as an interacting protein for the HCV NS5B. ATG5/NS5B interaction was confirmed by co-IP and metabolic labeling studies. Furthermore, ATG5 protein colocalizes with NS4B, a constituent of the membranous web. Importantly, immunofluorescence staining demonstrated a strong colocalization of ATG5 and NS5B within perinuclear regions of infected cells at 2 days postinfection. However, colocalization was completely lacking at 5DPI, suggesting that HCV utilizes ATG5 as a proviral factor during the onset of viral infection. Finally, inhibition of autophagy through ATG5 silencing blocks HCV replication.

Fig. 1

The NS5BΔ21 protein interacts with the full-length human ATG5 in yeast two-hybrid assay. NS5B and ATG5 fused to the Gal4 DNA binding and activation domains, respectively, were double-transformed into AH109 cells. Four independent colonies of recombinant AH109 cells were allowed to grow for a few days on −Leu, −Trp SD medium (left panel), after which they were replica-plated onto −Leu, −Trp, −His, −Ade + X-αgal plates (right panel).

Fig. 2

Specific interaction of HCV NS5BΔ21 protein with ATG5 as observed by co-IP. A. Soluble yeast extracts containing NS5BΔ21 (N-terminal c-myc tag) and ATG5 (N-terminal HA tag) were incubated with different monoclonal antibodies and the immunoprecipitates were pulled down using protein A/G beads. Precipitated proteins were revealed by Western blot using anti-c-myc (left panel) or anti-HA (right panel) monoclonal antibodies. Note that IP of ATG5 using anti-HA monoclonal antibody coprecipitated NS5BΔ21 (lane 2). Anti-HCV NS5A (lane 1) or no antibody (beads only, lane 3) did not precipitate NS5BΔ21. Soluble extract loaded on the gel was used as the size marker for NS5BΔ21. IP of NS5BΔ21 using anti-c-myc antibody precipitated ATG5 (lane 5) but not anti-HCV NS5A antibody (lane 6). Soluble extract loaded on the gel was used as the size marker for ATG5 (lane 7). B. Soluble [³⁵S]Met-labeled protein extract was immunoprecipitated using anti-HA (lanes 1 and 2) or anti-c-myc (lanes 3 and 4). Extracts were incubated in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of cupric sulfate, which induced NS5BΔ21 expression. Precipitated bands were analyzed by SDS–PAGE followed by autoradiography.

Fig. 3

Mapping of the NS5BΔ21 and ATG5 binding domains. Results are indicated in the column on the right and were obtained by yeast two-hybrid assay (first two columns) or by co-IP (third column). Deletion mutants of NS5B were assessed for interaction with full-length ATG5. The binding area covered the N-terminal amino acids 450–570 as indicated by yeast two-hybrid assay and co-IP. Note that none of the deletion mutants of NS5BΔ21 self-activated in the yeast two-hybrid screen, as shown in the second column. nd indicates not done.

Fig. 4

A. Huh7 and C5B cells were grown for 48 h and costained with antibodies specific to ATG5 and NS4B protein. All samples were examined via confocal microscopy. Magnified areas (a) are indicated by rectangles. Notice the colocalization of NS4B with ATG5 in C5B cells. B. Subcellular fractionation of clone A cells (subgenomic replicon) transfected with GFP-ATG5. Proteins were resolved on SDS–PAGE and visualized by Western blot using a polyclonal rabbit anti-NS5B (left) or a monoclonal anti-GFP (right). N, nucleus; C, cytoplasm; M/M, microsome and mitochondria.

Fig. 5

A. Subcellular distribution of NS5B and ATG5 in HCVcc-infected cells. JFH1-infected Huh7 cells were transfected with the GFP-ATG5 DNA construct and analyzed at 2 or 5 DPI. Colocalization of ATG5 and NS5B was observed at 2 DPI (magnified area in b) but not at 5 DPI (magnified area in c). B. Silencing ATG5 reduced viral replication in Huh7 cells. Huh7 cells were transfected with siRNA targeting ATG5 or with a scramble siRNA as control. The cells were then infected for 2 days and analyzed for the presence of HCV core protein by Western blot. As expected, scramble siRNA had no effect on HCV replication, whereas ATG5 greatly reduced HCV protein expression. C. Quantification of intracellular HCV genome from samples in panel B. Mock, mock-infected cells.