TIM-mediated inhibition of HIV-1 release is antagonized by Nef but potentiated by SERINC

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 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 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. HIV-1 Nef protein can overcome TIM-1–mediated restriction of Cellular inhibitory factors are frequently antagonized by lenti- HIV-1 release, especially that of Δnef HIV-1. To this end, we viral accessory proteins (e.g., Vif, Vpu, Vpr, Vpx, and Nef) or the transiently transfected HEK293T cells with increasing amounts viral envelope (Env) glycoprotein (12). To examine a possible (0.5 and 1.5 μg) of a plasmid encoding NL4-3 Nef, along with a role of HIV-1 accessory proteins in overcoming TIM-mediated proviral DNA construct encoding either NL4-3 WT or Δnef NL4-3, inhibition of viral release, we cotransfected HEK293T cells with in the presence or absence of TIM-1. In the absence of TIM-1 ex- HIV-1 proviral DNA constructs containing intact or defective pression, Nef caused only a small increase in virus release efficiency

Fig. 1. HIV-1 Nef counteracts the inhibitory effect of TIM-1 on HIV-1 production. (A) Effect of TIM-1 on viral production of different proviral HIV-1 NL4- 3 clones. HEK293T cells were transfected with HIV-1 proviral DNA plasmids encoding wild-type (WT) NL4-3 or nef, vpu, vif, vpr,orenv-deficient NL4-3, along with indicated amounts of TIM-1 expression plasmid. Forty-eight hours posttransfection, Western blotting was performed to measure cell-associated Gag (Cells) and cell-free viral particles (VPs) by using an anti–HIV-1 p24 antibody. TIM-1 expression in cell lysates was determined by using an anti–TIM-1 antibody. The values for NL4-3 Δnef are boxed to highlight the differences. Virion-associated p24 and cellular Gag (Pr55, p41, and p24) were quantified by using Quantity One (Bio-Rad) software. Viral release efficiency was determined as the ratio of virion-associated p24 vs. the total Gag (i.e., virion p24 plus cellular Pr55, p41, and p24). Relative virion release (indicated as p24/total Gag) was calculated by setting the value of WT in the absence of TIM-1 to 1.00. (B) Relative release efficiency of HIV-1 and mutants was determined by averaging results of three independent experiments, with means and SDs shown. *P < 0.05; **P < 0.01; ****P < 0.0001. (C) Effect of ectopic expression of an exogenous Nef on the production of HIV-1 WT and Δnef particles. HEK293T cells were cotransfected with HIV-1 NL4-3 WT or Δnef proviral DNA along with increasing amounts of NL4-3 Nef expression plasmid (0.5 or 1.5 μg) in the presence or absence of TIM-1. Viral release was determined as described for A. Note that an anti-Nef antibody was used to detect both the endogenous Nef (En) encoded by the provirus and an exogenous Nef (Ex) expressed in trans from an expression plasmid. (D) Relative release efficiency of HIV-1 WT and Δnef in the presence and absence of TIM-1 and exogenous Nef was determined by averaging results of three independent experiments, with means and SDs shown. **P < 0.01; ****P < 0.0001. (E–H) Effect of TIM-3 knockdown on HIV-1 release in MDMs derived from three donors. Viral release was measured by quantifying the viral p24 level of the cell supernatants harvested at 48-h postinfection. The fold increase of HIV-1 release caused by TIM-3 shRNA knockdown relative to thatof shRNA control was indicated. H shows a summary result of all three donors. *P < 0.05; **P < 0.01; ***P < 0.001.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1819475116 Li et al. Downloaded by guest on September 25, 2021 (Fig. 1 C and D); in contrast, in the presence of TIM-1, Nef over- WT and Δnef-producing cells so that de novo viral production could expression in trans markedly rescued TIM-1–mediated inhibition of be compared (TIM proteins are also known to promote viral entry). Δnef particle release while having only a small effect on WT particle In the absence of TIM-3 knockdown, the production of Δnef HIV- release (Fig. 1 C and D, boxed in red). Interestingly, we found that, 1 particles was ∼50% less than that of WT in MDMs of three do- with a higher dose of exogenous Nef plasmid (e.g., 1.5 μg), TIM- nors (Fig. 1 E–H; P < 0.001). Notably, upon TIM-3 knockdown, the 1 levels were significantly up-regulated in both WT- and Δnef HIV- efficiency of which (∼70%) was determined by qPCR (SI Appendix, 1–producing cells, despite the fact that viral production was increased Fig. S1 C–F), we found that the level of HIV-1 release, especially for both WT and Δnef viruses (see details below). that of Δnef particles, was significantly increased (P < 0.05 and P < MDMs express high levels of endogenous TIM-3 protein that 0.01 for both WT and Δnef, respectively); importantly, this led to an intrinsically blocks HIV release (9). We next attempted to knock almost comparable level of release between Δnef and WT HIV- down TIM-3 in human MDMs derived from three healthy donors 1particles(Fig.1E–H), which strongly suggests that the Nef pro- by shRNA and determined their effect on the release of HIV-1 WT tein of HIV-1 NL4-3 counteracts the inhibitory effect of endoge- and Δnef particles. We transduced MDMs with lentiviral shRNA nous TIM-3 on HIV-1 release in human MDMs. vectors targeting human TIM-3 or scramble control, and following puromycin selection we infected cells with VSV-G–pseudotyped The TIM-1–Antagonizing Activity of Nef Is Conserved Among Primate HIV-1 LAI particles, either lacking Env (labeled as “WT”)or Lentiviruses. Primary HIV-1 isolates are classified into M, N, O, lacking both Env and Nef (labeled as “Δnef”); the use of VSV-G and P groups that resulted from independent zoonotic trans- pseudotypes was to ensure a comparable level of initial infection in missions and share distinct and geographical MICROBIOLOGY

Fig. 2. The ability of Nef to antagonize TIM-1 is conserved among primate lentiviruses. (A) Effect of Nef proteins derived from different HIV-1 groups and SIV on HIV-1 production. HEK293T cells were transiently transfected with NL4-3 Δnef and TIM-1 plasmids, along with expression vectors encoding the Nef alleles derived from HIV-1 group M (NL4-3, JRCSF), N (YBF30, N-2693), O (O-13127, O-HJ162), P (P-14788, P-RBF168), as well as SIVgor (CP2139, CR8757) and SIVcpz (EK505, MB897). HIV-1 production was determined as described for Fig. 1A.(B) Effect of lentivirus Nef proteins on release of NL4-3 Δnef as determined by averaging results of three independent experiments. (C) Effect of SIVmac Nef on TIM-1 inhibition of SIVmac production. HEK293T cells were transfected with proviral DNAs encoding SIVmac239 WT or SIVmac239 Δnef, along with increasing amounts of TIM-1 expression plasmids (200 or 500 ng). Levels of cell- associated and virion-associated SIVmac239 Gag proteins were determined by Western blotting using anti-SIV p27 antibody. (D) Relative RT activities from experiments described in C. Viral particles released from transfected cells described in C were quantified by RT assay. Data are means ± SDs of three in- dependent experiments. ***P < 0.001; ****P < 0.0001. (E) Effect of HIV-2 Nef on the production of HIV-2 particles in the presence or absence of TIM-1. Western blotting was conducted to measure the expression of HIV-2 Gag in the cell lysates and in the purified virions. (F) Relative RT activities from ex- periments described in E. Shown are means ± SDs of three independent experiments by comparing with the RT activity of HIV-2 WT alone. ****P < 0.0001.

Li et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 25, 2021 distributions (31). We next examined whether the Nef proteins We examined additional Nef mutants for their effect on TIM- of different primary HIV-1 isolates, as well as the Nef proteins of 1–mediated inhibition of HIV-1 release and observed that, like HIV-2 and simian immunodeficiency virus (SIV), can also WT Nef, PP75AA and EE156QQ Nef fully counteracted the counteract TIM-1. Indeed, we observed that the Nef proteins of ability of TIM-1 to inhibit the release of HIV-1 Δnef (Fig. 3I and the HIV-1 M, N, O, and P isolates tested, as well as SIVcpz and SI Appendix, Fig. S4I). The PP75 motif is located in the proline- + SIVgor Nefs, all relieved the inhibitory effect of TIM-1 on re- rich SH3-binding domain and is known to be important for Nef lease of HIV-1 Δnef particles, based on the Western blotting virus growth but not CD4 down-regulation (35), whereas the analyses of viral p24 levels (Fig. 2A) and quantifications of the EE156 motif is located in the flexible loop region crucial for viral p24 band intensity vs. that of total viral and cellular Gag β-COP binding and CD4 down-regulation (36). Interestingly, the (Fig. 2B). In agreement with our initial finding shown above, the Nef LL165AA mutant, which is deficient for AP2 binding and Nef proteins of all HIV and SIV isolates also increased the total CD4 down-regulation (37), was still able to counteract the TIM- TIM-1 protein levels in the viral-producer cells (Fig. 2A). 1–mediated inhibition of HIV-1 Δnef release, although less ef- We next determined whether the Nef proteins of other pri- ficiently compared with WT, PP75AA, and EE156QQ (Fig. 3I mate lentiviruses can antagonize the TIM-1–mediated inhibition and SI Appendix, Fig. S4I). Immunostaining and imaging assays of release of their cognate viral particles. We found that, similar showed that the Nef PP75AA and EE156QQ mutants sequester to the results of different HIV-1 lineages shown above, the in- TIM-1 as efficiently as the WT, whereas the Nef LL165AA hibitory effect of TIM-1 on the release of Δnef SIVmac (Fig. 2 C mutant exhibits a much-reduced ability to sequester TIM-1 (SI and D) and HIV-2 (Fig. 2 E and F) variants was much stronger Appendix, Fig. S4 D, F, and G). These imaging data correlated compared with its inhibition of their respective WT counterparts. with the differential abilities of the Nef mutants to antagonize Thus, the antagonistic effect of Nef on TIM-mediated restriction TIM-1–mediated inhibition of viral production (SI Appendix, Fig. is conserved across primate lentiviruses. S4 H and I). Overall, these results demonstrate that expression of WT Nef protein increases TIM-1 internalization from the plasma Nef Promotes TIM-1 Internalization and Sequesters TIM-1 in Intracellular membrane and likely perturbs its endocytic trafficking; this leads Compartments. HIV-1 accessory genes often antagonize host re- to the accumulation and stabilization of TIM-1 in intracellular striction factors by downregulating their expression or sequestrating compartments. This internal retention of TIM-1 by Nef reduces them in intracellular compartments (32). As the first step to un- the capability of TIM-1 to block HIV-1 release. These data are derstand the possible mechanism by which Nef antagonizes TIM-1, reminiscent of the Vpu-mediated increase in overall tetherin we examined the steady-state level of TIM-1 expression on the levels in COS cells despite the ability of Vpu to counteract the plasma membrane, the kinetics of TIM-1 internalization, and its inhibitory effect of tetherin on virus release in this cell line (38). intracellular localization in the presence or absence of Nef. We found that, despite the significant increase in the total level of TIM- SERINC Proteins Potentiate Inhibition of HIV-1 Release by Stabilizing 1expressionintransfectedcells shown above (Figs. 1C and 2A), the TIM-1, the Effect of Which Is Counteracted by Nef. Recent studies surface expression of TIM-1 in Nef-expressing cells was relatively have demonstrated that Nef antagonizes SERINC-mediated in- low compared with cells expressing TIM-1 alone (Fig. 3 A and B). hibition of HIV-1 infectivity by downregulating SERINCs from We next performed immunostaining and imaging analyses and the plasma membrane, resulting in decreased SERINC incor- observed that, in the absence of Nef (HIV-1 ΔNef), TIM-1 was poration into virions and therefore enhanced viral infectivity (23, predominantly expressed on the plasma membrane of trans- 24). Given that both SERINCs and TIMs are antagonized by fected HEK293T cells (Fig. 3C, Bottom); however, when Nef was HIV-1 Nef, we sought to explore a possible interplay between coexpressed (HIV-1 ΔNef + Nef), a significant portion of TIM- SERINC and TIM proteins during HIV-1 production. We first 1 was detected within intracellular compartments (Fig. 3C, Top; used shRNA lentiviral transduction to deplete SERINC3 (two Pearson coefficient for Nef/TIM-1 colocalization, ∼0.74; Fig. shRNA clones; SI Appendix, Fig. S5A) in HEK293T cells, which 3D). Importantly, Nef also strongly colocalized with endogenous are known to express an endogenous albeit low level of SER- TIM-1 in an internal compartment in COS-7 cells, which express INC3 (SI Appendix, Fig. S5B) (23, 24). Depletion of SERINC3 high levels of TIM-1; the internal compartment in which Nef and more efficiently overcame the inhibition of TIM-1 on HIV-1 TIM-1 colocalized in COS-7 cells contained the autophagy Δnef production (by approximately threefold) compared with marker p62 (Pearson coefficient for TIM-1 and p62, 0.94) (Fig. 3 that of HIV-1 WT (∼30%) (Fig. 4A and SI Appendix, Fig. S5C). E and G) but not the TGN marker TGN-46 (Fig. 3F). Consistent As would be expected, the infectivity of HIV-1 Δnef was more with the increased overall levels of exogenous TIM-1 in Nef- dramatically rescued by knocking down SERINC3, approximately expressing HEK293T cells (see above), Nef expression mark- by fourfold, compared with that of WT, which was ∼30% (Fig. 4B edly increased the levels of endogenous TIM-1 in COS-7 cells, as and SI Appendix, Fig. S5D). These results suggested that endog- examined by Western blotting (SI Appendix, Fig. S2A). In enous SERINC3 protein in HEK293T cells potentiates the over- transfected HEK293T cells, TIM-1 also significantly colocalized expressed TIM-1 protein-mediated inhibition of HIV-1 release, with p62 (Pearson coefficient for TIM-1/p62 colocalization, particularly that of Nef-deficient virus. ∼0.73; SI Appendix, Fig. S2 B and F), an autophagy-related To further investigate the role of SERINC proteins in TIM-1– marker, but not with other intracellular compartmental mark- mediated inhibition of HIV-1 release, we cotransfected HEK293T ers CD63, LAMP-1, and TGN46 (SI Appendix, Fig. S2 C–F). cells with different amounts of SERINC3 or SERINC5 plasmids, Analysis of the TIM-1 internalization kinetics showed that along with a constant dose of TIM-1. We observed that coex- HIV-1 Nef substantially enhanced the rate of TIM-1 endocytosis pression of SERINC3 or SERINC5 with TIM-1 further decreased from the plasma membrane (Fig. 3H; see SI Appendix, Fig. S3 for the production of HIV-1 WT and Δnef particles that was inhibited flow cytometry profiles). We also examined two Nef mutants, by TIM-1 (Fig. 4 C and D), which correlated with the increased G2A and D123A, in which a myristoylation acceptor glycine (G) expression of TIM-1 on the cell surface as determined by flow and an acidic residue (D) were respectively replaced by an ala- cytometry (Fig. 4E and SI Appendix,Fig.S5E and F). Importantly, nine (A) resulting in a defect in membrane targeting (33) or SERINC3 or SERINC5 alone did not inhibit HIV-1 release (even AP2 binding (34). These mutants had a more modest effect on increased viral production in some cases) (SI Appendix,Fig.S5G). modulating TIM-1 internalization (Fig. 3H and SI Appendix, Fig. We next examined a series of Nef mutants harboring EDAA, S3) and sequestration (SI Appendix, Fig. S4 C and E) and were LLAA, ΔCAW, or Δ12–39 mutations, some of which have re- unable to antagonize TIM-1–mediated inhibition of HIV-1 cently been shown to less efficiently antagonize SERINCs (39). production (Fig. 3I and SI Appendix, Fig. S4I). We observed that these Nef mutants exhibited an intermediate

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1819475116 Li et al. Downloaded by guest on September 25, 2021 MICROBIOLOGY

Fig. 3. HIV-1 Nef promotes internalization of TIM-1 from the plasma membrane and sequesters TIM-1 in intracellular compartments. (A and B) HIV-1 Nef modestly down-regulates TIM-1 expression on the plasma membrane. HEK293T cells were transfected with a FLAG-tagged TIM-1 plasmid in the presence or absence of NL4-3 Nef (an equal DNA input for Nef and TIM-1 vector), and the expression of TIM-1 on the plasma membrane was determined by using an anti- FLAG antibody. A summary plot of the relative geometric means of TIM-1 from three independent experiments is shown in A.*P < 0.05. A representative flow cytometry profile demonstrating a modest decrease of TIM-1 in the presence of Nef is shown in B.(C and D) HIV-1 Nef sequesters TIM-1 in intracellular compartments. HEK293T cells cultured in chamber slides were transfected with pNL4-3ΔNef and the human TIM-1 expression vector in the absence or presence of a vector expressing HA-tagged Nef. At 24-h posttransfection, cells were permeabilized and incubated with primary and secondary antibodies to detect TIM-1 (green) and Nef (anti-HA, red). Cells were mounted with Vectashield mounting media with DAPI and examined with a Delta-Vision RT deconvolution microscope (C). Colocalization was quantified by calculating the Pearson correlation coefficients (R values) using the softWoRx colocalization module (D). (E–G) COS-7 cells cultured in chamber slides were transfected with a vector expressing HA-tagged Nef; cells were fixed, permeabilized, and incubated for 1 h with antibodies specific for TIM-1, HA, p62, or TGN46 appropriately diluted in 3% BSA–PBS. In this experiment, the primary antibodies were directly labeled with either Zenon Alexa Fluor 488 (TIM-1, green), Zenon Alexa Fluor 594 (p62 or TGN46, red), or Zenon Alexa Fluor 647 (anti-HA, blue) using the Zenon antibody labeling kit (Thermo Fisher). Colocalization between TIM-1 and p62 and between TIM-1 and Nef was performed; no colocalization between TIM-1 and TGN46 was detected. (H) Comparison of the internalization kinetics of TIM-1 in the presence of WT and Nef mutants. HEK293T cells stably expressing TIM-1 were transfected with plasmids encoding Nef-GFP (WT), Nef-G2A-GFP, Nef-D123A-GFP, or Vpr-YFP (negative control, Mock). The geometric means of the fluorescence intensity of TIM-1 at each time points were recorded, compared, and their relative values were plotted against time by setting the value of time point 0 for each Nef construct or mock as zero. Results are averaged percentages ± SDs of the internalized TIM-1 protein from three to five independent experiments. ***P < 0.001. (I) Effect of Nef mutants on TIM-1–mediated inhibition of HIV-1 release. HEK293T cells were cotransfected with NL4-3 Δnef proviral DNA plus plasmids encoding TIM-1, WT Nef, or Nef mutants. The relative viral release efficiency was determined as described in Fig. 1 and as indicated. A summary plot of the effect of Nef mutants on antagonizing TIM-1–mediated restriction of HIV-1 Δnef virus release from four independent experiments (SI Appendix, Fig. S4I).

Li et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 25, 2021 Fig. 4. SERINC3 and SERINC5 proteins potentiate TIM-mediated inhibition of HIV-1 production. (A) Effect of knockdown of SERINC3 on HIV-1 production. HEK293T cells stably expressing control shRNA or SERINC3 shRNAs (two clones) were transfected with proviral DNA plasmids encoding NL4-3 WT or Δnef together with TIM-1 plasmid. Viral production was evaluated by examining RT activity. Shown are means and SDs of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Effect of SERINC3 knockdown on HIV-1 infectivity as determined by infecting HeLa-TZM cells. *P < 0.05; ***P < 0.001. (C) Effect of coexpression of SERINC3 or SERINC5 with TIM-1 on HIV-1 production. HEK293T cells were transfected with plasmids encoding NL4-3 WT or ΔNef and a TIM-1 plasmid plus increasing amounts (200 and 500 ng) of pBJ-SERINC3-HA or SERINC5-HA plasmids. Forty-eight hours posttransfection, Western blotting was performed to examine Gag and SERINC (HA-tagged) expression in the cell lysates and purified virions using specific primary antibodies. (D) Effect of SERINC3 or SERINC5 coexpression with TIM-1 on HIV-1 release, as quantified by viral p24 vs. the total cellular Gag. Data are means ± SDs of three independent experiments relative to that of WT NL4-3 alone (“Vector”). (E) Effect of SERINC3 and SERINC5 on the expression of TIM-1 on the cell surface as determined by + flow cytometry using an anti–TIM-1 antibody. (F–J) Effect of SERINC3 and SERINC5 knockdown on HIV-1 WT and ΔNef release in PBMCs and CD4 T cells. The experimental design is shown in F. PBMCs (G and H) or CD4+ T cells (I and J) were transduced with VSV-G–pseudotyped lentiviral vectors encoding either control shRNA or shRNA targeting SERINC3 or SERINC5. Cells were then infected with HIV-1 LAI Δenv (WT) or LAI ΔenvΔnef (ΔNef) bearing VSV-G. The RT activities of newly produced HIV-1 virions were determined. Shown are means ± SDs of three independent experiments relative to the RT activity from PBMCs + or CD4 T cells transduced with control shRNA and infected with WT HIV-1. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant.

counteracting effect against TIM-1 relative to WT and Δnef (SI To determine the role of endogenous SERINCs in potentiat- Appendix,Fig.S6A and B). Altogether, these results suggest ing TIM-1 in CD4-positive T cells, we knocked down SERINC3 that SERINC proteins cooperate with TIM-1 to inhibit HIV- and SERINC5 either in activated human peripheral blood + 1 release, and that Nef likely antagonizes TIM-1, in part mononuclear cells (PBMCs) or purified CD4 T cells known to through SERINCs. express endogenous TIM-1 and TIM-3 proteins that intrinsically

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1819475116 Li et al. Downloaded by guest on September 25, 2021 inhibit HIV-1 release (9). To avoid the potential confounding release of HIV-1 particles that was inhibited by TIM-1 and that the effect of SERINC knockdown on HIV-1 infectivity in multiple effects were more pronounced for Δnef compared with WT (Fig. 6 rounds of replication, we applied HIV-1 LAI Δenv (expressing A and B). Moreover, MLV glycoGag, like HIV-1 Nef (Fig. 1C), up- Nef) and LAIΔenvΔnef (not expressing Nef) bearing VSV-G to regulated the TIM-1 expression levels in transfected cells (Fig. 6A). determine the effect of SERINC3 or SERINC5 knockdown on Similar results were obtained for EIAV S2 (Fig. 6 C and D). Im- a single-round HIV-1 production (see the procedure depicted portantly, neither MLV glycoGag nor EIAV S2 alone had an effect in Fig. 4F). Relative to shRNA control-transduced cells, we on HIV-1 release (Fig. 6 E and F). Results for direct comparisons observed roughly a threefold increase of HIV-1 production in between HIV-1 Nef, MLV glycoGag, and EIAV S2 for counteracting SERINC3 or SERINC5-depleted PBMCs (donors 1 and 2) TIM-1 are shown in SI Appendix,Fig.S8.Overall,theseresults + or CD4 T cells (donors 3 and 4), again more prominent for support a model by which HIV-1 Nef, MLV glycoGag, and EIAV HIV-1 LAIΔenvΔnef compared with LAIΔenv (Fig. 4 G–J). S2 share a common mechanism for antagonizing TIM-mediated in- Notably, in three of the four donors examined (donors 1, 2, hibition of HIV-1 release, which is in part through SERINCs. and 4), the production of LAIΔenvΔnef particles was ∼25– 50%lessthanthatofLAIΔenv particles, suggesting that Nef Discussion counteracts SERINC-mediated inhibition of HIV-1 LAI production HIV-1 Nef is a small, multifunctional protein that plays critical (Fig. 4 G, H,andJ). The knockdown efficiency of SERINC3 and roles in AIDS pathogenesis. Among other functions, Nef mod- + SERINC5 in PBMCs and CD4 T cells of these donors was about ulates cell surface expression of various receptors, including twofold to approximately threefold, based on quantitative RT-PCR CD4, MHC-I, CD8, CD28, and CD80, to promote viral immune (qRT-PCR) assays shown in SI Appendix,Fig.S7A–D.Overall,data evasion (reviewed in ref. 40). Consistent with its role in AIDS from these experiments revealed that endogenous SERINC3 and pathogenesis, SIVmac variants harboring deleted nef genes are + SERINC5 in human CD4 T cells potentiate TIM-mediated in- less pathogenic in rhesus macaques (41). Similarly, infection with hibition of HIV-1 release, especially that of Nef-deficient viruses. nef-defective HIV-1 strains is associated with low viral loads and delayed progression to AIDS in infected humans (42–44). An- SERINC5 Stabilizes TIM-1. One notable observation from the above other important function of Nef is to antagonize cellular re- experiments was that overexpression of SERINC3 and SERINC5 striction factors that intrinsically inhibit lentivirus replication. up-regulated the TIM-1 expression level in transfected HEK293T For example, the Nef proteins of HIV-1 group O and most SIV cells (Fig. 4C) and that knockdown of SERINC3 or SERINC5 strains are capable of counteracting tetherin by downregulating

decreased the TIM-1 expression in human PBMCs (SI Appendix, or sequestrating this antiviral protein, thereby enhancing lenti- MICROBIOLOGY Fig. S7E). To understand the underlying mechanism, we performed viral particle release (45–48). Recently, the Nef proteins of some pulse-chase labeling assays in HEK293T cells and compared the HIV-1 M group strains were also shown to counteract tetherin half-life of TIM-1 in the presence or absence of SERINC5 (Fig. 5). (49). Here, we provide evidence that the Nef proteins of primate We found that SERINC5 greatly stabilized TIM-1 expression, lentiviruses are capable of overcoming the inhibitory effect of extending its half-life from <2to∼5 h (Fig. 5). This likely accounts TIM proteins on the release of HIV-1, HIV-2, and SIVs. for the up-regulation of TIM-1 by SERINC5 (Fig. 4C)andalso Mechanistically, we find that Nef promotes the internalization of explains, at least in part, the potentiation of SERINCs on TIM- TIM-1 from the plasma membrane and sequesters TIM-1 in mediated inhibition of HIV-1 release. It remains to be determined intracellular compartments. While the exact nature of these how knockdown of SERINCs also down-modulates the RNA level compartments remains to be determined, our preliminary data of TIMs (SI Appendix,Fig.S7E). showed that Nef and TIM-1 are colocalized with p62, an autophagy-related marker, but not with TGN46, a marker for the Similar to Lentiviral Nef Proteins, MLV glycoGag and EIAV S2 Proteins cellular trans-Golgi network. Another intriguing finding of this also Counteract TIM-1. In addition to HIV-1 Nef, MLV glycoGag study is that SERINC proteins, which themselves do not block and EIAV S2 proteins have also been shown to antagonize SER- HIV-1 release but impair viral infectivity in a manner antago- INCs and enhance HIV-1 infectivity (23, 24, 28–30). We therefore nized by HIV-1 Nef, MLV glycoGag, and EIAV S2 (23, 24, 28– asked whether expression of these two proteins would overcome 30, 50), potentiate TIM-mediated restriction of virion release in TIM-mediated inhibition of HIV-1 release similarly to HIV-1 Nef. part by stabilizing TIM-1, revealing an intriguing interplay be- Indeed, we observed that expression of MLV glycoGag restored tween SERINCs and TIMs at the late stage of HIV-1 replication. How does Nef antagonize the function of TIM to inhibit HIV- 1 release? Given our previous finding that the TIM–PS in- teraction is essential for TIM-mediated inhibition of HIV-1 release (9), we have considered the following models: First, Nef may directly down-regulate TIM expression in the cell, in- cluding its expression on the cell surface. Second, Nef may act directly on PS, either by inhibiting its synthesis, trafficking, flip- ping to the outer leaflet of the plasma membrane, and/or re- ducing its incorporations into virions. Third, Nef may directly disrupt TIM–PS interaction, in particular as it occurs in the ex- Fig. 5. SERINC5 stabilizes TIM-1 as determined by pulse-chase labeling as- tracellular space where the budded viral particles accumulate say. HEK293T cells were transfected with pCIneo-FLAG-TIM-1 in the presence when TIMs are present. Last, Nef could act on some other or absence of pBJ-SERINC5-HA plasmid. Twenty-four hours after trans- protein(s) and/or lipid(s), thus indirectly counteracting the TIM- fection, cells were subjected to pulse-labeling for 1 h and chased for in- mediated inhibition of viral release. In this work, we have per- dicated periods of time. Cell were lysed at 24-h posttransfection, and lysates formed a series of experiments to test these possibilities. We were immunoprecipitated using an anti-FLAG antibody and resolved by SDS/ excluded the possible effect of Nef on PS by examining the PS PAGE. (A) A representative image of the pulse-chase labeling experiment. levels on the plasma membrane as well as in the viral particles by Proteins were detected by PhosphorImager analysis, and the TIM intensity in the presence or absence of SERINC5 was quantified by using Quantity One using FITC-labeled Annexin V or a nanoparticle plasmon coupling-based quantification (51). We also excluded the possi- (Bio-Rad) software. Relative TIM-1 intensity was determined by setting the – level of TIM-1 (time point 0) without SERINC5 to 1.00. (B) A summary plot of bility that HIV-1 Nef could directly disrupt the TIM-1 PS in- the half-life of TIM-1 in the absence or presence of SERINC5. Results are from teraction by performing an in vitro ELISA using a soluble form five independent experiments. of TIM-1 and liposome enriched with PS. We reasoned that the

Li et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 25, 2021 Fig. 6. MLV glycoGag and EIAV S2 antagonize TIM-1–mediated inhibition of HIV-1 production. (A) Effect of MLV glycoGag on HIV-1 production. HEK293T cells were transiently transfected with plasmids expressing WT or Δnef NL4-3 (1 μg each) and TIM-1 (200 ng), together with increasing amounts of glycoGag plasmid (200 or 500 ng). Viral release was determined as described in Fig. 1C.(B) Averaged HIV-1 release efficiency was determined from results of three in- dependent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Effect of EIAV S2 on HIV-1 production. The experimental procedure was the same as described for MLV glycoGag, except that 200 ng of an EIAV S2 plasmid was used. (D) Effect of EIAV S2 on TIM-1–mediated inhibition of HIV-1 release was determined from results of three independent experiments. *P < 0.05; ****P < 0.0001. (E and F) MLV glycoGag and EIAV S2 alone do not inhibit HIV-1 release. The experiments were performed as described in A and C, except that no TIM-1 plasmid was transfected; results from one representative experiment are shown.

interference by Nef of the TIM–PS interaction is topologically SERINCs on HIV-1 infectivity is counteracted by Nef via Nef- unfavorable, because Nef is essentially an intracellular protein mediated down-regulation of SERINCs from the plasma mem- despite the fact that it can be secreted into culture media and has brane (23, 24). In this work, we tested the hypothesis that been detected in exosomes (52, 53). Although more sensitive SERINCs could act on TIMs, thereby indirectly counteracting assays will be needed to precisely define the TIM–PS interaction TIM-mediated inhibition of HIV-1 release. Indeed, we find that in virus-producing cells and released viral particles, our current knockdown of endogenous SERINC3 in HEK293T cells, or de- data strongly suggest that mechanisms unrelated to PS (see be- pletion of SERINC3 and SERINC5 in human PBMCs or puri- + low) are involved in Nef antagonism of TIM-mediated restriction fied CD4 T cells, enhances the production of HIV-1 particles, of lentiviral production. particularly in the absence of Nef. Consistent with this finding, Our Western blotting analyses consistently show that lentiviral we showed that ectopic expression of SERINC3 or SERINC5 Nef proteins increase, rather than decrease, the total levels of enhances the ability of TIM-1 to block HIV-1 release, correlating TIM-1 expression in transfected cells or endogenous TIM-1 in with an up-regulation of TIM-1 expression by SERINCs, in- COS-7 cells. This up-regulation of TIM-1 by Nef could be due to cluding its expression on the cell surface. Additional lines of the transcriptional activity of Nef that activates NF-κB (54, 55) evidence that support a role for SERINCs in Nef-dependent or, alternatively, due to a slowed TIM-1 degradation during the antagonism of TIM restriction include the following: (i) Nef protein biosynthesis and trafficking process. In this regard, our mutants deficient in antagonizing SERINCs, such as G2A, preliminary results suggesting autophagy is associated with Nef D123A, LL165AA, are less potent in counteracting TIMs (Fig. and TIM-1 colocalization are very interesting. We found that the 3I and SI Appendix, Fig. S6 A and B); (ii) MLV glycoGag and internalization rate of TIM-1 is increased when Nef is ectopically EIAV S2 proteins, which effectively antagonize SERINCs, expressed and Nef mutants deficient for membrane targeting counteract TIM-mediated suppression of Nef-deficient lentivirus (G2A) and/or endocytic trafficking (D123A) are incapable of particle release (Fig. 6 A–D and SI Appendix, Fig. S8 A and B); + efficiently promoting TIM-1 internalization or antagonizing (iii) knockdown of SERINCs in PBMCs and CD4 T cells down- TIM-1. Consistent with this finding, we observed that TIM-1 is modulates TIM-1 and TIM-3 expression (SI Appendix, Fig. S7E); sequestered within intracellular compartments by Nef, in sharp and, finally, (iv) coexpression of SERINC5 in HEK293T cells contrast to the predominant plasma membrane localization of stabilizes TIM-1 expression (Fig. 5 A and B). Thus, SERINCs TIM-1 when Nef is absent. Moreover, Nef mutants deficient in appear to contribute to TIM-mediated inhibition of HIV- modulating TIM-1 internalization and trafficking are unable to 1 release by stabilizing TIMs, and lentiviral Nef proteins coun- antagonize the ability of TIM-1 to inhibit HIV-1 release. Hence, teract TIMs in part through acting on SERINCs. Nef appears to modulate the endocytic trafficking of TIM-1 to We must emphasize that, as has been previously reported (23, counteract its inhibitory effect on HIV-1 release. Additional 24), SERINC proteins alone do not affect HIV-1 release (SI experiments will be needed to dissect exactly how Nef modulates Appendix, Fig. S5G), and the potentiating effect of SERINCs on the trafficking of TIM-1, although this may be related to the TIM-mediated inhibition of lentivirus release is dependent on reported interactions between Nef with the cellular clathrin the levels of TIM proteins in viral-producer cells. This explains adaptor protein 2 (AP-2) (34, 56–58). why Nef does not significantly affect HIV-1 release in HEK293T SERINCs are multitransmembrane proteins that are normally cells, which express a very low level of TIMs (9). However, when expressed on the plasma membrane; the restrictive activity of TIM-1 is ectopically expressed in HEK293T cells, knockdown of

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1819475116 Li et al. Downloaded by guest on September 25, 2021 the endogenous SERINC3 led to an increase in HIV-1 pro- were then washed three times with PBS and maintained for an additional 18 h; duction, especially in the absence of Nef (Fig. 4A). In human HIV-1 release in the supernatant was monitored by measuring RT activity. + PBMCs and purified CD4 T cells, which are known to express endogenous levels of both TIMs and SERINCs, we observed that Western Blotting. Western blotting was performed as previously described (68, knockdown of SERINC3 or SERINC5 led to enhanced HIV- 69). In particular, we used a RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, Δnef G–J 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS), which disrupts membrane- 1 production, more so for than for WT (Fig. 4 ). While associated proteins, to lyse cells. The supernatants containing HIV-1 were SERINCs could modulate PS on the plasma membrane and/or in harvested, and viral particles were concentrated by ultracentrifugation at the virions, given that SERINCs have been previously implicated 32,000 rpm (Sorvall; Discovery 100SE, Rotor TH641) for 2 h at 4 °C. Cell lysates in the biosynthesis of serine-derived lipids including PS (59), our and purified virions were dissolved in 5× sample buffer, resolved on 10% SDS/ preliminary results show no significant difference in the levels of PAGE gel, and probed by antibodies against HIV-1 p24 (also for SIV and HIV-2 PS between SERINC-expressing and SERINC-depleted cells Gag), HIV-1 Nef, or TIM-1.

and in produced HIV-1 virions, consistent with a recent report + (39). Instead, we found that SERINC5 greatly stabilizes TIM- Knockdown of SERINC3 or SERINC5 in PBMCs and CD4 T Cells. PBMCs were from healthy, anonymous donors and were stimulated with 2 μg/mL PHA and 5 1 protein in coexpressing cells, extending the half-life of TIM- + 1 from <2 h to up to 6 h (Fig. 5). This explains, at least in part, U/mL IL-2 for 3 d. Subsequently, PBMCs or purified CD4 T cells were transduced with lentiviral vectors expressing SERINC3 or SERINC5 shRNA. The transduced + the potentiating effect of SERINCs on TIM-mediated inhibition PBMCs and CD4 cells were selected with 1 μg/mL puromycin for 1–3dbefore + of HIV-1 release. The link between TIMs and SERINCs, as well infection by HIV-1. The SERINC knockdown efficiency in PBMCs and CD4 Tcells as the capability of Nef to antagonize both of these proteins, as was determined by measuring the RNA level of SERINC3 or SERINC5 by qRT-PCR. revealed in this work (see working model in SI Appendix,Fig.S9), highlights an interesting but complex interplay between the HIV Internalization Assay. The internalization assay was performed as previously accessory protein Nef and host restriction by TIMs and SERINCs. described (48). Briefly, HEK293T cells were transiently transfected with plas- mids encoding a FLAG-tagged TIM-1 and Nef (including Nef mutants). Forty- Materials and Methods eight hours posttransfection, the TIM-1 expression level on the cell surface was Plasmids and Constructs. The pCIneo vector that expresses human TIM-1 with an determined by staining with anti-FLAG and FITC-conjugated secondary anti- N-terminal FLAG tag has been previously described (9). Molecular clones of HIV-1 mouse antibodies at 4 °C for 1 h. After three washes with PBS, the in- NL4-3 and SIVmac239 were obtained from the National Institutes of Health ternalization of TIM-1 was initiated by incubating cells at 37 °C for indicated (NIH) AIDS Reagent Program. HIV-1 proviral constructs NL4-3 Δnef (60), Δvpu periods of time. Cells were then split into two portions: one-half of the cells (61), Δvif (62), Δvpr (63), and Δenv (64) were generated by using PCR-based were washed with pH 2.0 buffer for 1 min; another half were left unwashed. mutagenesis based on the NL4-3 backbone. HIV-1 LAI Δenv, ΔenvΔnef,SIV- Cells were then fixed with 3.7% formaldehyde and analyzed together by flow MICROBIOLOGY mac239 Δnef, and HIV-2 Rod9 Δnef plasmids were gifts from Michael Emerman, cytometry using anti-FLAG and FITC-conjugated secondary antibodies at 4 °C Fred Hutchinson Cancer Research Center, Seattle. The HIV-2 Rod9 plasmid was for 1 h. The percentages of internalized TIM-1 were calculated by dividing the obtained from the NIH AIDS Reagent Program. The HIV-1 NL4-3 Nef expression intracellular fluorescent intensity by the total fluorescent intensity. plasmid was obtained from Yong-Hui Zheng, Michigan State University, East Lansing, MI. Plasmids encoding HIV-1 NL4-3 IRES-eGFP WT and mutants Δnef, Pulse-Chase Labeling. HEK293T cells were transfected with a TIM-1 expression Δvpu, ΔvpuΔnef,aswellasnef alleles of HIV-1 groups M, N, O, and P, and SIVs vector (pCIneo-FLAG-TIM-1) in the presence or absence of SERINC5 expression were kindly provided by Frank Kirchhoff and Daniel Sauter, Institute of Mo- vector (pBJ-SERINC5-HA). Twenty-four hours posttransfection, cells were lecular Virology, Ulm University Medical Center, Ulm, Germany (46, 48, 65, 66). starved with DMEM without cystine and methionine (MP Biomedicals) for 35 The human SERINC3, SERINC5, and MLV glycoGag expression plasmids were 30 min and pulse-labeled for 1 h in DMEM containing 62.5 μCi of S-Met/Cys. kindly provided by Heinrich Göttlinger, University of Massachusetts, Worcester, Cells were then chase-labeled for 0, 0.5, 1, 2, 4, and 6 h, followed by lysis in MA or Massimo Pizzato, University of Trento, Trento, Italy. The plasmids RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P- encoding Nef mutants (G2A, PP75AA, D123A, EE156QQ, and LL165AA, either 40, 0.1% SDS). The harvested cell lysates were immunoprecipitated with fused with IRES-GFP or tagged with HA) were obtained from Massimo Pizzato. anti-FLAG beads (Sigma) and were resolved by SDS/PAGE. The phosphor- The TIM-1–YFP construct was obtained from Jose Casasnovas, National Center images were collected by Typhoon FLA 9000 (GE Healthcare) and quantified for Biotechnology, Madrid. Plasmids encoding TIM-3 shRNA, SERINC3 shRNA, by Quantity One software (Bio-Rad). SERINC5 shRNA, and control shRNA were purchased from Sigma. HIV-1 NL4- 3 proviral DNA encoding Nef mutants EDAA, LLAA, ΔCAW, and Δ12–39 was Statistical Analysis. All statistical analyses were carried out in GraphPad Prism 5, kindly provided by Oliver Fackler, University Hospital Heidelberg, Heidelberg with Student’s t tests or one-way ANOVA, unless otherwise noted. Typically, (39). The EIAV S2 expression vector was obtained from Frederick Fuller, North data from at least three independent experiments were used for the analysis. Carolina State University, Raleigh, NC (67). Cells and Reagents, qRT-PCR, and Immunofluorescence Microscopy. Materials Virus Production and Infection. HEK293T cells were transfected with proviral and methods for cells and reagents, qRT-PCR, and immunofluorescence DNA plasmids encoding HIV-1, SIV, or HIV-2, along with a TIM-1 expression microscopy can be found in SI Appendix. plasmid in the absence or presence of HIV-1 Nef, MLV glycoGag, or EIAV S2 DNA by using calcium-phosphate. Twenty-four hours posttransfection, the ACKNOWLEDGMENTS. We thank Frank Kirchhoff and Daniel Sauter for supernatants were harvested and clarified through 0.2-μm filters. Virus providing valuable reagents and for providing critical comments on the production was quantified by measuring RT activity as previously described (9). manuscript. We thank Michael Emerman, Massimo Pizzato, Heinrich Göttlinger, HIV-1 release efficiency was also measured by quantifying the viral p24 signal Yong-Hui Zheng, Oliver Fackler, Jose Casasnovas, and the NIH AIDS Reagent on Western blots compared with total viral plus cellular Gag proteins. Alter- Program for reagents. This work was supported by NIH Grants R01AI112381 and R01GM132069 (to S.-L.L.), NIH Grant R01CA138509 (to B.M.R.), NIH natively, viral p24 levels were quantified by using an ELISA kit (catalog #5421; Grant R01AI132111 (to B.M.R. and S.G.), and NIH Grant R01AI064099 and ABL). Virus infectivity was examined by infecting HeLa-TZM-bl cells and firefly Providence/Boston Center for AIDS Research Grant P30AI042853 (to S.G.). luciferase activity was measured 48 h after infection according to the manu- + Research in the E.O.F. laboratory is supported by the Intramural Research facturer’s instructions (Promega). For virus production in PBMCs and CD4 Program of the Center for Cancer Research, National Cancer Institute, NIH, Tcells,cellswereinfectedwithHIV-1WTorΔnef bearing VSV-G for 6 h. Cells and by the Intramural AIDS Targeted Antiviral Program.

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