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TIM-Mediated Inhibition of HIV-1 Release Is Antagonized by Nef but Potentiated by SERINC Proteins

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TIM-Mediated Inhibition of HIV-1 Release Is Antagonized by Nef but Potentiated by SERINC Proteins TIM-mediated inhibition of HIV-1 release is antagonized by Nef but potentiated by SERINC proteins Minghua Lia,b,1,2, Abdul A. Waheedc,1, Jingyou Yua,b,1,3, Cong Zenga,b, Hui-Yu Chend, Yi-Min Zhenga,b, Amin Feizpoure, Björn M. Reinharde, Suryaram Gummuluruf, Steven Lind, Eric O. Freedc, and Shan-Lu Liua,b,g,h,4 aCenter for Retrovirus Research, The Ohio State University, Columbus, OH 43210; bDepartment of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210; cVirus–Cell Interaction Section, HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702; dInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; eDepartment of Chemistry and The Photonics Center, Boston University, Boston, MA 02215; fDepartment of Microbiology, Boston University School of Medicine, Boston, MA 02118; gDepartment of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210; and hViruses and Emerging Pathogens Thematic Program, Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210 Edited by Michael Emerman, Fred Hutchinson Cancer Research Center, Seattle, WA, and accepted by Editorial Board Member Stephen P. Goff February 3, 2019 (received for review November 15, 2018) The T cell Ig and mucin domain (TIM) proteins inhibit release of HIV-1 Vif binds to apolipoprotein B mRNA-editing enzyme cata- HIV-1 and other enveloped viruses by interacting with cell- and lytic polypeptide-like 3 (APOBEC3), thereby inducing its proteaso- virion-associated phosphatidylserine (PS). Here, we show that the mal degradation and enhancing viral infectivity (14). To enable Nef proteins of HIV-1 and other lentiviruses antagonize TIM- efficient virus release, HIV-1 Vpu counteracts tetherin (also known mediated restriction. TIM-1 more potently inhibits the release of as BST2 and CD317), either by preventing its trafficking to the Nef-deficient relative to Nef-expressing HIV-1, and ectopic expres- plasma membrane or by targeting it to endolysosomal compartments sion of Nef relieves restriction. HIV-1 Nef does not down-regulate for degradation (15–17). The strategies and modes of action by the overall level of TIM-1 expression, but promotes its internali- which HIV-1 accessory proteins antagonize cellular restrictions have zation from the plasma membrane and sequesters its expression in provided critical molecular and genetic insights into our under- intracellular compartments. Notably, Nef mutants defective in standing of virus–host interaction and coevolution (13, 18–20). modulating membrane protein endocytic trafficking are incapable Unlike typical cellular restriction factors (12), the expression of antagonizing TIM-mediated inhibition of HIV-1 release. Intrigu- of TIM proteins is not induced by type I IFN (9). However, TIM MICROBIOLOGY ingly, depletion of SERINC3 or SERINC5 proteins in human periph- proteins, especially TIM-1 and TIM-3, are expressed in activated + eral blood mononuclear cells (PBMCs) attenuates TIM-1 restriction human CD4 T cells and monocyte-derived macrophages (MDMs), of HIV-1 release, in particular that of Nef-deficient viruses. In contrast, respectively (9, 21, 22), and they constitutively inhibit HIV-1 coexpression of SERINC3 or SERINC5 increases the expression of TIM- production (9). Similarly, SERINC proteins, recently discovered 1 on the plasma membrane and potentiates TIM-mediated inhibition cellular “restriction factors” that diminish the infectivity of HIV of HIV-1 production. Pulse-chase metabolic labeling reveals that the and other lentiviruses (23, 24), are not induced by type I IFN, nor half-life of TIM-1 is extended by SERINC5 from <2to∼6 hours, sug- are they under positive selection (25). Because the antiviral gesting that SERINC5 stabilizes the expression of TIM-1. Consistent function of SERINCs is specifically antagonized by the lentivirus with a role for SERINC protein in potentiating TIM-1 restriction, we find that MLV glycoGag and EIAV S2 proteins, which, like Nef, antag- Significance onize SERINC-mediated diminishment of HIV-1 infectivity, also effec- tively counteract TIM-mediated inhibition of HIV-1 release. Collectively, TIM proteins inhibit release of HIV-1 and other enveloped our work reveals a role of Nef in antagonizing TIM-1 and high- viruses. However, it is currently unknown whether and how lights the complex interplay between Nef and HIV-1 restriction by the virus counteracts this restriction. In this work, we demon- TIMs and SERINCs. strate that Nef proteins of HIV-1 and other lentiviruses function as antagonists to overcome the TIM-mediated restriction. TIM- HIV | TIM | SERINC | Nef 1 is more potent at inhibiting release of Nef-deficient HIV- 1 relative to wild-type (WT) HIV-1, and ectopic expression of uman T cell Ig and mucin domain (TIM) proteins, which Nef relieves this restriction. Interestingly, we find that SERINC Hinclude TIM-1, TIM-3, and TIM-4, bind to phosphati- proteins potentiate TIM-mediated inhibition of HIV-1 release dylserine (PS) via a conserved IgV domain and regulate the host likely by stabilizing TIM-1 expression. Our work reveals a role immune response (1, 2). In a manner dependent on their expres- for lentiviral Nef in antagonizing TIMs, in part through SERINCs. sion patterns in different cell types, TIM-family proteins play dis- tinct roles in cell proliferation, apoptosis, immune tolerance, and Author contributions: M.L., A.A.W., J.Y., C.Z., H.-Y.C., Y.-M.Z., A.F., B.M.R., S.G., E.O.F., and T-cell activation (2). Furthermore, TIM-1 polymorphisms have S.-L.L. designed research; M.L., A.A.W., J.Y., C.Z., H.-Y.C., Y.-M.Z., A.F., and S.-L.L. per- – formed research; E.O.F. contributed new reagents/analytic tools; M.L., A.A.W., J.Y., C.Z., been associated with some allergic human diseases (3 5). Although H.-Y.C., Y.-M.Z., A.F., B.M.R., S.G., S.L., E.O.F., and S.-L.L. analyzed data; and M.L., A.A.W., expression of TIM proteins in target cells has been shown to pro- J.Y., E.O.F., and S.-L.L. wrote the paper. mote entry of a wide range of enveloped viruses (6–8), we recently The authors declare no conflict of interest. found that all three human TIM-family proteins also inhibit the This article is a PNAS Direct Submission. M.E. is a guest editor invited by the Editorial Board. release of HIV-1 and other enveloped viruses, including murine Published under the PNAS license. leukemia virus (MLV) and Ebola virus (EBOV). This inhibition of 1M.L., A.A.W., and J.Y. contributed equally to this work. particle release is achieved by TIM binding to PS present on the 2Present address: Department of Microbiology, University of Pennsylvania, Philadelphia, surface of viral producer cells and newly budded virions (9). PA 19104. Upon viral infection, cells produce type I interferons (IFNs) 3Present address: Center for Virology and Vaccine Research, Beth Israel Deaconess Med- that up-regulate expression of hundreds of IFN-stimulated genes ical Center, Harvard Medical School, Boston, MA 02115. (ISGs). Many of these ISGs execute antiviral activities (10) and are 4To whom correspondence should be addressed. Email: [email protected]. collectively referred to as cellular “restriction factors” (11). As a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. countermeasure, many viruses, including HIV, have evolved effective 1073/pnas.1819475116/-/DCSupplemental. strategies to overcome cellular restrictions (12, 13). For example, www.pnas.org/cgi/doi/10.1073/pnas.1819475116 PNAS Latest Articles | 1of10 Downloaded by guest on September 25, 2021 Nef protein (23, 24, 26, 27), an important question is whether, and nef, vpu, vif, vpr,orenv genes, together with increasing amounts how, HIV-1 may counteract TIM-mediated restriction to facilitate of a TIM-1 expression plasmid. TIM-1 inhibited release of these viral production and replication. Here, we provide evidence that HIV-1 variants, similarly to wild-type (WT) and in a dose- lentiviral Nef proteins are capable of antagonizing the ability of TIM- dependent manner, based on the quantification of Western 1 to inhibit viral release, in part by promoting TIM-1 internalization blotting bands for the virion p24 versus the total HIV-1 Gag, from the plasma membrane and sequestering TIM-1 within in- which includes the virion p24 plus all cellular Gag fractions, that tracellular compartments. Interestingly, we find that SERINC is, p24, p41, and Pr55 (Fig. 1 A and B). Notably, TIM-1 more po- proteins potentiate TIM-mediated inhibition of HIV-1 release, tently inhibited the release of Δnef virus relative to WT virus or the in part by stabilizing TIM-1 protein expression. Moreover, two other variants (Fig. 1 A and B), indicating that Nef likely counter- other SERINC antagonists, that is, MLV glycoGag and equine acts TIM-1 inhibition of HIV-1 release. Expression of TIM-1 did infectious anemia virus (EIAV) S2 proteins (23, 24, 28–30), can not change HIV-1 Gag (Pr55) levels in viral producer cells, al- also effectively relieve TIM-mediated inhibition of HIV-1 release, though, as we have reported previously, cell-associated p24 levels further supporting the involvement of SERINC proteins in TIM- were increased in a TIM-1 dose-dependent manner (Fig. 1A), in mediated restriction. Our work unveils a mechanism by which part because of the accumulation of newly budded HIV-1 virions on lentiviral Nef proteins counteract restriction by TIM in part theplasmamembrane(9).Similarresultswereobtainedusinga through SERINC to facilitate HIV-1 release and replication. WT HIV-1 NL4-3-IRES-eGFP construct and mutants thereof lacking vpu and/or nef genes (SI Appendix,Fig.S1A and B). Results We next assessed whether ectopic expression of an exogenous HIV-1 Nef Antagonizes TIM-1–Mediated Restriction of Viral Release.
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