SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef

Abstract

HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4+ T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Inhibition of incorporation of SERINC proteins into HIV-1 virions by Nef correlates with infectivity enhancement.
Figure 2: Effects of exogenous SERINCs on HIV-1 infectivity.
Figure 3: Effects of depleting SERINCs in virus producer cells.
Figure 4: Effects of SERINC knockout and reconstitution on HIV-1 infectivity.
Figure 5: Nef counteracts inhibition of HIV-1 replication by SERINC3 and SERINC5.

References

  1. 1

    Kestier, H. W. III et al. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65, 651–662 (1991)

    Article  Google Scholar 

  2. 2

    Deacon, N. J. et al. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270, 988–991 (1995)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Zou, W. et al. Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes. Retrovirology 9, 44 (2012)

    CAS  Article  Google Scholar 

  4. 4

    Kim, S., Ikeuchi, K., Byrn, R., Groopman, J. & Baltimore, D. Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc. Natl Acad. Sci. USA 86, 9544–9548 (1989)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Miller, M. D., Warmerdam, M. T., Gaston, I., Greene, W. C. & Feinberg, M. B. The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. J. Exp. Med. 179, 101–113 (1994)

    CAS  Article  Google Scholar 

  6. 6

    Spina, C. A., Kwoh, T. J., Chowers, M. Y., Guatelli, J. C. & Richman, D. D. The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. J. Exp. Med. 179, 115–123 (1994)

    CAS  Article  Google Scholar 

  7. 7

    Garcia, J. V. & Miller, A. D. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350, 508–511 (1991)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Aiken, C., Konner, J., Landau, N. R., Lenburg, M. E. & Trono, D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 76, 853–864 (1994)

    CAS  Article  Google Scholar 

  9. 9

    Rhee, S. S. & Marsh, J. W. Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4. J. Virol. 68, 5156–5163 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Chaudhuri, R., Lindwasser, O. W., Smith, W. J., Hurley, J. H. & Bonifacino, J. S. Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor. J. Virol. 81, 3877–3890 (2007)

    CAS  Article  Google Scholar 

  11. 11

    Veillette, M. et al. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity. J. Virol. 88, 2633–2644 (2014)

    Article  Google Scholar 

  12. 12

    Haller, C. et al. HIV-1 Nef and Vpu are functionally redundant broad-spectrum modulators of cell surface receptors, including tetraspanins. J. Virol. 88, 14241–14257 (2014)

    Article  Google Scholar 

  13. 13

    Schwartz, O., Marechal, V., Le Gall, S., Lemonnier, F. & Heard, J. M. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nature Med. 2, 338–342 (1996)

    CAS  Article  Google Scholar 

  14. 14

    Collins, K. L., Chen, B. K., Kalams, S. A., Walker, B. D. & Baltimore, D. HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391, 397–401 (1998)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Cohen, G. B. et al. The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10, 661–671 (1999)

    CAS  Article  Google Scholar 

  16. 16

    Schindler, M. et al. Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell 125, 1055–1067 (2006)

    CAS  Article  Google Scholar 

  17. 17

    Chowers, M. Y., Pandori, M. W., Spina, C. A., Richman, D. D. & Guatelli, J. C. The growth advantage conferred by HIV-1 nef is determined at the level of viral DNA formation and is independent of CD4 downregulation. Virology 212, 451–457 (1995)

    CAS  Article  Google Scholar 

  18. 18

    Münch, J. et al. Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses. J. Virol. 81, 13852–13864 (2007)

    Article  Google Scholar 

  19. 19

    Aiken, C. & Trono, D. Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J. Virol. 69, 5048–5056 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Schwartz, O., Marechal, V., Danos, O. & Heard, J. M. Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell. J. Virol. 69, 4053–4059 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Miller, M. D., Warmerdam, M. T., Page, K. A., Feinberg, M. B. & Greene, W. C. Expression of the human immunodeficiency virus type 1 (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp160 or viral entry. J. Virol. 69, 579–584 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Forshey, B. M. & Aiken, C. Disassembly of human immunodeficiency virus type 1 cores in vitro reveals association of Nef with the subviral ribonucleoprotein complex. J. Virol. 77, 4409–4414 (2003)

    CAS  Article  Google Scholar 

  23. 23

    Ross, T. M., Oran, A. E. & Cullen, B. R. Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein. Curr. Biol. 9, 613–621 (1999)

    CAS  Article  Google Scholar 

  24. 24

    Lama, J., Mangasarian, A. & Trono, D. Cell-surface expression of CD4 reduces HIV-1 infectivity by blocking Env incorporation in a Nef- and Vpu-inhibitable manner. Curr. Biol. 9, 622–631 (1999)

    CAS  Article  Google Scholar 

  25. 25

    Goldsmith, M. A., Warmerdam, M. T., Atchison, R. E., Miller, M. D. & Greene, W. C. Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef. J. Virol. 69, 4112–4121 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Pizzato, M. MLV glycosylated-Gag is an infectivity factor that rescues Nef-deficient HIV-1. Proc. Natl Acad. Sci. USA 107, 9364–9369 (2010)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Prats, A. C., De Billy, G., Wang, P. & Darlix, J. L. CUG initiation codon used for the synthesis of a cell surface antigen coded by the murine leukemia virus. J. Mol. Biol. 205, 363–372 (1989)

    CAS  Article  Google Scholar 

  28. 28

    Pillemer, E. A., Kooistra, D. A., Witte, O. N. & Weissman, I. L. Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane. J. Virol. 57, 413–421 (1986)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Usami, Y., Popov, S. & Gottlinger, H. G. The Nef-like effect of murine leukemia virus glycosylated gag on HIV-1 infectivity is mediated by its cytoplasmic domain and depends on the AP-2 adaptor complex. J. Virol. 88, 3443–3454 (2014)

    Article  Google Scholar 

  30. 30

    Usami, Y. & Gottlinger, H. HIV-1 Nef Responsiveness Is Determined by Env Variable Regions Involved in Trimer Association and Correlates with Neutralization Sensitivity. Cell Rep. 5, 802–812 (2013)

    CAS  Article  Google Scholar 

  31. 31

    Pizzato, M. et al. Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef. Proc. Natl Acad. Sci. USA 104, 6812–6817 (2007)

    ADS  CAS  Article  Google Scholar 

  32. 32

    Craig, H. M., Pandori, M. W. & Guatelli, J. C. Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity. Proc. Natl Acad. Sci. USA 95, 11229–11234 (1998)

    ADS  CAS  Article  Google Scholar 

  33. 33

    Inuzuka, M., Hayakawa, M. & Ingi, T. Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis. J. Biol. Chem. 280, 35776–35783 (2005)

    CAS  Article  Google Scholar 

  34. 34

    Grossman, T. R., Luque, J. M. & Nelson, N. Identification of a ubiquitous family of membrane proteins and their expression in mouse brain. J. Exp. Biol. 203, 447–457 (2000)

    CAS  PubMed  Google Scholar 

  35. 35

    Day, J. R., Munk, C. & Guatelli, J. C. The membrane-proximal tyrosine-based sorting signal of human immunodeficiency virus type 1 gp41 is required for optimal viral infectivity. J. Virol. 78, 1069–1079 (2004)

    CAS  Article  Google Scholar 

  36. 36

    Cavrois, M., De Noronha, C. & Greene, W. C. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nature Biotechnol. 20, 1151–1154 (2002)

    CAS  Article  Google Scholar 

  37. 37

    Aiken, C. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A. J. Virol. 71, 5871–5877 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Luo, T., Douglas, J. L., Livingston, R. L. & Garcia, J. V. Infectivity enhancement by HIV-1 Nef is dependent on the pathway of virus entry: implications for HIV-based gene transfer systems. Virology 241, 224–233 (1998)

    CAS  Article  Google Scholar 

  39. 39

    Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nature Methods 10, 957–963 (2013)

    CAS  Article  Google Scholar 

  40. 40

    Waheed, A. A. & Freed, E. O. The Role of Lipids in Retrovirus Replication. Viruses 2, 1146–1180 (2010)

    CAS  Article  Google Scholar 

  41. 41

    Lundquist, C. A., Tobiume, M., Zhou, J., Unutmaz, D. & Aiken, C. Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. J. Virol. 76, 4625–4633 (2002)

    CAS  Article  Google Scholar 

  42. 42

    Tobiume, M., Lineberger, J. E., Lundquist, C. A., Miller, M. D. & Aiken, C. Nef does not affect the efficiency of human immunodeficiency virus type 1 fusion with target cells. J. Virol. 77, 10645–10650 (2003)

    CAS  Article  Google Scholar 

  43. 43

    Cavrois, M., Neidleman, J., Yonemoto, W., Fenard, D. & Greene, W. C. HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion. Virology 328, 36–44 (2004)

    CAS  Article  Google Scholar 

  44. 44

    Cohen, F. S. & Melikyan, G. B. The energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement. J. Membr. Biol. 199, 1–14 (2004)

    CAS  Article  Google Scholar 

  45. 45

    Schaeffer, E., Geleziunas, R. & Greene, W. C. Human immunodeficiency virus type 1 Nef functions at the level of virus entry by enhancing cytoplasmic delivery of virions. J. Virol. 75, 2993–3000 (2001)

    CAS  Article  Google Scholar 

  46. 46

    Brandenberg, O. F., Magnus, C., Rusert, P., Regoes, R. R. & Trkola, A. Different infectivity of HIV-1 strains is linked to number of envelope trimers required for entry. PLoS Pathog. 11, e1004595 (2015)

    Article  Google Scholar 

  47. 47

    Sougrat, R. et al. Electron tomography of the contact between T cells and SIV/HIV-1: implications for viral entry. PLoS Pathog. 3, e63 (2007)

    Article  Google Scholar 

  48. 48

    Chojnacki, J. et al. Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy. Science 338, 524–528 (2012)

    ADS  CAS  Article  Google Scholar 

  49. 49

    Rodenburg, C. M. et al. Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents. AIDS Res. Hum. Retroviruses 17, 161–168 (2001)

    CAS  Article  Google Scholar 

  50. 50

    Collman, R. et al. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J. Virol. 66, 7517–7521 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Dorfman, T., Popova, E., Pizzato, M. & Gottlinger, H. G. Nef enhances human immunodeficiency virus type 1 infectivity in the absence of matrix. J. Virol. 76, 6857–6862 (2002)

    Article  Google Scholar 

  52. 52

    Dorfman, T., Mammano, F., Haseltine, W. A. & Gottlinger, H. G. Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J. Virol. 68, 1689–1696 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Akagi, T., Shishido, T., Murata, K. & Hanafusa, H. v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation. Proc. Natl Acad. Sci. USA 97, 7290–7295 (2000)

    ADS  CAS  Article  Google Scholar 

  54. 54

    Dettenhofer, M. & Yu, X. F. Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions. J. Virol. 73, 1460–1467 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Chesebro, B., Wehrly, K., Nishio, J. & Perryman, S. Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism. J. Virol. 66, 6547–6554 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Kelstrup, C. D., Young, C., Lavallee, R., Nielsen, M. L. & Olsen, J. V. Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer. J. Proteome Res. 11, 3487–3497 (2012)

    CAS  Article  Google Scholar 

  57. 57

    Accola, M. A., Strack, B. & Gottlinger, H. G. Efficient particle production by minimal gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. J. Virol. 74, 5395–5402 (2000)

    CAS  Article  Google Scholar 

  58. 58

    Abacioglu, Y. H. et al. Epitope mapping and topology of baculovirus-expressed HIV-1 gp160 determined with a panel of murine monoclonal antibodies. AIDS Res. Hum. Retroviruses 10, 371–381 (1994)

    CAS  Article  Google Scholar 

  59. 59

    Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols 3, 1101–1108 (2008)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank J. Leszyk and S. Shaffer for protein microsequencing, BGI Americas for RNA-seq, R. Maehr for sgRNA and Cas9 expression plasmids, J. Sodroski for HIVec2.GFP, T. Akagi for pCXbsr, and the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH for p89.6, indinavir, maraviroc, the monoclonal antibodies 183-H12-5C and Chessie 8, and TZM-bl cells. This work was supported by NIAID/NIH grant R01AI029873 and by NIDA/NIH grant DP1DA038034.

Author information

Affiliations

Authors

Contributions

Y.U., Y.W. and H.G.G. designed the experiments and analysed the data. Y.U. carried out the analysis of virions and the SERINC overexpression and depletion experiments. Y.W. generated and characterized the SERINC knockout cells, carried out all experiments involving knockout cells and primary cells, and performed the qRT–PCR experiments and the BlaM-Vpr-based fusion assays. H.G.G. wrote the manuscript.

Corresponding author

Correspondence to Heinrich G. Göttlinger.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Identification of SERINC3 as a candidate target of Nef and glycoGag.

a, Anti-HIV-1 CA immunoblot of Nef+, Nef and glycoMA+ HIV-1 virions collected from the indicated fractions of OptiPrep gradients. b, Proteins identified by mass spectrometry in Nef but not in Nef+ or glycoMA+ virion lysates. The data are from two independent experiments.

Extended Data Figure 2 MLV glycoGag inhibits the incorporation of SERINC3 and SERINC5 into HIV-1 virions.

a, b, Western blots showing the effects of wild-type or mutant glycoMA on the incorporation of SERINC3–HA (a) or SERINC5–HA (b) into Nef HIV-1 virions. The NL4-3/glycoMA proviral construct expresses untagged glycoMA in cis. In all other cases, HA-tagged (a) or Flag-tagged (b) glycoMA proteins were expressed in trans. The white bands marked by asterisks are caused by co-migrating HIV-1 Pr55gag. Both experiments were performed twice.

Extended Data Figure 3 Nef and glycoGag downregulate SERINC5 from the cell surface.

a, SERINC5 re-localizes from the plasma membrane to perinuclear vesicles in the presence of glycoGag. HeLa or U2-OS cells transiently expressing SERINC5–mCherry alone or together with glycoGag were examined by live-cell fluorescence microscopy. b, Nef and glycoGag both downregulate SERINC5. JTAg cells transiently expressing SERINC5(iHA), either alone or together with NefSF2 or glycoGag, were surface-stained with anti-HA antibody and analysed by flow cytometry. Per cent fractions of cells expressing SERINC5(iHA) on the surface are indicated. This experiment was performed twice.

Extended Data Figure 4 SERINC mRNA expression levels.

a, Expression of SERINC family members in uninfected and HIV-infected Jurkat E6.1 cells. RNA was extracted at the peak of infection with wild-type (Nef+) or Nef HIV-1NL43, and gene expression was quantified by RNA-seq as reads per kilobase of coding sequence per million reads (RPKM) (n = 1). The HIV-1 budding factor TSG101 and the housekeeping gene HPRT1 are included for comparison. b, Levels of SERINC3 and SERINC5 mRNA (arbitrary units) in cell lines and primary cells, as measured by qRT–PCR (n = 3). PBMC were left unstimulated or stimulated with 0.5 μg ml−1 phytohemagglutinin (PHA) and 20 U ml−1 IL-2 for 2 days. c, SERINC5 mRNA expression is not induced by INF-α. PBMC were left untreated or treated with 1,000 U ml−1 human INF-α 2a (PBL Assay Science) for 14 h (n = 2). Data are mean and s.d. NS, not significant (P > 0.05) two-tailed unpaired t-test.

Extended Data Figure 5 Exogenous SERINC5 inhibits the fusion of progeny virions with target cells.

TZM-bl or A549/CD4/CXCR4 cells were exposed to equal amounts of virus containing BlaM-Vpr, and fusion was analysed by measuring the Env-dependent increase in blue fluorescence using multiparameter flow cytometry. Virions were produced in 293T cells transfected with an Env HIV-1 provirus, a vector expressing EnvHXB2 (Env+) or a frameshift mutant (Env), a vector expressing BlaM-Vpr, and a vector expressing SERINC5 (1 μg or 100 ng) or an equimolar amount of the empty vector (0.7 μg or 70 ng). The percentage of cells displaying increased blue fluorescence is indicated.

Extended Data Figure 6 Exogenous SERINC5 reduces the infectivity of Nef HIV-1 progeny virions for primary target cells.

In two independent experiments, PHA-stimulated PBMC from different donors were infected with equal amounts of single-cycle GFP–HIV-1 virions produced in 293T cells in the absence or presence of exogenous SERINC5. Per cent fractions of infected (GFP-positive) cells are indicated.

Extended Data Figure 7 SERINC5 incorporation into HIV-1 virions that differ in Nef responsiveness.

Recombinant virions were produced in 293T cells co-transfected with the HXB/Env/Nef provirus and vectors expressing the poorly Nef-responsive EnvJRFL or the highly Nef-responsive JR(SF V1/V2) Env chimaera, along with a vector expressing SERINC5–HA. Empty pBJ5 vector or a version expressing HA-tagged Nef97ZA012 was also co-transfected. SERINC5–HA in purified virions was detected by western blotting. This experiment was performed twice.

Extended Data Figure 8 Characterization of JTAg knockout cells.

a, Mutant SERINC3 alleles identified in SERINC3 knockout clones. b, Mutant SERINC5 alleles identified in SERINC5 knockout and SERINC3/5 double-knockout clones. The single-guide RNA (sgRNA) target sites are highlighted, and the predicted Cas9 target sites are indicated by arrowheads. Inserted nucleotides are in red. One of the two mutated SERINC5 alleles in JTAg S3−/− S5−/− (1) cells has an inversion between sgRNA target sites A and B. JTAg S5−/− (2) cells contain three mutated SERINC5 alleles. All mutations cause frameshifts and/or large deletions of coding sequence. No wild-type alleles were detected in any of the knockout clones.

Extended Data Figure 9 SERINC3 and SERINC5 expression levels in reconstituted double-knockout cells

a, SERINC3 protein levels in parental, double-knockout, and reconstituted double-knockout JTAg cells were compared by western blotting. SERINC3 migrated close to a prominent background band that was also recognized by the anti-SERINC3 antibody. b, SERINC5 mRNA levels in parental and reconstituted double-knockout JTAg cells were compared by qRT–PCR (n = 3).

Extended Data Figure 10 Effects of SERINC knockout and reconstitution on HIV-1 replication.

Parental, double-knockout and SERINC3+SERINC5-reconstituted double-knockout CD4high JTAg cells were analysed by immunoblotting with anti-HIV CA at days 9 and 11 after infection with equal amounts (2 ng ml−1p24) of HIV-1NL43 encoding either wild-type or disrupted versions of NefNL43 or Nef97ZA012.

Supplementary information

Supplementary Figure 1

The file contains the full scans of Western blot data with molecular weight markers for Figures 1a, 1b, 2b, 2e, 3a, 3f, 5a, 5b and Extended Data Figures 1a, 2a, 2b, 7, 9a and 10. (PDF 1387 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Usami, Y., Wu, Y. & Göttlinger, H. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef. Nature 526, 218–223 (2015). https://doi.org/10.1038/nature15400

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.