HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation

Abstract

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

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: Nef counteracts an HIV-1 inhibitor.
Figure 2: SERINC5 and SERINC3 inhibit HIV-1.
Figure 3: Determinants of Nef activity against SERINC5 and conservation across different retroviruses.
Figure 4: Nef and glycoGag promote relocalization of SERINC5 to an endosomal compartment and prevent its incorporation into virions.
Figure 5: SERINC5 inhibits an early step of virus infection.

Accession codes

Primary accessions

Sequence Read Archive

Referenced accessions

GenBank/EMBL/DDBJ

Data deposits

RNA-seq fatsq data have been deposited in NCBI Sequence Read Archive (SRA) under accession code SRP062444.

References

  1. 1

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

    CAS  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

    Kirchhoff, F., Greenough, T. C., Brettler, D. B., Sullivan, J. L. & Desrosiers, R. C. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N. Engl. J. Med. 332, 228–232 (1995)

    CAS  Article  Google Scholar 

  4. 4

    Landi, A., Iannucci, V., Nuffel, A. V., Meuwissen, P. & Verhasselt, B. One protein to rule them all: modulation of cell surface receptors and molecules by HIV Nef. Curr. HIV Res. 9, 496–504 (2011)

    CAS  Article  Google Scholar 

  5. 5

    Baur, A. S. et al. HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. Immunity 1, 373–384 (1994)

    CAS  Article  Google Scholar 

  6. 6

    Schrager, J. A. & Marsh, J. W. HIV-1 Nef increases T cell activation in a stimulus-dependent manner. Proc. Natl Acad. Sci. USA 96, 8167–8172 (1999)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Alexander, L., Du, Z., Rosenzweig, M., Jung, J. U. & Desrosiers, R. C. A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation. J. Virol. 71, 6094–6099 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Simmons, A., Aluvihare, V. & McMichael, A. Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity 14, 763–777 (2001)

    CAS  Article  Google Scholar 

  9. 9

    Stolp, B. et al. HIV-1 Nef interferes with host cell motility by deregulation of Cofilin. Cell Host Microbe 6, 174–186 (2009)

    CAS  Article  Google Scholar 

  10. 10

    Chowers, M. Y. et al. Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. J. Virol. 68, 2906–2914 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Munch, 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 

  12. 12

    Carl, S. et al. Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS. J. Virol. 75, 3657–3665 (2001)

    CAS  Article  Google Scholar 

  13. 13

    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 

  14. 14

    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 

  15. 15

    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 

  16. 16

    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 

  17. 17

    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 

  18. 18

    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 

  19. 19

    Xu, J. et al. Cloning and expression of a novel human C5orf12 gene, a member of the TMS_TDE family. Mol. Biol. Rep. 30, 47–52 (2003)

    CAS  Article  Google Scholar 

  20. 20

    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 

  21. 21

    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 

  22. 22

    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 

  23. 23

    Saksela, K., Cheng, G. & Baltimore, D. 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. EMBO J. 14, 484–491 (1995)

    CAS  Article  Google Scholar 

  24. 24

    Piguet, V. et al. Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of β-COP in endosomes. Cell 97, 63–73 (1999)

    CAS  Article  Google Scholar 

  25. 25

    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 

  26. 26

    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 

  27. 27

    Chazal, N., Singer, G., Aiken, C., Hammarskjold, M. L. & Rekosh, D. Human immunodeficiency virus type 1 particles pseudotyped with envelope proteins that fuse at low pH no longer require Nef for optimal infectivity. J. Virol. 75, 4014–4018 (2001)

    CAS  Article  Google Scholar 

  28. 28

    Pizzato, M., Popova, E. & Gottlinger, H. G. Nef can enhance the infectivity of receptor-pseudotyped human immunodeficiency virus type 1 particles. J. Virol. 82, 10811–10819 (2008)

    CAS  Article  Google Scholar 

  29. 29

    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 

  30. 30

    Lai, R. P. et al. Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41. PLoS Pathog. 7, e1002442 (2011)

    CAS  Article  Google Scholar 

  31. 31

    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 

  32. 32

    Campbell, E. M., Nunez, R. & Hope, T. J. Disruption of the actin cytoskeleton can complement the ability of Nef to enhance human immunodeficiency virus type 1 infectivity. J. Virol. 78, 5745–5755 (2004)

    CAS  Article  Google Scholar 

  33. 33

    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 

  34. 34

    Zhou, J. & Aiken, C. Nef enhances human immunodeficiency virus type 1 infectivity resulting from intervirion fusion: evidence supporting a role for Nef at the virion envelope. J. Virol. 75, 5851–5859 (2001)

    CAS  Article  Google Scholar 

  35. 35

    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 

  36. 36

    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 

  37. 37

    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 

  38. 38

    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 

  39. 39

    van der Aa, L. M. et al. A large new subset of TRIM genes highly diversified by duplication and positive selection in teleost fish. BMC Biol. 7, 7 (2009)

    Article  Google Scholar 

  40. 40

    Duggal, N. K. & Emerman, M. Evolutionary conflicts between viruses and restriction factors shape immunity. Nature Rev. Immunol. 12, 687–695 (2012)

    CAS  Article  Google Scholar 

  41. 41

    Briggs, J. A., Wilk, T., Welker, R., Kräusslich, H. G. & Fuller, S. D. Structural organization of authentic, mature HIV‐1 virions and cores. EMBO J. 22, 1707–1715 (2003)

    CAS  Article  Google Scholar 

  42. 42

    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 

  43. 43

    Razinkov, V. I. & Cohen, F. S. Sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus. Biochemistry 39, 13462–13468 (2000)

    CAS  Article  Google Scholar 

  44. 44

    Ciechonska, M. & Duncan, R. Lysophosphatidylcholine reversibly arrests pore expansion during syncytium formation mediated by diverse viral fusogens. J. Virol. 88, 6528–6531 (2014)

    CAS  Article  Google Scholar 

  45. 45

    Chen, A. et al. Fusion-pore expansion during syncytium formation is restricted by an actin network. J. Cell Sci. 121, 3619–3628 (2008)

    CAS  Article  Google Scholar 

  46. 46

    Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L. & Trono, D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature Biotechnol. 15, 871–875 (1997)

    CAS  Article  Google Scholar 

  47. 47

    Pizzato, M. et al. A one-step SYBR Green I-based product-enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture supernatants. J. Virol. Methods 156, 1–7 (2009)

    CAS  Article  Google Scholar 

  48. 48

    Simon, J. H., Gaddis, N. C., Fouchier, R. A. & Malim, M. H. Evidence for a newly discovered cellular anti-HIV-1 phenotype. Nature Med. 4, 1397–1400 (1998)

    CAS  Article  Google Scholar 

  49. 49

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

    Article  Google Scholar 

  50. 50

    Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods 5, 621–628 (2008)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank the Centre for AIDS Reagents, NIBSC, and NIH AIDS Research and Reference Reagent Program, Division of AIDS, for cell lines, plasmids and antibodies. We thank V. Adami and the CIBIO high-throughput screening and the Advanced Imaging facilities staff for technical assistance, G. De Silvestro, G. Mattiuzzo, C. Reinhard and L. Conti for reagents, G. Melikian, S. Basmaciogullari, P. Cherepanov, O. Fackler, N. Segata, F. Demichelis, A. Marcello, T. Fedrizzi and A. Helander for critical discussions. This work was supported by the Biotechnology Program of University of Trento, FP7 Marie Curie Career Integration grant number 322130 and Caritro ‘Ricerca Biomedica’ grant number 2013.0248 to M.P., National Institute of Health grant DP1DA034990 to J.L. and European Research Council grant 249968 to S.E.A.

Author information

Affiliations

Authors

Contributions

A.R., A.C., S.Z., V.D.S., R.B., S.E.A., J.L., F.A.S. and M.P. designed the experiments. A.R., S.Z., A.C., V.D.S., R.B., S.L.G., S.M.M., A.N., F.A.S. and M.P. performed the experiments. All authors contributed to the assembly and writing of the manuscript. A.R., A.C. and S.Z. contributed equally to the study.

Corresponding author

Correspondence to Massimo Pizzato.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 SERINC5 is an inhibitor of HIV-1 infectivity.

a, Mapping of the INDELS in the genomic locus spanning SERINC5 exon 2 in JTAg cell clonal populations from Fig. 2a. b, Infectivity of HIV-1 from JTAg cells stably transduced with lentiCRISPR targeting GFP or SERINC5 in three different exons (n = 4, experiment replicated twice). c, Relative expression of SERINC5 in primary cells and in cell lines measured by qPCR normalized by expression of ACTB (n = 3). d, Infectivity of HIV-1 from the indicated cell lines expressing SERINC5 (n = 4, experiments were replicated twice). Mean ± s.d., unpaired two-tailed t-test, ***P < 0.001 e, Expression levels of the five SERINC genes in JTAg cells obtained from RNA-seq.

Extended Data Figure 2 Nef and glycoGag expression result in relocalization of SERINC5 to an endosomal compartment and prevent its incorporation into virions.

a, Single round Nef-defective NL4-3 produced by cotransfection of HEK293T cells with plasmids expressing Nef proteins or the empty vector control, and PBJ6-SERINC5–HA: immunoblotting of virions and cell lysates from producer cells. b, Immunofluorescence staining of JTAg cells transfected to express SERINC5–GFP, Nef–HA from HIV-1 isolate 97ZA012 (clade C), from SIVmac239, HA–glycoGag or an empty vector control. Scale bar, 10 μm.

Extended Data Figure 3 SERINC5 inhibits cytoplasmic delivery of virion content.

a, Immunodetection of Cre-recombinase (38 kDa) and p24 in HIV-1 particles. b, Effect of 1 μM AZT or 100 nM T20 on Cre-delivery and virus infectivity (TU, transducing units). c, Immunoblotting of HIV-1 virus particles produced from HEK293T expressing increasing levels of SERINC5–HA. d, Effect of SERINC5 on virus fusion measured with BLAM assay T20 served as a negative control. (n = 4, experiment replicated twice). e, Cre delivery by EBOV-GP pseudotyped HIV-1 particles. f, Inhibition of Cre delivery and counteraction by Nef on HIV-1 from HEK293T expressing SERINC5. Mean ± s.d., n = 4, unpaired two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 100 μm.

Extended Data Figure 4 SERINC3 and SERINC5 expression is not induced by interferon nor LPS treatments.

ad, Relative gene expression levels of SERINC3, SERINC5 and CXCL10 in response to treatment with IFN-β and LPS in Jurkat (a), monocyte-derived dendritic cells from two donors (MDDC, b), CD4+ primary T cells unstimulated (c) or stimulated with PHA (d) from two donors. Expression of the housekeeping gene OAZ1 was used as a normalization control. Mean ± s.d., n = 3.

Extended Data Table 1 Description of the cells lines used in Fig. 1a

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rosa, A., Chande, A., Ziglio, S. et al. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature 526, 212–217 (2015). https://doi.org/10.1038/nature15399

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.