Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4+ T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.
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Friedrich, B.M. et al. Host factors mediating HIV-1 replication. Virus Res. 161, 101–114 (2011).
Fätkenheuer, G. et al. Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1. Nat. Med. 11, 1170–1172 (2005).
Gulick, R.M. et al. Maraviroc for previously treated patients with R5 HIV-1 infection. N. Engl. J. Med. 359, 1429–1441 (2008).
Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370, 901–910 (2014).
Hütter, G. et al. Long-term control of HIV by CCR5 Δ32/Δ32 stem cell transplantation. N. Engl. J. Med. 360, 692–698 (2009).
Glass, W.G. et al. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J. Exp. Med. 203, 35–40 (2006).
Srivastava, A., Pandey, S.N., Choudhuri, G. & Mittal, B. CCR5-Δ32 polymorphism: associated with gallbladder cancer susceptibility. Scand. J. Immunol. 67, 516–522 (2008).
Singh, H., Sachan, R., Jain, M. & Mittal, B. CCR5-Δ32 polymorphism and susceptibility to cervical cancer: association with early stage of cervical cancer. Oncol. Res. 17, 87–91 (2008).
Eri, R. et al. CCR5-Δ32 mutation is strongly associated with primary sclerosing cholangitis. Genes Immun. 5, 444–450 (2004).
König, R. et al. Global analysis of host–pathogen interactions that regulate early-stage HIV-1 replication. Cell 135, 49–60 (2008).
Brass, A.L. et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science 319, 921–926 (2008).
Zhou, H. et al. Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe 4, 495–504 (2008).
Bassik, M.C. et al. Rapid creation and quantitative monitoring of high-coverage shRNA libraries. Nat. Methods 6, 443–445 (2009).
Shao, D.D. et al. ATARiS: computational quantification of gene suppression phenotypes from multisample RNAi screens. Genome Res. 23, 665–678 (2013).
Zhu, J. et al. Comprehensive identification of host modulators of HIV-1 replication using multiple orthologous RNAi reagents. Cell Rep. 9, 752–766 (2014).
Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).
Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).
Keele, B.F. et al. Identification and characterization of transmitted and early-founder virus envelopes in primary HIV-1 infection. Proc. Natl. Acad. Sci. USA 105, 7552–7557 (2008).
Ochsenbauer, C. et al. Generation of transmitted and founder HIV-1 infectious molecular clones, and characterization of their replication capacity in CD4 T lymphocytes and monocyte-derived macrophages. J. Virol. 86, 2715–2728 (2012).
Lai, M.M. Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244, 1–12 (1998).
Rolando, M. & Buchrieser, C. Legionella pneumophila type IV effectors hijack the transcription and translation machinery of the host cell. Trends Cell Biol. 24, 771–778 (2014).
Egan, E.S. et al. Malaria. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion. Science 348, 711–714 (2015).
Cavrois, M., De Noronha, C. & Greene, W.C. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat. Biotechnol. 20, 1151–1154 (2002).
Kamiyama, S. et al. Molecular cloning and identification of 3′-phosphoadenosine 5′-phosphosulfate transporter. J. Biol. Chem. 278, 25958–25963 (2003).
Beisswanger, R. et al. Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase 2. Proc. Natl. Acad. Sci. USA 95, 11134–11139 (1998).
Baeuerle, P.A. & Huttner, W.B. Chlorate—a potent inhibitor of protein sulfation in intact cells. Biochem. Biophys. Res. Commun. 141, 870–877 (1986).
Rosmarin, D.M. et al. Attachment of Chlamydia trachomatis L2 to host cells requires sulfation. Proc. Natl. Acad. Sci. USA 109, 10059–10064 (2012).
Connell, B.J. & Lortat-Jacob, H. Human immunodeficiency virus and heparan sulfate: from attachment to entry inhibition. Front. Immunol. 4, 385 (2013).
Farzan, M. et al. Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell 96, 667–676 (1999).
Seibert, C., Cadene, M., Sanfiz, A., Chait, B.T. & Sakmar, T.P. Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence. Proc. Natl. Acad. Sci. USA 99, 11031–11036 (2002).
Wu, L. et al. Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding. J. Exp. Med. 186, 1373–1381 (1997).
Bowen, M.A. et al. Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand. J. Exp. Med. 181, 2213–2220 (1995).
Swart, G.W. Activated leukocyte cell adhesion molecule (CD166 or ALCAM): developmental and mechanistic aspects of cell clustering and cell migration. Eur. J. Cell Biol. 81, 313–321 (2002).
Iolyeva, M. et al. Novel role for ALCAM in lymphatic network formation and function. FASEB J. 27, 978–990 (2013).
Williams, D.W., Anastos, K., Morgello, S. & Berman, J.W. JAM-A and ALCAM are therapeutic targets to inhibit diapedesis across the BBB of CD14+CD16+ monocytes in HIV-infected individuals. J. Leukoc. Biol. 97, 401–412 (2015).
Te Riet, J. et al. Dynamic coupling of ALCAM to the actin cortex strengthens cell adhesion to CD6. J. Cell Sci. 127, 1595–1606 (2014).
van Kempen, L.C. et al. Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)–ALCAM interaction. J. Biol. Chem. 276, 25783–25790 (2001).
Gartner, Z.J. & Bertozzi, C.R. Programmed assembly of 3-dimensional microtissues with defined cellular connectivity. Proc. Natl. Acad. Sci. USA 106, 4606–4610 (2009).
Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl. Acad. Sci. USA 112, 10437–10442 (2015).
Dustin, M.L. & Springer, T.A. T cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341, 619–624 (1989).
Sabatos, C.A. et al. A synaptic basis for paracrine interleukin-2 signaling during homotypic T cell interaction. Immunity 29, 238–248 (2008).
Zumwalde, N.A., Domae, E., Mescher, M.F. & Shimizu, Y. ICAM-1-dependent homotypic aggregates regulate CD8 T cell effector function and differentiation during T cell activation. J. Immunol. 191, 3681–3693 (2013).
Kaelin, W.G. Jr. Use and abuse of RNAi to study mammalian gene function. Science 337, 421–422 (2012).
Sasaki, N. et al. A mutation in Tpst2, encoding tyrosylprotein sulfotransferase, causes dwarfism associated with hypothyroidism. Mol. Endocrinol. 21, 1713–1721 (2007).
Ding, Z.M. et al. Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration. J. Immunol. 163, 5029–5038 (1999).
Ghosh, S., Chackerian, A.A., Parker, C.M., Ballantyne, C.M. & Behar, S.M. The LFA-1 adhesion molecule is required for protective immunity during pulmonary Mycobacterium tuberculosis infection. J. Immunol. 176, 4914–4922 (2006).
Clément, A. et al. Regulation of zebrafish skeletogenesis by ext2 (dackel) and papst1 (pinscher). PLoS Genet. 4, e1000136 (2008).
Jäger, S. et al. Global landscape of HIV–human protein complexes. Nature 481, 365–370 (2011).
Singh, P.K. et al. LEDGF (p75) interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes. Genes Dev. 29, 2287–2297 (2015).
Sowd, G.A. et al. A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin. Proc. Natl. Acad. Sci. USA 113, E1054–E1063 (2016).
Desfosses, Y. et al. Regulation of human immunodeficiency virus type 1 gene expression by clade-specific Tat proteins. J. Virol. 79, 9180–9191 (2005).
Gardner, M.R. et al. AAV-expressed eCD4–Ig provides durable protection from multiple SHIV challenges. Nature 519, 87–91 (2015).
Seibert, C. et al. Sequential tyrosine sulfation of CXCR4 by tyrosylprotein sulfotransferases. Biochemistry 47, 11251–11262 (2008).
Kajumo, F., Thompson, D.A., Guo, Y. & Dragic, T. Entry of R5X4 and X4 human immunodeficiency virus type 1 strains is mediated by negatively charged and tyrosine residues in the amino-terminal domain and the second extracellular loop of CXCR4. Virology 271, 240–247 (2000).
Lin, G., Baribaud, F., Romano, J., Doms, R.W. & Hoxie, J.A. Identification of gp120-binding sites on CXCR4 by using CD4-independent human immunodeficiency virus type 2 Env proteins. J. Virol. 77, 931–942 (2003).
Ingulli, E., Mondino, A., Khoruts, A. & Jenkins, M.K. In vivo detection of dendritic cell antigen presentation to CD4+ T cells. J. Exp. Med. 185, 2133–2141 (1997).
Hommel, M. & Kyewski, B. Dynamic changes during the immune response in T cell–antigen-presenting cell clusters isolated from lymph nodes. J. Exp. Med. 197, 269–280 (2003).
Inaba, K., Witmer, M.D. & Steinman, R.M. Clustering of dendritic cells, helper T lymphocytes, and histocompatible B cells during primary antibody responses in vitro. J. Exp. Med. 160, 858–876 (1984).
Sourisseau, M., Sol-Foulon, N., Porrot, F., Blanchet, F. & Schwartz, O. Inefficient human immunodeficiency virus replication in mobile lymphocytes. J. Virol. 81, 1000–1012 (2007).
Agosto, L.M., Uchil, P.D. & Mothes, W. HIV cell-to-cell transmission: effects on pathogenesis and antiretroviral therapy. Trends Microbiol. 23, 289–295 (2015).
Chen, P., Hübner, W., Spinelli, M.A. & Chen, B.K. Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses. J. Virol. 81, 12582–12595 (2007).
Sigal, A. et al. Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature 477, 95–98 (2011).
Doitsh, G. et al. Abortive HIV infection mediates CD4 T cell depletion and inflammation in human lymphoid tissue. Cell 143, 789–801 (2010).
Santangelo, P.J. et al. Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral-therapy-treated macaques. Nat. Methods 12, 427–432 (2015).
Lorenzo-Redondo, R. et al. Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature 530, 51–56 (2016).
Yusuf-Makagiansar, H., Anderson, M.E., Yakovleva, T.V., Murray, J.S. & Siahaan, T.J. Inhibition of LFA-1–ICAM-1 and VLA-4–VCAM-1 as a therapeutic approach to inflammation and autoimmune diseases. Med. Res. Rev. 22, 146–167 (2002).
Hourmant, M. et al. A randomized multicenter trial comparing leukocyte function-associated antigen 1 monoclonal antibody with rabbit antithymocyte globulin as induction treatment in first kidney transplantations. Transplantation 62, 1565–1570 (1996).
Brockman, M.A., Tanzi, G.O., Walker, B.D. & Allen, T.M. Use of a novel GFP reporter cell line to examine replication capacity of CXCR4- and CCR5-tropic HIV-1 by flow cytometry. J. Virol. Methods 131, 134–142 (2006).
Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Wang, T., Lander, E.S. & Sabatini, D.M. Large-scale single guide RNA library construction and use for CRISPR–Cas9-based genetic screens. Cold Spring Harb. Protoc. 2016, pdb.top086892 (2016).
McKinley, K.L. et al. The CENP–L–N complex forms a critical node in an integrated meshwork of interactions at the centromere–kinetochore interface. Mol. Cell 60, 886–898 (2015).
Weber, K., Bartsch, U., Stocking, C. & Fehse, B. A multicolor panel of novel lentiviral 'gene ontology' (LeGO) vectors for functional gene analysis. Mol. Ther. 16, 698–706 (2008).
Salzberger, W. et al. Influence of glycosylation inhibition on the binding of KIR3DL1 to HLA-B*57:01. PLoS One 10, e0145324 (2015).
We would like to thank the Ragon Institute Virology, Imaging, and Flow Cytometry cores, as well as the Center for Computational and Integrative Biology (CCIB) DNA Core Facility at Massachusetts General Hospital. We would like to thank A. McKeon, P. Jani, N.W. Hughes, and B.X. Liu for superb technical assistance, and A. Brass, G. Gaiha, and J.S. Park for helpful discussions. pMM310 was obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH from M. Miller (Merck Research Laboratories). All plasmid reagents generated in this study have been deposited in Addgene. This work was supported by the Howard Hughes Medical Institute (D.M.S. and B.D.W.), the National Institutes of Health (grants CA103866 (D.M.S.), F31 CA189437 (T.W.), P50 GM082250 (A.M. and N.J.K.), U19 AI106754 (J.F.H. and N.J.K.), and P01 AI090935 (N.J.K.)), the National Human Genome Research Institute (grant 2U54HG003067-10; E.S.L.), the National Science Foundation (T.W.), the MIT Whitaker Health Sciences Fund (T.W.), the UCSF Sandler Fellowship (A.M.), a gift from J. Aronov (A.M.), the UCSF MPHD T32 Training Grant (J.F.H.), and the Deutsche Forschungsgemeinschaft (grant SCHU3020/2-1; K.S.). Support was also provided by NIH-funded Centers for AIDS Research (grant P30 AI027763, UCSF Center for AIDS Research (N.J.K.) and grant P30 AI060354, Harvard University Center for AIDS Research (B.D.W.)), which are supported by the following NIH co-funding and participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. D.M.S. and B.D.W. are investigators of the Howard Hughes Medical Institute. R.J.P. is a Howard Hughes Medical Institute Research Fellow.
T.W., D.M.S., and E.S.L. are inventors on a patent application for functional genomics using the CRISPR–Cas system (US 15/141,348), T.W. and D.M.S. are founders of KSQ Therapeutics, a CRISPR functional genomics company, and D.M.S. is a scientific advisor for KSQ Therapeutics. A patent has been filed on the use of Cas9–RNPs to edit the genome of human primary T cells (A.M. and K.S.). A.M. serves as an advisor to Juno Therapeutics, and the laboratory of A.M. has had sponsored research agreements with Juno Therapeutics and Epinomics.
Integrated supplementary information
Gene scores were calculated by using mean log2 fold change in the abundance of all sgRNAs for each gene.
Supplementary Figure 2 mRNA expression analysis of SLC35B2 and TPST2 in GXRCas9 cells subcloned following CRISPR-based knockout.
Wild-type SLC35B2 and TPST2 mRNA expression levels in WT GXRCas9 cells and TPST2- and SLC35B2-knockout clones as assessed by qRT-PCR, using primers that overlap the sgRNA target site to selectively amplify wild-type cDNA. Error bars, s.d. from triplicate reactions.
Supplementary Figure 3 ALCAM-null GXRCas9 cells are protected from a multi-round, spreading JR-CSF infection.
Low MOI (MOI = 0.1) virus challenge. Six days following JR-CSF infection, viable, GFP– cells were counted and cell number was normalized to that under a mock-infected condition. Error bars, s.d. from triplicate wells; *P < 0.0001, Welch’s t test.
Sulfation of surface CCR5 in primary CD4+ T cells following JR-CSF or Rejo.C challenge. Intracellular HIV Gag (p24) and total and sulfated surface CCR5 expression are shown as assessed by flow cytometry. Error bars, s.d. from triplicate wells; *P < 0.01, Welch’s t test. All P < 0.0001, except as follows: Donor 1 uninfected vs. Rejo.C p24+, P = 0.0005; Donor 2 uninfected vs. JRCSF p24–, P = 0.0003; uninfected vs. Rejo.C p24–, P = 0.0033; Rejo.C p24– vs. p24+, P = 0.0001.
mRNA expression of ALCAM, LFA-1, and the ICAM family in primary CD4+ T cells and GXRCas9 cells as assessed by RNA sequencing.
Supplementary Figure 6 Antibody blockade of cell adhesion factors attenuates HIV spread in primary human CD4+ T cells.
(a) CRISPR-mediated knockout of the LFA-1 subunit (encoded by ITGAL) only blocks cell-to-cell transmission if donor and acceptor cells are both CD11a-null. (b,c) Cell-to-cell HIV transmission assay in primary CD4+ T cells following blockade with antibody to ICAM-1/LFA-1 (b) or CD45, as a control (c). The assay is set up as in Figure 5 except that donor cells are infected 36 h prior to co-culture and co-culture is for 2 d. Readout is by flow cytometry following intracellular staining for HIV Gag. Antibodies against ICAM-1, CD11a, and CD18 are added 15 min prior to co-culture. Error bars, s.d. from triplicate wells; *P < 0.01, Welch’s t test. P values were as follows: (a) n.s., P = 0.836; *P = 0.0036; (b) *P = 0.002; (c) n.s., P = 0.72.
Supplementary Figure 7 Loss of RELA, a candidate HDF identified by three previous RNAi screens, does not protect GXR cells from JR-CSF viral challenge.
Virus challenge assay (JR-CSF, MOI = 1) of GXRCas9 cells transduced with sgRELA (left) and immunoblot demonstrating depletion of RelA (right). RagC is a loading control. Error bars, s.d. from triplicate wells; n.s., P = 0.787, Welch’s t test.
Screen for essential genes in the Raji B cell line (Wang et al., 2015). For every gene in the human genome, the mean of the individual log2-transformed fold change values in the abundance of each of the sgRNAs targeting that gene is shown. Screen hits and selected putative HIV HDFs that are among the 10% most depleted (i.e., cell-essential) genes are highlighted.
(a,b) mRNA expression of TPST2, SLC35B2, PAPSS1, and select paralogs in GXRCas9 cells (a) and activated, primary CD4+ T cells and GXRCas9 cells (b) as assessed by RNA sequencing.
Supplementary Figures 1–9 and Supplementary Note. (PDF 1104 kb)
Genome-wide human sgRNA library annotation. (XLSX 10841 kb)
CRISPR gene scores. (XLSX 518 kb)
RNA sequencing analysis of GXRCas9 and activated, primary CD4+ T cells. (XLSX 1261 kb)
Nucleotide sequences. (XLSX 8 kb)
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Park, R., Wang, T., Koundakjian, D. et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat Genet 49, 193–203 (2017). https://doi.org/10.1038/ng.3741
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