Fig 1.
MLV-glycoGag antagonizes Ser5 much more effectively than HIV-1 Nef.
A) The MLV-Gag precursor (p65) is translated from a full-length genomic RNA and cleaved into the matrix (MA), p12, capsid (CA), and nucleocapsid (NC). The glycoGag (p80) translation is initiated from a CUG codon upstream of Gag, containing three N-glycosylation sites (N113, N480, N505, highlighted in red). This glycoprotein is predicted to function as a type II transmembrane protein, as suggested by the transmembrane hidden Markov model (TMHMM). B) Nef-defective HIV-1 were produced from HEK293T cells after transfection with 1 μg Nef and Env-deficient HIV-1 proviral pNL-∆E∆N vector, 500 ng HIV-1 Env expression vector pNLnΔBS, 50 ng pCMV6-Ser5, and 3 μg pcDNA3.1-Nef or pcDNA3.1-glycoMA. After being normalized by p24Gag ELISA, viral infectivity was measured via infection of TZM-bI cells. Results were shown as relative values, with the infectivity of viruses produced alone set as 100. C) HEK293T cells were transfected with a Ser5, Nef, and glycoMA expression vector, and protein expression was determined by western blotting (WB) using an anti-HA antibody. Levels of Ser5, Nef, and glycoMA protein expression were quantified by ImageJ and are shown as relative values, with the value of Ser5 alone or Nef set as 1. Error bars represent standard error of measurements (SEMs) calculated from two or three experiments. n = 2 (B) or n = 3 (C); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 2.
MLV glycoGag is rapidly turned over in proteasomes.
A) NIH3T3 cells were infected with MLV. After 24 hrs of infection, cells were treated with 50 μM CHX or dimethyl sulfoxide (DMSO) as a vehicle control. Cells were collected at the indicated time points and the protein expression was analyzed by WB. Levels of viral proteins (glycoGag, p30, p65) were quantified using ImageJ. Results are shown as relative values, with the values at time zero set as 1. B) Nef, glycoMA, and glycoGag were expressed in HEK293T cells and treated with 50 μM CHX or DMSO. Their expression and stability were determined and quantified similarly. C) Nef, glycoMA, and glycoGag were expressed in HEK293T cells and treated with 10 mM 3-methyladenine (3-MA), 20 μM LY294002 (Ly), 100 nM bafilomycin A1 (Baf-A1), or 20 μM MG132. Their expression was analyzed by WB and quantified by Image J. Results are shown in relative values, with the values in the absence of CHX treatment set as 1. D) Nef, glycoMA, and glycoGag were expressed with Cas9 in HEK293T cells in the presence of Cul3- and/or KLHL20-specific sgRNAs. Protein expression was determined by WB. Error bars represent SEMs calculated from three experiments. n = 3 (C); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 3.
Ser5 stabilizes glycoGag by targeting it to the plasma membrane.
A) Nef with a C-terminal GFP-tag, glycoMA with a C-terminal HA-tag, and glycoGag with a Myc-tag in the p12-coding region were expressed in HeLa cells. After 24 hrs, cells were stained with DAPI for the nuclei and with anti-HA or anti-Myc followed by Alexa Fluor 488-conjugated goat anti-mouse antibodies. Nef, glycoMA, and glycoGag expression were detected by confocal microscopy (scale bar 5 µm). B) Ser5-FLAG was expressed with Nef-HA or glycoMA-HA in HEK293T cells. Cells were separated into cytoplasmic (Cyto) and plasma membrane (Mem) fractions, where Ser5, Nef, and glycoMA expression were detected by WB. C) Experiments were repeated as in (B). Proteins were immunoprecipitated (IP) from Mem and Cyto fractions by anti-FLAG beads and detected by WB. D) Nef, glycoMA, and glycoGag were expressed alone or with Ser5 in HEK293T cells. After treatment with 50 μM CHX, cells were collected at the indicated time points and protein expression was analyzed by WB. Levels of Nef, glycoMA, and glycoGag expression were quantified using ImageJ. Results are shown as relative values, with the values at time zero in the presence of Ser5 set as 1. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 4.
MLV glycoGag downregulates Ser5 in the cytoplasm.
A) Ser5 and mSer5 amino acid sequences containing the conserved lysine (K130, K131) are aligned. The ICL1 and TMD3 regions are indicated. Dots indicate identical residues. B) Ser5 and mSer5 WT proteins and their lysine mutants (K130R, K131R) with a GFP-tag were expressed in HeLa cells and detected by confocal microscopy (scale bar 5 µm). C) Ser5 and mSer5 proteins and their lysine mutants were expressed in HEK293T cells and their expression in Mem and Cyto fractions were analyzed by WB. D) MLV∆glycoGag and HIV-1∆Nef luciferase-reporter viruses were produced from HEK293T cells in the presence of indicated Ser5 proteins. After infection of NIH3T3 or TZM-bI cells, their infectivity was compared by measuring intracellular firefly luciferase activity. Results are normalized by the p30Gag or p24Gag protein levels of virions and presented as relative values, with the infectivity of viruses produced in a control (Ctrl) vector set as 100. Error bars indicate SEMs calculated from two independent experiments. E) Ser5, mSer5, and their lysine mutants with a FLAG-tag were expressed with Nef or glycoMA with an HA-tag in HEK293T cells. Protein expression was analyzed by WB. 19K/R, Ser5 with all 19 lysine residues replaced with arginine; 23K/R, mSer5 with all 23 lysine residues replaced with arginine. F) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells, where Cas9 and KLHL20- or Cul3-specific sgRNA was also expressed. Protein expression was detected by WB. G) Ser5 K130R-FLAG was expressed with Nef-HA or glycoMA-HA, and alternatively, mSer5 and its K131R mutant with a FLAG-tag were expressed with glycoGag-HA in HEK293T cells. Proteins were pulled down by anti-FLAG and analyzed by WB. H) Ser5 and its S249A and S360A mutants were expressed with Nef or glycoMA in HEK293T cells. Protein expression was analyzed by WB. I) Ser5 was expressed with a CCNK-shRNA or CDK13-shRNA expression vector and a Nef or glycoMA expression vector in HEK293T cells. Protein expression was analyzed by WB. J) Ser5-GFP and its mutants S360A andS249A were expressed with HA-tagged Nef or glycoMA in HeLa cells. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) for the nuclei (blue) and with anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies for viral proteins and observed by confocal microscopy (scale bar 5 µm). Error bars represent SEMs calculated from two independent experiments. n = 2 (C); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 5.
MLV glycoGag targets cytoplasmic Ser5 to lysosomes for degradation.
A) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells and treated with 20 μM MG132 or 100 nM Baf-A1. Protein expression was analyzed by WB. B) Ser5-GFP or K130R-GFP was expressed with LAMP1-mCherry in HeLa cells in the presence of Nef or glycoMA. Their co-localization with LAMP1 was determined by confocal microscopy (scale bar 5 μm). Pearson’s Correlation Coefficient (PCC) was calculated and shown below. C) Nef and Ser5 with a C-terminal GFP-tag were expressed with calreticulin (CALR) with a mCherry-tag in HeLa cells. Alternatively, glycoMA and glycoGag with an HA-tag were expressed with CALR with a GFP-tag in HeLa cells. GlycoMA and glycoGag were stained with mouse anti-HA followed by Alexa Fluor 594-conjugated goat anti-mouse antibodies. Co-localization of Nef, Ser5, glycoMA, and glycoGag with CALR was determined by confocal microscopy (scale bar 5 µm). PCC was calculated and shown below. D) Indicated proteins with a FLAG or HA-tag were expressed in HEK293T cells and immunoprecipitated with anti-FLAG or anti-HA beads. Affinity-purified (AP) samples were analyzed by mass spectrometry (MS). Identified ER-phagy receptors are listed. Error bars represent SEMs calculated from three experiments. n = 3 (B) (C); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 6.
RETREG1 enhances glycoGag downregulation of Ser5.
A) Ser5 was expressed with Nef in the presence of indicated ER-phagy receptors in HEK293T cells, and their expression was determined by WB. B) Ser5 was expressed with glycoGag in the presence of indicated ER-phagy receptors in HEK293T cells, and their expression was determined by WB. C) Ser5 expression was quantified from (A) and (B) by ImageJ and presented. Error bars represent SEMs calculated from three experiments. n = 3 (C); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 7.
RETREG1 is required for glycoGag downregulation of cytoplasmic Ser5.
A) Ser5 K130R was expressed with glycoGag in the indicated ER-phagy receptor-knockout (KO) cells, and their expression was determined by WB. B) K130R was expressed with glycoGag in HEK293T cells in the presence of small interfering RNA (siRNA) #1 and #2 that specifically silence RETREG2. Ser5 and glycoGag expression were determined by WB. C) K130R was expressed with glycoGag in HEK293T RETREG1 (R1)-KO cells in the presence of indicated ectopic ER-phagy receptors, and their expression was determined by WB. D) Ser5, mSer5, K130R, and K131R with a FLAG-tag were expressed with glycoGag-HA in HEK293T WT or R1-KO cells. Protein expression was analyzed by WB. E) Nef-HA and glycoGag-HA were expressed alone or with Ser5-FLAG in HEK293T cells. Alternatively, glycoGag-HA was expressed mSer5-FLAG in HEK293T cells. Proteins were pulled down by anti-FLAG or anti-HA beads. The endogenous RETREG1 in these samples was detected by WB using a specific antibody. Protein expression in (A), (C), and (D) was quantified and presented. Error bars represent SEMs calculated from two or three experiments. n = 2 (C) or n = 3 (A) (D); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 8.
RETREG1 is required for MLV antagonism of Ser5.
A) NIH3T3 cells were transfected with 40 nM siRNAs, and after 12 hrs, these cells were transfected with 200 ng Ser5 expression vector. These cells were infected with MLV, and 6 hrs later, protein expression was determined by WB 24 hrs post-infection. B) HEK293T WT and RETREG1-KO cells were transfected with 1 μg pNL-∆E∆N vector, 500 ng pNLnΔBS, 50 ng pCMV6-Ser5, and 3 μg pcDNA3.1-Nef or pcDNA3.1-glycoMA. Viral infectivity was analyzed in TZM-bI cells and presented as before. Ser5 expression in (A) was quantified and presented as relative values. Error bars represent SEMs calculated from two or three experiments. n = 3 (A) or n = 6 (B); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.
Fig 9.
MLV glycoGag targets Ser5 to micro-ER-phagy for degradation.
A) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T WT and indicated knockout cells that do not express ATG3, ATG5, ATG7, BECN1, SQSTM1/p62, or PIK3C3. Protein expression was analyzed by WB. B) Ser5 and K130R were expressed with Nef or glycoMA in HEK293T cells and treated with 10 mM 3-MA, 20 μM Ly, or 100 nM Baf-A1. Protein expression was analyzed by WB. C) A proposed model for MLV glycoGag downregulation of Ser5. GlycoGag is expressed as a type II integral membrane protein in the ER, where it interacts with Ser5. This interaction leads to the recruitment of RETREG1, resulting in Ser5 degradation via micro-ER-phagy (1). If Ser5 is not cleared within the ER, it proceeds to the Golgi apparatus and is subsequently delivered to the cell surface after undergoing polyubiquitination. Throughout this process, glycoGag is also recruited to the cell surface by Ser5. At the cell surface, both glycoGag and HIV-1 Nef can internalize Ser5 through early endosomes (EE) and late endosomes (LE), culminating in degradation within endolysosomes (2). Ser5 expression in (B) was quantified and presented. Error bars represent SEMs calculated from three experiments. n = 3 (B); One-way ANOVA test: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Unless indicated, all experiments were repeated three times, and representative experiments are shown.