Fig 2: Impacts of serine incorporator 5 (SERINC5)-induced non-glycosylated LHB on virion secretion was similar to those of glycan mutant. HepG2 cells were co-transfected with pHBV1.3-ENV– and LHB-flag proteins or the indicated glycan mutant N146 plus a negative control vector VR1012 or the SERINC5-HA. Cells and culture supernatant were harvested at day 5 post-transfection. (A) Immunoblot analysis of SERINC5, LHB wild-type (WT), and N146 mutant expression using anti-HA or anti-flag antibodies; tubulin served as a loading control. Quantitation of bands corresponding to the proteins was analyzed with ImageJ software and was shown below each panel. (B) The culture supernatants were monitored by hepatitis B surface antigen (HBsAg) ELISA. HepG2 cells co-transfected with pHBV1.3-ENV– and LHB WT proteins plus a negative control vector VR1012 was set as 100% (n = 3, mean ± SD, ***P < 0.001, paired t-test). (C) Virion DNA from culture supernatant was detected by qPCR following immunoprecipitation with anti-S Abs, HepG2 cells co-transfected with pHBV1.3-ENV– and LHB WT proteins plus a negative control vector VR1012 were set as 100% (n = 3, mean ± SD, **P < 0.01, ***P < 0.001, paired t-test). (D) Quantification of intracellular hepatitis B virus (HBV) DNA and the total RNA in cell lysates were detected by Southern blot and Northern blot. (E) LHB and the glycan mutant of LHB proteins interacted with SERINC5 by a co-immunoprecipitation (co-IP) assay. HEK293T cells were co-transfected as indicated. At 48 h post-transfection, the cells were harvested and co-IP with anti-flag plus protein G beads. The cell lysates and co-IP products were analyzed by immunoblotting.

Fig 3: The effect of serine incorporator 5 (SERINC5) on hepatitis B virus (HBV) production in HepG2.2.15 cells. (A–D) HepG2.2.15 cells were transfected with a negative control vector VR1012 or SERINC5-HA and harvested for detection at day 3. (E–H) The depletion of SERINC5 increased HBV production in HepG2 cells. HepG2 cells were co-transfected with pHBV1.3 and the negative control siRNA (siNC) or siRNA targeting SERINC5 (siSERINC5). (I–L) The effect of SERINC5 on HBV production in HepG2-NTCP. HepG2-NTCP cells were transfected with a negative control vector VR1012 or SERINC5-HA at day 1 post-infection with HBV virions from HepAD38. The cells were harvested for detection at day 5. (A,E,I) Immunoblot analysis of SERINC5 using anti-HA antibody or anti-SERINC5 antibody, tubulin served as a loading control. (B,J) The culture supernatants were monitored by hepatitis B surface antigen (HBsAg) ELISA. HBsAg in the absence of SERINC5 was set as 100%. (C,K) Virion DNA from culture supernatant was detected by qPCR following the immunoprecipitation with anti-S Abs conjugated to protein G beads. HBV virions DNA in the absence of SERINC5 were set as 100%. (D,H,L) Core particle-associated HBV DNA and the total RNA in cell lysates were detected by Southern blot and Northern blot. All of the above results are representative of three independent experiments and presented as the mean ± SD (P-values: ** < 0.01 and *** < 0.001). (F) The knockdown of SERINC5 increased the levels of secreted HBsAg in the supernatant. HBsAg in the supernatant treated with siNC was set as 100% (n = 3, mean ± SD, ***P < 0.001, paired t-test). (G) Virion DNA from culture supernatant was detected by qPCR following the immunoprecipitation with anti-S Abs conjugated to protein G beads. HBV DNA in siNC group was set as 100% (n = 3, mean ± SD, **P < 0.01, paired t-test).

Fig 4: The effect of serine incorporator 5 (SERINC5) on hepatitis B virus (HBV) envelope proteins. (A) SERINC5 induced the non-glycosylated LHB, MHB, and SHB proteins in HepG2 cells. HepG2 cells were co-transfected with LHB-flag, MHB-flag, or SHB-flag protein plus a negative control vector VR1012 or the SERINC5-HA. Immunoblot analysis of SERINC5, LHB, MHB, and SHB expression using anti-HA or anti-flag antibodies. The glycosylated (gp or ggp) and non-glycosylated (p) forms of LHB (L), MHB (M), and SHB (S) proteins are indicated. Tubulin served as a loading control. Quantitation of bands corresponding to the proteins of interest was performed using the ImageJ software and were shown below each panel (n = 3, mean ± SD). (B) Inhibitors MG132 and thapsigargin had no effect on the function of SERINC5-induced the non-glycosylation of LHB and MHB proteins in HepG2 cells. Twelve hours prior to cell harvest, the cells were treated with 10 µM of the proteasome inhibitor MG132 and 3 µM of the autophage-lysosome inhibitor thapsigargin. (C) The effect of SERINC5 on HIV-1 Env expression. HEK293T cells were co-transfected with Env or NL4-3 plus a negative control vector VR1012 or SERINC5-HA. Immunoblot analysis of SERINC5 and Env expression using anti-HA or anti-Env antibodies. (D) SERINC1 had no effect on the glycosylation of LHB, MHB, and SHB proteins. HepG2 cells were co-transfected with LHB-flag, MHB-flag, or SHB-flag protein plus a negative control vector VR1012 or the SERINC1-HA. Immunoblot analysis of SERINC1, LHB, MHB, and SHB expression using anti-HA or anti-flag antibodies. Tubulin served as a loading control.

Fig 5: The functional domain of serine incorporator 5 (SERINC5) required for hepatitis B virus (HBV) inhibition. (A) Schematic diagram of the transmembrane domains of SERINC5 and its mutants. The structure was predicted by Transmembrane Prediction using Hidden Marow Models (TMHMM) on a public server (http://www.cbs.dtu.dk/services/TMHMM-2.0/). (B–D) The effect of wild-type (WT) SERINC5 and SERINC5 truncated mutants on hepatitis B surface antigen (HBsAg) and HBV virion secretion. HepG2 cells were co-transfected as indicated. (B) Immunoblot analysis of SERINC5 and SERINC5 mutants using anti-HA antibody, tubulin served as a loading control. (C) The culture supernatants were monitored by HBsAg ELISA. HBsAg in the absence of SERINC5 was set as 100%. (D) Virion DNA from culture supernatant was detected by qPCR following the immunoprecipitation with anti-S Abs conjugated to protein G beads. HBV virion DNA in the absence of SERINC5 was set as 100% (n = 3, mean ± SD, **P < 0.01, ***P < 0.001, paired t-test). The effect of WT SERINC5 and SERINC5 mutants on the glycosylation of LHB (E) and MHB (F) proteins. HEK293T cells were co-transfected as indicated and then harvested for immunoblot analysis 48 h post-transfection. Immunoblot analysis of SERINC5, LHB, and MHB expression using anti-HA or anti-flag antibodies. The glycosylated (gp or ggp) and non-glycosylated (p) forms of LHB (L), and MHB (M) proteins are indicated. Tubulin served as a loading control. Quantitation of bands corresponding to the proteins of interest was performed using the ImageJ software and was shown below each panel (n = 3, mean ± SD).