RNA-Protein Interaction Analysis of SARS-CoV-2 5′ and 3′ Untranslated Regions Reveals a Role of Lysosome-Associated Membrane Protein-2a during Viral Infection

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  FIG 1. Establishment of RaPID assay to identify the host interaction partners of SARS-CoV-2 5′-UTR and 3′-UTR RNA. (A) Schematic of predicted secondary structure of SARS-CoV-2 5′-UTR RNA. An asterisk denotes an unannotated stem-loop in the 5′ UTR. SL-A and SL-B represent BirA-binding RNA motifs, TRS denotes the transcriptional regulatory sequence, and SL-I to SL-V denote the stem-loops present in the 5′-UTR RNA. (B) Schematic of predicted secondary structure of SARS-CoV-2 3′-UTR RNA. SL-A and SL-B represent BirA-binding RNA motifs, HVR denotes the hypervariable region, S2M denotes the stem-loop II-like motif, and SL-I to SL-IV denote the stem-loops present in the 3′-UTR RNA. (C) Schematic of RaPID assay workflow. B, biotin; A, agarose; S, streptavidin. (D) Western blot detection of BirA ligase and GAPDH level in HEK293T cells transfected with the RaPID plasmid for 48 h. (E) Western blot detection of biotinylated protein in HEK293T cells transfected with BirA ligase and treated with biotin for different times, as indicated. (F) Western blot detection of CUGBP1 protein in the whole-cell extract of HEK293T cells (WCE) and in streptavidin agarose pulldown of EDEN15 RNA-interacting…

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  FIG 2. Identification of SARS-CoV-2 5′-UTR and 3′-UTR RNA-interacting host proteins by RaPID assay. (A) Clustering analysis of correlation between biological replicates used in LC-MS/MS. (B) Venny analysis of 5′-UTR and 3′-UTR RNA-interacting proteins. (C) Schematic of RNA-protein interaction network of the 5′-UTR + 3′-UTR SARS-CoV-2 RNAs. Black nodes, 5′ UTR or 3′ UTR; blue or red nodes, host protein. Host proteins that interact with each other are indicated by yellow or green color inside the node. Common host proteins that interact with both 5′-UTR and 3′-UTR RNA are represented by dual (red and blue) colors.

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  FIG 3. Lamp2 associates with the 5′ end of the SARS-CoV-2 genome and modulates the level of viral RNA in infected Vero E6 cells. (A) Immunofluorescence staining of nucleocapsid protein (red) and nucleus (blue) in SARS-CoV-2-infected Vero E6 cells, 48 h postinfection. Anti-N denotes incubation with nucleocapsid antibody, and NS denotes incubation with normal rabbit serum (preimmune serum control), followed by incubation with Alexa Fluor 594 conjugated secondary antibody. (B) Western blot detection of nucleocapsid protein (upper image) and GAPDH (lower image) in SARS-CoV-2-infected Vero E6 cells, 48 h postinfection. (C) Level of SARS-CoV-2 RNA in the culture medium (secreted, normalized to that of RNase P [RP]) and inside Vero E6 cells (intracellular, normalized to that of RNA Pol II [RP II]) that are infected with SARS-CoV-2 for the indicated periods. Real-time PCRs were performed using a SYBR green-based protocol. Data are the mean ± SEM of triplicate samples. (D). Level of SARS-CoV-2 RNA in Vero E6 cells in culture medium that are infected with SARS-CoV-2 for the indicated periods. Real-time PCRs were performed using a TaqMan probe-based protocol. N1, N2, and RP represents two regions…

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  FIG 4. Silencing of Lamp2a promotes SARS-CoV-2 infection in mammalian cell culture. (A) Fluorescence in situ hybridization of SARS-CoV-2 5′-UTR RNA and Lamp2, at indicated periods postinfection. Lamp2 protein, SARS-CoV-2 RNA, and nucleus are denoted by green, red, and blue, respectively. Scale bar, 20 μM. (B) Western blot detection of Lamp2 (Lamp2 antibody, upper panel) and GAPDH (lower panel) proteins in Vero E6 cells transfected for 72 h with nontargeting (NT) siRNA or Lamp2 siRNA. (C) Level of SARS-CoV-2 RNA (normalized to that of RP) in culture medium of Vero E6 cells transfected with NT siRNA or pan-Lamp2 siRNA (Lamp2 siRNA) and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM. P value was calculated using a two-tailed Student t test. (D) Intracellular level of SARS-CoV2 RNA (normalized to that of RP II) in Vero E6 cells treated with pan-Lamp2 siRNA (Lamp2 siRNA) and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM. P value was calculated using a two-tailed Student t test. (E) Western blot detection of nucleocapsid protein (N, upper image) and GAPDH (lower image) in nontargeting (NT) or pan-Lamp2 (Lamp2) siRNA-treated SARS-CoV-2-infected Vero E6 cells, 48 h…

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  FIG 5. Lamp2a modulates viral RNA level in SARS-CoV-2-infected cells. (A) SARS-CoV-2 RNA level (normalized to that of RP) in culture medium of Vero E6 cells transfected with the indicated plasmids and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM of triplicate samples. P values were calculated using a two-tailed Student t test. (B) Intracellular level of SARS-CoV-2 RNA (normalized to that of RP II) in Vero E6 cells transfected with the indicated plasmids and infected with SARS-CoV-2 for 48 h. Data are the mean ± SEM of triplicate samples. P values were calculated using a two-tailed Student t test. (C) Western blot detection of Lamp2a protein level (using anti-HA antibody [upper panel] and anti-Lamp2 antibody [middle panel]) and GAPDH protein level (lower panel) in pCDNA3-transfected or pcDNA Lamp2a-transfected Vero E6 Cells. (D) Western blot detection of Lamp2b protein level using anti-Lamp2 antibody (upper panel) and GAPDH protein level (lower panel) in pCDNA3-transfected or pcDNA Lamp2b-transfected Vero E6 cells. (E) Western blot detection of Lamp2c protein level using anti-Lamp2 antibody (upper panel) and GAPDH protein level (lower panel) in pCDNA3-transfected or pcDNA…

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  FIG 6. Increased Lamp2 and LC3-II levels in SARS-CoV-2-infected Vero E6 and Huh7 cells do not promote autophagolysosome formation. (A) Western blot detection of indicated proteins in SARS-CoV-2-infected Vero E6 and Huh7 cells, 48 h postinfection. (B) Percent viability of Vero E6 cells treated for 48 h with 5 mM 3-MA or dimethylformamide (DMF) (vehicle). The value for the vehicle-treated sample was considered to be 100%, and other values were calculated with reference to that. Values are the mean ± SEM of triplicate samples. (C) Level of SARS-CoV-2 RNA (normalized to that of RP) in the culture medium of Vero E6 cells infected with SARS-CoV-2 and treated with 5 mM 3-MA or DMF (vehicle) for 48 h. Data are the mean ± SEM of triplicate samples. P value was calculated using a two-tailed Student t test. (D) Intracellular level of SRS-CoV-2 RNA (normalized to that of RP II) in Vero E6 cells treated with 5 mM 3-MA or DMF (vehicle) for 48 h. Data are the mean ± SEM of triplicate samples. P value was calculated using a two-tailed Student t test. (E) Percent viability of the Huh7 cells treated for 48 h with 5 mM 3-MA or DMF (vehicle). The value for the vehicle-treated sample was considered to be…