Ahmad I et al. (2019), The retroviral accessory proteins S2, Nef, and ...

XB-ART-55765

J Biol Chem 2019 Apr 26;29417:7013-7024. doi: 10.1074/jbc.RA119.007662.

Show Gene links Show Anatomy links

The retroviral accessory proteins S2, Nef, and glycoMA use similar mechanisms for antagonizing the host restriction factor SERINC5.

Ahmad I , Li S , Li R , Chai Q , Zhang L , Wang B , Yu C , Zheng YH .

???displayArticle.abstract???
Serine incorporator 5 (SERINC5) is a recently identified restriction factor that blocks virus entry but is antagonized by three unrelated retroviral accessory proteins. The S2 protein from equine infectious anemia virus (EIAV) has been reported to reduce SERINC5 expression at steady-state levels likely via the endocytic pathway; however, the precise mechanism is still unclear. Here, we investigated how EIAV S2 protein down-regulates SERINC5 compared with down-regulation induced by Nef from HIV-1 and glycoMA proteins from murine leukemia virus (MLV). Using bimolecular fluorescence complementation (BiFC) assay and immunoprecipitation (IP), we detected an interaction between S2 and SERINC5. We found that this interaction relies on the S2 myristoylation site, indicating that it may occur on the plasma membrane. S2 internalized SERINC5 via receptor-mediated endocytosis and targeted it to endosomes and lysosomes, resulting in a ubiquitination-dependent decrease in SERINC5 expression at steady-state levels. Both BiFC and IP detected a glycoMA-SERINC5 interaction, but a Nef-SERINC5 interaction was detected only by BiFC. Moreover, S2 and glycoMA down-regulated SERINC5 more effectively than did Nef. We further show that unlike Nef, both S2 and glycoMA effectively down-regulate SERINC2 and also SERINC5 from Xenopus tropicalis (xSERINC5). Moreover, we detected expression of the equine SERINC5 (eSERINC5) protein and observed that its expression is much weaker than expression levels of SERINC5 from other species. Nonetheless, eSERINC5 had a strong antiviral activity that was effectively counteracted by S2. We conclude that HIV-1, EIAV, and MLV share a similar mechanism to antagonize viral restriction by host SERINC5.

???displayArticle.pubmedLink??? 30862674
???displayArticle.pmcLink??? PMC6497950
???displayArticle.link??? J Biol Chem
???displayArticle.grants??? [+]

Species referenced: Xenopus tropicalis
Genes referenced: lamp1 rab11a rab5a rab5b rab5c rab5d rab7a rab7b serinc5

???attribute.lit??? ???displayArticles.show???

	Figure 1. S2 down-regulation of Ser5. A, WT and ΔN HIV-1 pseudoviruses were produced from 293T cells after transfection of 1 μg of Env expression vector pNLnΔBS and 1 μg of Env-deficient HIV-1 proviral vector pNLenCAT or pNLenCAT-Xh in the absence or presence of 1 μg of pBJ5-mSer5-FLAG and/or 2 μg of pcDNA3.1-S2-HA. Viruses were normalized by p24Gag ELISA, and viral infectivity was determined after infection of TZM-bI cells. The infectivity of WT viruses produced in the absence of Ser5 was set as 100%. B, 293T cells were transfected with 2 μg of pBJ5-mSer5-FLAG and 3 μg of pcDNA3.1-S2-HA or its empty vector. After 24 h, protein expression was detected by Western blotting. C, 293T cells were transfected with 0.1 μg of pCMV6-mSer5-FLAG and 2 μg of pcDNA3.1-S2-HA or its empty vector. After 24 h, cells were treated with MG132 (20 μm) or NH4Cl (20 μm) for 12 h, and the protein expression was analyzed by Western blotting. D, HeLa cells were transfected with 1 μg of pEGFP-N1-mSer5-FLAG and/or 2 μg of pcDNA3.1-S2-HA or pcDNA3.1-NefSF2-HA. The Ser5- and S2-cotransfected cells were treated with bafilomycin A1 (100 nm) for another 12 h. S2 and Nef proteins were stained with anti-HA followed by Alexa Fluor 647–conjugated goat anti-mouse and detected by confocal microscopy. E, HeLa cells were transfected with pEGFP-N1-mSer5-FLAG and pcDNA3.1-S2-HA or pcDNA3.1-NefSF2-HA. Cells were treated with bafilomycin A1 and stained with anti-HA as described (D). The frequency of S2/Ser5 and Nef/Ser5 double-positive cells was calculated by confocal microscopy, with that in treated cells set as 100%. Error bars in A, C, and E indicate S.E. from three independent experiments. **, p < 0.01.
	Figure 2. Crucial S2 residues for Ser5 down-regulation. A, S2 protein sequences from six different EIAV strains are aligned, including AAC03764 (WY), U01888 (PV), AAK21109 (LIA), GU385359 (FDD), AFW99166 (IRE), and AFV61766 (MIY). Red, blue, and black represent residues completely, partially, or not conserved, and dashes indicate deletions. Targeted residues for mutagenesis including Gly2, Trp10, Ser15, Glu22, and Leu26 are indicated by arrowheads. The putative EXXXLL, YXXL, and PXXP motifs are also indicated. B, 293T cells were transfected with 2 μg of pBJ5-iFLAG-mSer5 and 3 μg of pEGFP-N1 vector expressing the indicated S2 proteins. Levels of Ser5 expression on the surface of EGFP-positive cells were analyzed by flow cytometry. Results are shown as relative values, with the value of Ser5 in the presence of the pEFGP-N1 vector set as 100%. C, HeLa cells were transfected with 1 μg of pEGFP-N1-mSer5-FLAG and 3 of μg pcDNA3.1 vector expressing the indicated S2 proteins. Doubly transfected cells were treated with bafilomycin A1. Ser5 and S2 localizations were detected by confocal microscopy as in Fig. 1D. D, 293T cells were transfected with 0.1 μg of pCMV6-mSer5-FLAG and 3 μg of pcDNA3.1 vector expressing the indicated S2 proteins. The Ser5 protein expression was detected by Western blotting. The relative Ser5 expression levels are shown by quantifying their intensity on Western blots, with the value of Ser5 in the presence of pcDNA3.1 vector set as 100%. Error bars in B and D indicate S.E. from three independent experiments. , p < 0.01; *, p < 0.001.
	Figure 3. Detection of the S2–Ser5 interaction. A, 293T cells were transfected with 1 μg of pcDNA3.1-S2-HA, pcDNA3.1-glycoMA-HA, or pcDNA3.1-NefSF2-VN-HA alone or together with 100 ng of pBJ5-iHA-Ser5. Levels of S2, glycoMA, and Nef expression inside cells and levels of Ser5 expression on the cell surface were measured by flow cytometry. The relative Ser5 expression on the cell surface was calculated, with the value in the presence of pcDNA3.1 vector set as 100%. B, 293T cells were transfected with 0.5 μg of pCMV6-mSer5-FLAG and 12 μg of pcDNA3.1-S2-HA, pcDNA3.1-glycoMA-HA, or pcDNA3.1-NefSF2-HA. After immunoprecipitation by anti-FLAG, proteins in cell lysate (Input) and pulldown (IP) samples were analyzed by Western blotting. The relative Ser5 expression in cell lysate was calculated by quantifying the intensity on Western blots, with the value in the presence of the pcDNA3.1 vector set as 100%. C, 293T cells were transfected with 0.5 μg of pCMV6-mSer5-FLAG and 12 μg of pcDNA3.1 expressing WT or the indicated mutant S2 proteins. Proteins were pulled down and analyzed similarly. Error bars in A and B indicate S.E. from three independent experiments. ***, p < 0.001.
	Figure 4. Detection of Ser5 endocytosis. A, 293T cells were transfected with 0.1 μg of pCMV6-mSer5-FLAG and 3 μg of pcDNA3.1-S2-HA in the presence of 1 μg of AP-2α or AP-2σ expression vector. AP-2α and AP-2σ were detected by anti-V5, Ser5 was detected by anti-FLAG, and S2 was detected by anti-HA. The relative Ser5 expression was calculated by quantifying the intensity on Western blots, with the value in the presence of pcDNA3.1 vector only set as 100%. B, 293T cells were transfected with 0.1 μg of pCMV6-mSer5-FLAG and 3 μg of pcDNA3.1-S2-HA in the presence of 4 μg of AP-2σ shRNA expression vector. Ser5 was detected by Western blotting. C, 293T cells were transfected with 1 μg of pBJ5-iFLAG-mSer5 and 3 μg of pcDNA3.1-S2-HA, and Ser5 endocytosis was detected at 4 and 37 °C. The levels of Ser5 endocytosis in the presence of S2 at 37 °C were set as 100%. Error bars in A and C indicate S.E. from three independent experiments. , p < 0.05; , p < 0.01; **, p < 0.001.
	Figure 5. Ser5 is targeted to endosomes. A, HeLa cells were transfected with 1 μg of pcDNA3.1-mSer5-FLAG-VC and 3 μg of pcDNA3.1-S2-VN-HA. After 24 h, cells were incubated with anti-FLAG or anti-HA followed by staining with Alexa Fluor 647–conjugated goat anti-mouse. The BiFC and immunofluorescence signals were detected by a confocal microscopy. B, HeLa cells were transfected with 1 μg of pCMV-mRFP-HA-Rab5a, pCMV-DsRed-2xHA-Rab7a, or pCMV-DsRed-2xHA-Rab11a in the presence of 1 μg of pEGFP-N1-mSer5-FLAG or 1 μg of pcDNA3.1-mSer5-FLAG-VC plus 3 μg of pcDNA3.1-S2-VN-HA. Fluorescence signals were detected by confocal microscopy. C, the colocalization of Ser5 with Rab small GTPases in B was statistically analyzed. Error bars indicate S.E. from three independent experiments. D and E, 293T cells were transfected with 0.1 μg of pCMV6-mSer5-FLAG and 3 μg of pcDNA3.1-S2-HA or its control vector in the presence of either 4 μg of Rab5, Rab7, and Rab11 shRNA expression vectors (D) or 1 μg of their expression vectors (E). Protein expressions were determined by Western blotting. ***, p < 0.001.
	Figure 6. Ser5 is targeted to lysosomes. A, 293T cells were transfected with 0.1 μg of pCMV6-mSer5-FLAG and 3 μg of pcDNA3.1-S2-HA or its control vector in the presence of 1 μg of WT Ub or its mutant (UbK48R or UbK63R) expression vectors. Protein expressions were detected by Western blotting. B, Ser5 and the indicated S2 proteins were expressed, and proteins were pulled down and analyzed as in Fig. 3B. C, HeLa cells were transfected with 1 μg of pCMV-LAMP1-mRFP in the presence of 1 μg of pEGFP-N1-mSer5-FLAG or 1 μg of pcDNA3.1-mSer5-VN-HA plus 3 μg of pcDNA3.1-S2-FLAG-VC. Fluorescence signals were detected by confocal microscopy.
	Figure 7. Analysis of eSer5 antiviral activity and sensitivity to S2. A, WT and ΔN HIV-1 pseudoviruses were produced from 293T cells in the absence or presence of 0.2 μg of pcDNA3.1-mSer5-FLAG or pcDNA3.1-eSer5-FLAG. Viral infectivity was measured and presented as in Fig. 1A. B, ΔN HIV-1 pseudoviruses were produced from 293T cells in the presence of pcDNA3.1-mSer5-FLAG or pcDNA3.1-eSer5-FLAG and the indicated S2 expression vectors. Viral infectivity was measured and presented as in Fig. 1A. C, 293T cells were transfected with 1 μg of pcDNA3.1-eSer5-FLAG, pcDNA3.1-mSer5-FLAG, or pcDNA3.1-hSer5-FLAG. Lysate from cells expressing human and murine Ser5 was diluted as indicated, and Ser5 expressions were compared by Western blotting. D, 293T cells were transfected with 3 μg of pcDNA3.1-S2-HA and 4 μg of pCMV6-eSer5-FLAG or 0.2 μg of pCMV6-mSer5-FLAG. Ser5 and S2 expressions were determined by Western blotting. Error bars in A and B indicate S.E. from three independent experiments. ***, p < 0.001.
	Figure 8. Broadness of the S2 antagonism. A, 293T cells were transfected with 0.2 μg of pCMV6-mSer1-FLAG, pCMV6-mSer2-FLAG, pCMV6-mSer3-FLAG, or pCMV6-hSer5-FLAG in the presence of 3 μg of pcDNA3.1-S2-HA or its control vector. SERINC and S2 expressions were determined by Western blotting. B, 293T cells were transfected with 3 μg of pcDNA3.1-S2-HA, pcDNA3.1-glycoMA-HA, pcDNA3.1-NefSF2-HA, or its control vector and 0.2 μg of pCMV6-mSer5-FLAG, pCMV6-Ser5-xICL4-FLAG, or pCMV6-xSer5-FLAG, and their expressions were detected by Western blotting.

References [+] :

Ahi, Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins. 2016, Pubmed
Alvarado, Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment. 2014, Pubmed
Bentham, Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein. 2006, Pubmed
Chande, S2 from equine infectious anemia virus is an infectivity factor which counteracts the retroviral inhibitors SERINC5 and SERINC3. 2016, Pubmed
Cook, Equine infectious anemia and equine infectious anemia virus in 2013: a review. 2013, Pubmed
Craig, Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity. 1998, Pubmed
Dai, A Long Cytoplasmic Loop Governs the Sensitivity of the Anti-viral Host Protein SERINC5 to HIV-1 Nef. 2018, Pubmed
Deacon, Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. 1995, Pubmed
de Sousa-Pereira, The antiviral activity of rodent and lagomorph SERINC3 and SERINC5 is counteracted by known viral antagonists. 2019, Pubmed
Fagerness, The S2 accessory gene of equine infectious anemia virus is essential for expression of disease in ponies. 2006, Pubmed
Greenberg, A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4. 1998, Pubmed
Inuzuka, Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis. 2005, Pubmed
Kestler, Importance of the nef gene for maintenance of high virus loads and for development of AIDS. 1991, Pubmed
Kirchhoff, Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. 1995, Pubmed
Li, Murine Leukemia Virus Glycosylated Gag Reduces Murine SERINC5 Protein Expression at Steady-State Levels via the Endosome/Lysosome Pathway to Counteract SERINC5 Antiretroviral Activity. 2019, Pubmed
Li, The S2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies. 2000, Pubmed
Li, A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses. 2003, Pubmed
Park, Adaptor protein complexes and intracellular transport. 2014, Pubmed
Payne, Characterization of infectious molecular clones of equine infectious anaemia virus. 1994, Pubmed
Rosa, HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. 2015, Pubmed
Saksela, Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. 1995, Pubmed
Shi, HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System. 2018, Pubmed
Sood, SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins. 2017, Pubmed
Trautz, The Antagonism of HIV-1 Nef to SERINC5 Particle Infectivity Restriction Involves the Counteraction of Virion-Associated Pools of the Restriction Factor. 2016, Pubmed
Usami, SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef. 2015, Pubmed
Zhang, Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction. 2017, Pubmed
Zheng, Mutations occurring during serial passage of Japanese equine infectious anemia virus in primary horse macrophages. 2000, Pubmed
Zheng, Insertions, duplications and substitutions in restricted gp90 regions of equine infectious anaemia virus during febrile episodes in an experimentally infected horse. 1997, Pubmed
Zheng, In vivo dynamics of equine infectious anemia viruses emerging during febrile episodes: insertions/duplications at the principal neutralizing domain. 1997, Pubmed