Avian sarcoma leukosis virus receptor-envelopesystem for simultaneous dissection of multipleneural circuits in mammalian brainMakoto Matsuyamaa, Yohei Ohashia, Tadashi Tsubotaa, Masae Yaguchia, Shigeki Katob, Kazuto Kobayashib,and Yasushi Miyashitaa,c,1

aDepartment of Physiology, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo 113-0033, Japan; bDepartment of Molecular Genetics, Institute ofBiomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan; and cCore Research for Evolutional Science andTechnology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Edited by Joshua R. Sanes, Harvard University, Cambridge, MA, and approved April 17, 2015 (received for review December 15, 2014)

Pathway-specific gene delivery is requisite for understanding complexneuronal systems in which neurons that project to different targetregions are locally intermingled. However, conventional genetic toolscannot achieve simultaneous, independent gene delivery into multi-ple target cells with high efficiency and low cross-reactivity. In thisstudy, we systematically screened all receptor–envelope pairs result-ing from the combination of four avian sarcoma leukosis virus (ASLV)envelopes (EnvA, EnvB, EnvC, and EnvE) and five engineered avian-derived receptors (TVA950, TVBS3, TVC, TVBT, and DR-46TVB) in vitro.Four of the 20 pairs exhibited both high infection rates (TVA–EnvA,99.6%; TVBS3–EnvB, 97.7%; TVC–EnvC, 98.2%; and DR-46TVB–EnvE,98.8%) and low cross-reactivity (<2.5%). Next, we tested these fourreceptor–envelope pairs in vivo in a pathway-specific gene-transfermethod. Neurons projecting into a limited somatosensory area werelabeled with each receptor by retrograde gene transfer. Three of thefour pairs exhibited selective transduction into thalamocortical neu-rons expressing the paired receptor (>98%), with no observed cross-reaction. Finally, by expressing three receptor types in a single animal,we achieved pathway-specific, differential fluorescent labeling ofthree thalamic neuronal populations, each projecting into differentsomatosensory areas. Thus, we identified three orthogonal pairs fromthe list of ASLV subgroups and established a new vector system thatprovides a simultaneous, independent, and highly specific genetictool for transferring genes into multiple target cells in vivo. Our ap-proach is broadly applicable to pathway-specific labeling and func-tional analysis of diverse neuronal systems.

avian sarcoma leukosis virus | pseudotyped lentiviral vector |pathway-specific gene transfer

In the mammalian brain, neurons projecting to different targetregions are locally intermingled, and even adjacent neurons

can have different connectivities and functions (1–5). To disentanglesuch complex networks, anatomical mapping and individual ma-nipulation of each neural pathway are both critical. To date, con-ventional genetic tools, such as site-specific recombinase (Cre/loxP)(6), prokaryotic repressor (Tet-On/Off system) (7), and a viralreceptor–envelope pair (TVA–EnvA) (8) have been used togenetically control specific neural pathways in the mammalianbrain, usually in combination with retrograde viral infection (9).Despite their usefulness, these technologies can simultaneously

manipulate no more than two neural pathways—a number that isfar from sufficient for a dissection of natural brain circuitry.Therefore, it is necessary to increase the number of availabletools. One challenge to doing so is that all such tools need to beorthogonal; in other words, the biological reactions on whichthey are based must not cross-react.A number of biochemical mechanisms are potentially appli-

cable to orthogonal gene expression systems. One of the mostpromising candidates is the use of specific combinations of virusenvelope (Env) proteins and corresponding receptors. Becauseviral vectors pseudotyped with Env proteins exclusively infect

cells that express compatible receptors, exogenous expression ofreceptors in target cells provides a specific guide for viral entry.However, some viruses can use more than one molecular speciesas receptors, and these receptors provide a variety of functionsessential for viral entry. In simple situations, receptors bind tovirus envelopes and initiate endocytic uptake of viruses; alterna-tively, receptors affect cellular signaling pathways that facilitatevirus entry, or they directly activate fusion/penetration processes byinducing conformational changes in Env proteins (10).Among the enveloped viruses, we focused on avian sarcoma

leukosis virus subgroups (ASLVs) for three reasons. First, ASLV’snatural host range is restricted to birds. Therefore, it is likely thatASLV-pseudotyped viral vectors will exhibit low, nonspecificinfectivity toward mammalian neurons via endogenous receptors.Second, ASLVs are reported to require single molecular speciesas receptors, and this simple mode of infection is suitable foradaptation as a conditional gene-delivery system. Third, manydifferent Env and receptor proteins are available from among sixdistinct ASLV subgroups (A, B, C, D, E, and J) and 10 differentreceptors (11), and there is evidence that some ASLV Envproteins exhibit specific binding to disparate receptor sequences.For example, chicken TVA protein belongs to the family of low-density lipoprotein receptors and determines susceptibility toASLV-A (12). The tumor necrosis factor receptor-related pro-teins TVBS1 and TVBS3 confer susceptibility to ASLV-B/-D/-E,

Significance

Genetic dissection of multiple neural pathways remains chal-lenging because of the limited number of genetic methods thatcan be used simultaneously. To overcome this limitation, weused modified avian sarcoma and leukosis virus envelopes andreceptors to develop highly orthogonal genetic tools that canachieve expression of different genes in different target cells.From in vitro and in vivo screens, we identified tools that canspecifically transfer genes of interest into mammalian neuronsvia engineered receptors, with minimal unintended interactions.Using this approach, we achieved pathway-specific, differentialfluorescent labeling of three thalamic neuronal populations, eachprojecting into different cortical regions. Thus, our approachprovides independent, simultaneous, and specific genetic toolsfor manipulating intermingled neural pathways in vivo.

Author contributions: M.M., Y.O., T.T., and Y.M. designed research; M.M. and M.Y. per-formed research; M.M., Y.O., T.T., S.K., and K.K. contributed new reagents/analytic tools;M.M., Y.O., T.T., and Y.M. analyzed data; and M.M., Y.O., T.T., S.K., K.K., and Y.M. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: yasushi_miyashita@m.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1423963112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1423963112 PNAS | Published online May 19, 2015 | E2947–E2956

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and ASLV-B/-D, respectively (13). ASLV-C uses the TVCprotein of the butyrophilin family, which contains two Ig-likedomains (14). TVBT, a turkey homolog of TVB, is an ASLV-E–specific receptor (15). Finally, ASLV-J uses the chicken multi-membrane-spanning cell-surface protein Na+/H+ exchanger type1 (chNHE1) as a receptor (16).However, no study to date has systematically and quantita-

tively determined which types of ASLVs can infect with highefficiency mammalian cells expressing a single receptor, and nostudy has shown which combination of envelopes and receptorsof ASLV specifically interact with each other. In this study, wefirst performed in vitro and in vivo screens to identify orthogonalreceptor–envelope pairs. We then conducted a proof-of-conceptstudy in vivo to demonstrate that these orthogonal pairs canachieve pathway-specific differential fluorescent labeling ofmultiple neuronal populations, each projecting to different cor-tical regions. Our findings expand the repertoire of genetic toolsthat can be used to dissect and manipulate the complex neuralnetworks created by intermingled neurons projecting to differenttarget regions.

ResultsPutative Orthogonal ASLV Receptor–Envelope Pairs. The variousASLV subgroups exhibit different infectious properties inchicken cell lines (CEFs and DF-1 cells) (12–17). Based on thesedata, we estimated the feasibility of six envelopes and seven re-ceptors as genetic tools, and eliminated several (TVBS1, ASLV-Denvelope, ASLV-J envelope, and chNHE1) for the following reasons:TVBS1 is a nonspecific cellular receptor for ASLV-B, -D, and -E(13, 17); ASLV-D can infect a variety of mammalian cell lines inthe absence of exogenous receptors (18–20); and chNHE1, the re-ceptor for ASLV-J (16), may alter neuronal membrane potential.Additionally, we selected TVA950, a transmembrane form, fromtwo splice-variant forms of TVA, even though the other formof TVA [TVA800, a chicken glycophosphatidylinositol (GPI)-anchored form] has been used in previous studies (8, 21, 22). Wechose the transmembrane form because GPI-anchored proteinscontain a signal peptide in the C terminus that is cleaved off andreplaced by the GPI-anchor, precluding the use of C-terminalepitope tags. This process narrowed down the set of candidatesto four receptors (TVA950, TVBS3, TVC, and TVBT) and fourenvelopes (EnvA, EnvB, EnvC, and EnvE) (Fig. 1A).

Measurement of Orthogonality Between the Receptor–Envelope Pairsin Vitro. To examine the orthogonality of the receptor–envelopepairs, we tested the various combinations to determine whichones afforded specific viral entry and transgene expression inhuman embryonic kidney (HEK) 293T cells. To mitigate theunwanted side effects associated with the expression of an ex-ogenous transmembrane protein in mammalian cells, we deleteda part of the original intracellular domains from each receptor(Fig. 1B). In TVA950, the original intracellular domain, ex-cept for four amino acid residues, was deleted (12). In TVBS3,TVBT, and DR-46TVB, the intracellular domain, except for 107amino acid residues, was deleted (13, 17). In TVC, the intracellulardomain remained. Epitope tags (HA, c-Myc, V5, and 3× FLAG)were C-terminally fused to the receptors to allow detection byimmunohistochemistry. To increase protein half-life according tothe mammalian N-end rule (23), the N termini of the receptorswere also modified, such that valine was the second amino acid.We then developed two types of recombinant lentiviral vec-

tors: FuGB2-pseudotyped bicistronic vectors that coexpressedthe engineered receptor and Aequorea coerulescens green fluo-rescent protein (AcGFP1) and ASLV Env-pseudotyped vectorsthat expressed mCherry (Fig. 2A). The FuGB2-pseudotypedlentiviral vector can transduce neurons by retrograde axonaltransport in vivo (24). Some dividing cells can be also transducedin vitro (25). In the present study, this vector was used to express

ASLV receptors in both in vitro and in vivo experiments. Thecytomegalovirus (CMV) promoter was selected because it drivesa high level of protein expression in the mammalian brain (26)and has been used previously for TVA expression (8, 21, 27). Wetested all possible receptor–envelope combinations by seriallyinfecting receptor-expressing and Env-pseudotyped vectors intoHEK 293T cells. mCherry expression was observed in only fivepairs: TVA950–EnvA, TVBS3

–EnvB, TVC–EnvC, TVBT–EnvB,

and TVBT–EnvE (Fig. 2 B–E). Although the results obtained

with TVA950–EnvA, TVBS3–EnvB, and TVC–EnvC were con-

sistent with those obtained in DF-1 cells, the infection of EnvBvector into TVBT-expressing cells observed in our assays was in-consistent with previous reports.We next sought to rationally design an alternative EnvE-spe-

cific receptor for TVBT. The chimeric protein DR-46TVBwas engineered by exchanging the first cysteine-rich domain of

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Fig. 1. Selection and construction of ASLV-pseudotyped lentiviral vectorsand avian-derived receptors. (A) Schematic illustration of receptors expressed onthe cell surface and their specificity for ASLV envelope-pseudotyped lentiviralentry. The ASLV receptors and envelopes used in the following experimentswere underlined. (B) Schematic illustration of the altered ASLV receptors andenvelopes constructs used in this study. Numbers indicate the position of aminoacid residues in mature proteins; signal peptide residues have positive numbers.SU, surface envelope subunit; TM, transmembrane envelope subunit; VSV-G,vesicular stomatitis virus G protein; SP, signal peptide; EC, extracellular domain;MS, transmembrane spanning domain; IC, intracellular domain; HA, epitope tagfrom influenza virus HA protein; His, histidine residue tag; Ser–Glyx4 linker,5-amino acid peptide normally used as the linker; DR5, DEATH RECEPTOR 5.

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chicken TVBS1, which is critical for ASLV-B infection (28), withthose of the human TVB homolog DEATH RECEPTOR 5.HEK 293T cells expressing this chimeric receptor expressedmCherry in combination with EnvE vector, but not EnvB vector(Fig. 2F).

These results were quantitatively confirmed by flow cytometry.A progressive shift to the right was observed only in the histo-grams for the following six pairs: TVA950–EnvA, TVBS3

–EnvB,TVC–EnvC, TVBT

–EnvB, TVBT–EnvE, and DR–46TVB–EnvE

(Fig. 3A). Furthermore, the infection rates (the ratio of AcGFP1/

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Fig. 2. Characterization of ASLV receptor–envelope pairs in vitro. (A) Schematic representation of the lentiviral vector constructs used in vitro and theexperimental outline of serial infections of bicistronic vectors and Env vectors in HEK 293T cells. cPPT, central polypurine tract; CTS, central terminationsequence; LTR, long terminal repeat; RRE, Rev responsive element; Ψ, packaging signal; CMV, CMV promoter; P2A, picornaviral 2A peptide; WPRE, Wood-chuck hepatitis virus posttranscriptional regulatory element. (B–G) Fluorescence images of HEK 293T cells expressing TVA950 (B), TVBS3 (C), TVC (D), TVBT (E),and DR-46TVB (F) after transfection with one of the Env vectors. Receptor proteins were coexpressed with AcGFP1. (G) A vector expressing only AcGFP1 wasused as a negative control. (Scale bars: 50 μm.)

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Fig. 3. Quantitative comparison of in vitro infection rates of all ASLV receptor–envelope pairs. (A) Representative histograms of mCherry fluorescence in-tensity for every possible receptor–envelope pair expressed in HEK 293T cells. Receptor-expressing cells infected by EnvA-, EnvB-, EnvC-, or EnvE-pseudotypedvectors (blue, green, yellow, and red histograms) are compared with receptor-expressing cells infected with no Env vectors (purple histograms). In the controlcondition, HEK 293T cells were infected with AcGFP1-expressing vector instead of the bicistronic vector. The vertical axis represents the total number ofcounted cells, and the horizontal axis represents Logicle scaling (29). (B) Histograms showing the infection rates of Env vectors into HEK 293T cells positive foreach receptor. Cells expressing AcGFP1 were considered receptor-positive. Thus, the infection rate is estimated by calculating the ratio of mCherry- andAcGFP1-positive cells to all AcGFP1-positive cells in flow-cytometry analysis. Three independent experiments were conducted, and the data were analyzed byusing one-way ANOVA followed by Scheffé’s F-test. *P < 0.001. The average mean values of three experiments are shown, with the SD of the data indicatedby error bars.

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mCherry double-positive cells to all AcGFP1-positive cells undereach condition) for TVA950–EnvA, TVBS3

–EnvB, TVC–EnvC,and DR-46TVB–EnvE were significantly higher than those ofthe other pairs [one-way analysis of variance (ANOVA) andScheffé’s F-test, P < 0.001] except for TVBT

–EnvB and TVBT–

EnvE (Fig. 3B and Table S1). Together, these data demon-strate that these four pairs (TVA950–EnvA, TVBS3

–EnvB,TVC–EnvC, and DR-46TVB–EnvE) are orthogonal in HEK293T cells.

Orthogonality of the TVA950–EnvA Pair in Vivo. Having demon-strated that the four aforementioned orthogonal pairs were ca-pable of transferring genes into cultured mammalian cells, wenext examined the gene-transfer capability and the orthogonalityof our receptors and envelopes in vivo using the thalamocorticalsystem of rats. FuGB2-pseudotyped lentiviral vector shows ahigh efficiency of gene transfer via retrograde axonal transport.In these in vivo experiments, the receptor genes were transferredinto both thalamocortical and cortico-cortical neurons projectingto the injection site (the primary somatosensory cortex) (Fig. S1).

By contrast, fluorescent protein genes were expressed only in thethalamocortical neurons by the injection of Env vectors into thethalamus (Fig. S2). In a pilot study, we injected FuGB2–TVCvector along with a mixture of EnvA/EnvB/EnvC/EnvE vectorsinto 12 rats. We did not observe any sign of infection by theEnvC vector in these rats in vivo. Hence, the TVC–EnvC pairwas not used in the following experiment.We first tested the specificity of the TVA950–EnvA pair (Fig.

4A). In these experiments, the retrograde vector expressingTVA950 (FuGB2–TVA950) was injected into the primary so-matosensory cortex (S1) (Fig. 4B), and a mixture of EnvA/EnvB/EnvE vectors that expressed different fluorescent proteins(XFPs) was injected into the thalamus [EnvA vector expressingblue fluorescent protein (BFP), EnvB vector expressing en-hanced green fluorescent protein (EGFP), and EnvE vectorexpressing tdTomato]. The Env vectors were injected 3 wk afterthe injection of the FuGB2-pseudotyped vector. This timing wasnecessary to allow time for TVA950 expression, which mediatesthe EnvA vector infection. In sections in the immediate vicinityof either the FuGB2–TVA950 or Env vectors’ injection site,

EnvA-BFP

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Fig. 4. Orthogonality of the TVA950–EnvA pair in vivo, demonstrated in rat thalamocortical neurons. (A) Schematic representation of retrograde targetingof TVA950 expression through projection terminals and the selective entry of EnvA vectors from TVA950-expressing neuronal cell bodies. For clarity, EnvA,EnvB, and EnvE are depicted as different shapes. (B) Stereotaxic coordinates of two-step viral injection. First, mutant rabies virus glycoprotein (FuGB2)-pseudotyped TVA950 vectors were injected into primary somatosensory cortex (S1). Three weeks after the first injection, a mixture of Env vectors was injectedinto the thalamus. (C) Overview of the rat brain sagittal sections counterstained with NeuN antibody. Merged images of NeuN (blue) and BFP (cyan) areshown. BFP-positive cells were restricted to the thalamus (C, Right) and BFP-positive axons innervated S1 (C, Left). (Scale bars: 1,000 μm.) (D) A coronal sectionnear the first injection site. The merged images show the distribution of BFP-positive axons. The boxed area (D, Left) is magnified (D, Right) to show thedistribution of BFP-positive axons in the layers of S1. (Scale bars: D, Left, 1,000 μm; D, Right, 250 μm.) (E) A coronal section near the second injection site. Theboxed area in E, Left, is magnified in E, Upper Right, to show BFP-positive neurons in the ventral posteromedial (VPM) and posteromedian (POm) thalamicnuclei. The boxed area in E, Upper Right is further magnified in E, Lower Right), to show double labeling of BFP- and NeuN-positive cells. (Scale bars: E, Left,1,000 μm; E, Upper Right, 250 μm; E, Lower Right, 50 μm.) (F) A representative coronal section stained with anti-HA antibody. The merged image of BFP (cyan)and TVA950–HA (purple) shows that BFP-positive neurons were a subpopulation of TVA950 (HA)-positive neurons. (Scale bar: 250 μm.) (G) Confocal images ofa section stained with anti-HA antibody. BFP expression was observed in a subset of TVA950-HA–positive neurons, whereas expression of enhanced GFP(EGFP) or tdTomato expression was not observed. (Scale bar: 50 μm.) Str, striatum; WM, white matter.

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many BFP-positive axons in S1 or cells in the thalamus wereobserved, respectively (Fig. 4C). Near the FuGB2–TVA950vector injection site, dense projections of BFP-positive axonswere observed in layers I, IV, and VI (Fig. 4D), consistent withthe projection pattern of thalamocortical neurons (30–34). Bycontrast, around the injection sites of the Env vectors, BFP-positive cells were detected in the ventral posteromedial tha-lamic nucleus (VPM) and the posteromedian thalamic nucleus(POm); all BFP-positive cells were neurons, as evidenced bydouble labeling with an anti-NeuN antibody (Fig. 4E). Next, wecompared the distribution of TVA950-positive neurons with thatof the BFP-positive neurons in brain sections, using an anti-HAantibody to immunohistochemically detect the HA-taggedTVA950 protein. Notably, BFP expression was observed only inthese thalamic neurons, even though HA-tagged TVA950 pro-tein expression was also observed in cortices with neurons pro-jecting to the injection site of the TVA950-expressing vector,indicating that our vector system selectively visualized a singlepathway (Fig. S1A). Most of the TVA950-positive neurons wereretrogradely transduced via their axon terminals, because theFuGB2-pseudotyped lentiviral vector showed at least 14.4 timeshigher preference for transducing neurons via axon terminalsover via soma (Fig. S3).A quantitative analysis performed by using sections stained for

the HA epitope tag revealed that >99% of BFP-positive neuronswere TVA950-positive (ratio of BFP and TVA950 double-posi-tive neurons to all BFP-positive neurons: 1,942/1,959) (Fig. 4F),whereas none were EGFP- or tdTomato-positive neurons (Fig.4G). Similar results were observed for the other two rats (974/982 and 572/580; Table 1), showing that the EnvA vector leads tospecific transduction of TVA950-positive neurons, and otherEnvB/EnvE vectors do not transduce TVA950-positive or -neg-ative neurons in vivo.

Orthogonality of the TVBS3–EnvB Pair in Vivo. We also tested thespecificity of the TVBS3

–EnvB pair in vivo (Fig. 5A). In thisexperiment, we injected FuGB2–TVBS3 vector into the S1 regionand a mixture of EnvA/EnvB/EnvE vectors into the thalamus3 wk later (Fig. 5B). The injection coordinates for the FuGB2–TVBS3 vector were 2.5 mm posterior to those used for theFuGB2–TVA950 vector in the rats shown in Fig. 4. We observedmany EGFP-positive axons from thalamic neurons innervatingthe S1 cortex (Fig. 5C). The EGFP-positive axons projected intocortical layers I, IV, and VI in the S1 cortex near the FuGB2–TVBS3 vector injection site (Fig. 5D). To compare the distribu-tion of TVBS3-positive cells with that of EGFP-positive cells, weperformed an immunohistochemical analysis using an anti–c-Myc antibody to detect the c-Myc–tagged TVBS3 protein in brain

sections; EGFP-positive cells were detected only in the VPM andPOm (Fig. 5E).We then performed a quantitative immunohistochemical

analysis using the c-Myc epitope tag and found that 96.7% ofEGFP-positive neurons were also TVBS3-positive (ratio ofEGFP and TVBS3 double-positive neurons to all EGFP-positiveneurons: 397/410) (Fig. 5F). By contrast, neither BFP- nortdTomato-positive neurons were observed (Fig. 5G). Similarresults were obtained from the other two rats (1,212/1,224 and964/974; Table 1). These results confirmed that the EnvB vectorleads to specific transduction of TVBS3-positive neurons, and otherEnvA/EnvE vectors do not transduce TVBS3-positive or -negativeneurons in vivo.

Orthogonality of the DR-46TVB–EnvE Pair in Vivo.We next tested thespecificity of the DR-46TVB–EnvE pair in vivo (Fig. 6A). Inthese experiments, FuGB2–DR-46TVB vector was injected intothe S1 region, and a mixture of EnvA/EnvB/EnvE vectors wasinjected into the thalamus 3 wk later (Fig. 6B). The injectioncoordinates for the FuGB2–DR-46TVB vector in the rats were2.5 mm posterior to those used for the FuGB2–TVBS3 vector inthe rats shown in Fig. 5. We observed many tdTomato-positiveaxons from thalamic neurons innervating the S1 cortex (Fig. 6C).Many tdTomato-positive axons projected into S1 cortical layersI, IV, and VI near the FuGB2–DR-46TVB vector injection site(Fig. 6D). To compare the distribution of the DR-46TVB–pos-itive cells with that of tdTomato-positive cells in brain sections,we performed an immunohistochemical analysis using an anti-FLAG antibody to detect the 3× FLAG-tagged DR-46TVBprotein. The tdTomato-positive cells were detected only in theVPM and POm (Fig. 6E).We then performed a quantitative analysis using immunohis-

tochemistry with the 3× FLAG epitope tag and found that 99.9%of tdTomato-positive neurons were also DR-46TVB–positive(ratio of tdTomato and DR-46TVB double-positive neurons toall tdTomato-positive neurons: 502/503) (Fig. 6F), whereas nonewere BFP- or EGFP-positive (Fig. 6G). Among three rats, theaverage specificity of the DR-46TVB–EnvE pair was 99.7%, thehighest among all three receptor–envelope pairs (vs. 99.1% forTVA950–EnvA and 98.7% for TVBS3

–EnvB; Table 1). Theseresults confirmed that the EnvE vector leads to the specifictransduction of DR-46TVB–positive neurons, and other EnvA/EnvB vectors do not transduce DR-46TVB–positive or –negativeneurons in vivo.We also examined the orthogonality of the three ASLV re-

ceptor–envelope pairs in corticocortical projection neurons inthe primary motor cortex (M1) (Fig. S4). In this experiment,FuGB2 vector encoding the ASLV receptors was first injected to

Table 1. The orthogonality of the receptor–envelope combinations in vivo

Receptor Rat no. No. of sections No. of BFP+ neurons No. of EGFP+ neurons No. of tdTomato+ neurons

TVA950+ TVA950− TVA950+ TVA950− TVA950+ TVA950−TVA950 A1 5 572 8 0 0 0 0

A2 5 974 8 0 0 0 0A3 5 1,942 17 0 0 0 0

TVBS3+ TVBS3− TVBS3+ TVBS3− TVBS3+ TVBS3−TVBS3 B1 5 0 0 1,200 12 0 0

B2 5 0 0 384 13 0 0B3 5 0 0 954 10 0 0

DR-46TVB+ DR-46TVB− DR-46TVB+ DR-46TVB− DR-46TVB+ DR-46TVB−DR-46TVB D1 5 0 0 0 0 400 2

D2 5 0 0 0 0 501 1D3 5 0 0 0 0 222 0

Env vectors are EnvA–BFP, EnvB–EGFP, and EnvE–tdTomato. Number of BFP-, EGFP-, tdTomato-, and receptor-positive neurons in rats are shown. Note: Thistable is related to Figs. 4, 5, and 6: Distribution of fluorescent-positive neurons in individual animals.

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M1, and then a second injection of Env vectors was administeredto the contralateral M1. The results showed the orthogonality ofthe three ASLV receptor–envelope pairs in corticocortical neu-rons of M1 (Table S2), as well as in thalamocortical neurons(Table 1).

Simultaneous Gene Transfer in Cultured Mammalian Cells. We nextexamined whether these three receptor–envelope pairs could beused simultaneously to deliver three different genes into threedifferent cell populations in a single culture. For this purpose, weinfected a mixture of EnvA/EnvB/EnvE vectors, each expressingdifferent fluorescent proteins, into an intermingled population ofHEK 293T cells in which each cell expressed one of the threereceptor proteins (TVA950, TVBS3, or DR-46TVB) (Fig. 7A).The infected cells expressed only one of the three fluorescentproteins in a mutually exclusive manner, creating a three-colorcellular mosaic (Fig. 7B). This result confirmed that the infectionspecificity of the three receptor–envelope pairs is preserved, even inthe presence of nonoptimal receptors and Env vectors in vitro.

ASLV Receptor–Envelope Pairs Enable Triple Pathway-Specific GeneTransfer in Vivo. To demonstrate the selective and simultaneouslabeling of three neuronal populations—each projecting to dif-ferent target regions—we injected three types of receptor-expressing vectors into the different cortical areas in S1 and amixture of Env vectors into the thalamus (Fig. 8 A and B). Theinjection coordinates for each receptor-expressing vector werethe same as those shown in Figs. 4–6. In a confocal tiling of asagittal section, we distinguished three XFPs in different axons(Fig. 8C). BFP-, EGFP-, and tdTomato-positive axons inner-vated areas near each injection site. In the thalamus, neuronspositive for all three types of fluorescence were present. Al-though the three Env vectors were injected at the same co-ordinates, the three types of XFP-positive neurons exhibiteddifferent distributions in the thalamus (Fig. 8D). Most of theBFP-positive neurons were medial to the EGFP-positive neuronsin the thalamus, and the EGFP-positive neurons were moremedial than the tdTomato-positive neurons. Furthermore, in-dividual neurons distinctly expressed BFP, EGFP, or tdTomatoin a mutually exclusive manner (Fig. 8 E and F).

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Fig. 5. Orthogonality of the TVBS3–EnvB pair in vivo, demonstrated in rat thalamocortical neurons. (A) Schematic representation of retrograde targeting of

the TVBS3 expression through projection terminals and selective entry of EnvB vectors from TVBS3-expressing neuronal cell bodies. (B) Stereotaxic coordinatesof two-step viral injection. First, FuGB2-pseudotyped TVBS3 vectors were injected into S1. Three weeks after the first injection, a mixture of Env vectors wasinjected into the thalamus. (C) Overview of the rat brain sagittal sections counterstained with NeuN antibody. The merged images of NeuN (blue) and EGFP(green) are shown. EGFP-positive cells were restricted to the thalamus (C, Right), whereas EGFP-positive axons innervated S1 (C, Left). (Scale bars: 1,000 μm.)(D) Coronal section near the first injection site. Merged images show the distribution of EGFP-positive axons. The boxed area (D, Left) is magnified (D, Right)to show the distribution of EGFP-positive axons in the layers of S1. (Scale bars: D, Left, 1,000 μm; D, Right, 250 μm.) (E) Coronal section near the secondinjection site. The boxed area in E, Left, is magnified in E, Right, to show EGFP-positive neurons in the VPM and POm thalamic nuclei. (Scale bars: E, Left,1,000 μm; E, Right, 250 μm.) (F) Representative coronal section stained with anti–c-Myc antibody. The merged image of EGFP and TVBS3

–c-Myc (purple) showsthat EGFP-positive neurons were a subpopulation of the TVBS3 (c-Myc)–positive neurons. (Scale bar: 500 μm.) (G) Confocal images of a section stained withanti–c-Myc antibody. EGFP expression was observed in a subset of TVBS3

–c-Myc–positive neurons, whereas BFP or tdTomato expression was not observed.(Scale bar: 50 μm.)

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In conclusion, we identified three ASLV receptor–envelopepairs that are orthogonal in mammalian cells and rat brains. Thesepairs could be used simultaneously in single cultures or individualrats to fluorescently label three distinct subgroups of neurons.

DiscussionBy experimentally identifying and characterizing ASLV recep-tor–envelope pairs in mammalian cells, we engineered a novelmultitargeted gene-transfer system and conducted a proof-of-concept study demonstrating that this system can geneticallydissect intermingled neural connections in rat brains. The highorthogonality of three artificial ASLV receptors and their cor-responding ASLV envelopes was key to the success of this systemand permitted the ASLV-pseudotyped lentiviral vectors to se-lectively transduce mammalian cells expressing specific receptorsin vitro and in vivo.We combined FuGB2-pseudotyped lentiviral vector (35) with

our receptor–envelope system to identify neural populationsprojecting to a target region in the brain. This retrograde gene-

transfer method was selected based on its high efficiency ofretrograde transduction via axon terminals (24, 35). Varioustypes of pseudotyped lentiviral vectors, including FuGB2 vector,were developed to achieve retrograde gene transfer by fusingrabies virus glycoprotein to vesicular stomatitis virus glycoprotein(VSV-G) (25, 35–38). The FuGB- and FuGB2-pseudotypedlentiviral vectors show highly efficient retrograde gene transfer(HiRet series) (25, 35). Furthermore, the FuGC- and FuGE-pseudotyped lentiviral vectors show neuron-specific retrogradegene transfer (NeuRet series), but exhibit lower efficiency thanthat of the HiRet series (37, 38). Therefore, although the Neu-Ret series was developed after the HiRet series, FuGB2-pseu-dotyped lentiviral vector is the system that yields the highestefficiency of retrograde gene transfer. Based on these reports, weselected the FuGB2 vector in this study. Although we believe thethree ASLV receptor–envelope pairs would also work with theNeuRet series, in vivo tests are necessary to check the NeuRetefficiency for each experimental design. Importantly, to useNeuRet series instead of HiRet, it is necessary to optimize the

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Fig. 6. Orthogonality of the DR-46TVB–EnvE pair in vivo, demonstrated in rat thalamocortical neurons. (A) Schematic representation of retrograde targetingof DR-46TVB expression through projection terminals and selective entry of EnvE vectors from DR-46TVB–expressing neuronal cell bodies. (B) Stereotaxiccoordinates of two-step viral injection. First, FuGB2-pseudotyped DR-46TVB vectors were injected into S1. Three weeks after the first injection, a mixture ofEnv vectors was injected into the thalamus. (C) Overview of rat brain sagittal sections counterstained with NeuN antibody. Merged images of NeuN (blue) andtdTomato (red) are shown. tdTomato-positive cells were restricted to the thalamus (C, Right), and tdTomato-positive axons innervated S1 (C, Left). (Scale bars:C, Left, 1,000 μm; C, Right, 250 μm.) (D) Coronal section near the first injection site shows the distribution of tdTomato-positive axons. The boxed area (D, Left)is magnified (D, Right) to show tdTomato-positive axons innervate layers I, IV, and VI in the S1. (Scale bars: D, Left, 1,000 μm; D, Right, 250 μm.) (E) Coronalsection near the second injection site shows the distribution of tdTomato-positive neurons in detail. The boxed area (E, Left) is magnified (E, Right) to showtdTomato-positive neurons in the VPM and POm thalamic nuclei. (Scale bars: E, Left, 1,000 μm; E, Upper Right, 250 μm; E, Lower Right, 100 μm.) (F) Rep-resentative coronal section stained with anti-FLAG antibody. The merged image of tdTomato (red) and DR-46TVB–3× FLAG (purple) shows that tdTomato-positive neurons were a subpopulation of DR-46TVB (3× FLAG)-positive neurons. (Scale bar: 500 μm.) (G) Confocal images of a section stained with anti-FLAGantibody. tdTomato expression was observed in a subset of DR-46TVB–3×-FLAG–positive neurons, whereas BFP (cyan) or EGFP (green) expression was notobserved. (Scale bar: 50 μm.) S2, secondary somatosensory cortex; Rt, reticular thalamic nucleus.

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titers and the waiting time between the injection of NeuRet andEnv vectors beforehand.Our system can also be combined with other retrograde gene-

transfer methods, such as recombinant rabies virus (5, 27, 39–43)and adeno-associated virus (AAV) vectors (44–47). However,ASLV pseudotyping may change the pH tolerance and stabilityof viral particles (48–50); consequently, the infectious titer ofviral vectors should be checked before use. Reports about ret-rograde transduction by AAV vector appear inconsistent; it hasbeen reported as weak or ineffective in some cases (51, 52) andeffective in others (44, 45). In addition to this inconsistency,AAV serotypes 8 and 9, which are known to undergo retrogradeaxonal transport, also anterogradely transduce second-orderneurons in vivo (53). This anterograde transport of AAV vectoris not suitable for pathway-specific gene expression. Related, theHiRet or NeuRet series selectively undergo retrograde, but notanterograde, axonal transport (24, 25, 36–38).ASLVs are divided into six viral subgroups, designated A–E

and J, based on their cellular receptors (11). From these ASLVsubgroups and receptors, we screened for specificity and effi-ciency using HEK 293T cells and rat brains. In our in vitroassays, the infection rate of the TVBT

–EnvB pair was lowerthan that of TVBT

–EnvE pair, but much higher than that ofcontrol (Fig. 3). However, TVBT is reported to permit entry ofretroviral vector pseudotyped with EnvE, but not EnvB (15).Because we used EnvB-pseudotyped lentiviral vectors at highMOI (MOI = 100), weak receptor–envelope interaction wasperhaps detected in our experimental condition. With theTVC–EnvC pair, EnvC vector transduced TVC-expressingneurons only in the absence of other Env vectors (Fig. S3); thereason for these conflicting results remains unclear. Someviruses, such as HIV and hepatitis C virus, use multiple cell-surface components to enter host cells (10). Hence, we specu-late that ASLV(C)-pseudotyped lentiviral vectors might requirefactors other than TVC that are occupied by the other Envvectors (EnvA, EnvB, and EnvE).In our system, the main factor that determines the trans-

duction efficiency is the expression level of the receptor thatinteracts with the Env vectors. To increase the efficiency, a 2- to

3-wk period between the injection of FuGB2 and Env vectors isnecessary to achieve sufficient expression of the receptors. Toincrease receptor expression, two refinements are necessary:codon optimization for mammals and shortening of the re-ceptor sequence. Both refinements are expected to increaseprotein expression level.To apply our system into optogenetic tools, the insert length

will become a problem. The RNA titer of lentiviral vectorsdecreases with increasing insert length (54). In addition, theability of ASLV envelope to pseudotype lentiviral vectors ispoor relative to that of VSV-G envelope glycoprotein, asdemonstrated by its relatively low physical and infectious titers(49). However, we produced Env vectors containing tdTomato(1,475 bp) at high titers [EnvB–tdTomato 8.8 × 1010 trans-duction units (TU)/mL; EnvE–tdTomato 6.15 × 1010 TU/mL),and they worked well in in vivo experiments (Figs. 6 and 8).Possible candidates of frequently used optogenetic tools in-clude hChR2–EYFP (1,662 bp), eNpHR3.0–EYFP (1,683 bp),ChETA–EYFP (1,662 bp), GCaMP6f (1,353 bp), and ArchT–GFP(1,475 bp) (55–59). In GCaMP6f and ArchT–GFP, because thelengths of their DNA sequences are similar to tdTomato, we couldproduce Env vectors expressing these tools at sufficiently high ti-ters to be used in practice. In hChR2–, eNpHR3.0–, and ChETA–EYFP, because their DNA sequences are not so much longerthan that of tdTomato, the titer of Env vectors expressing themcould not decrease so much. We will proceed to the next stepto apply our receptor–envelope pairs to the frequently usedoptogenetic tools.One advantage of this receptor–envelope system is its compati-

bility with other gene-expression systems. Combining the Cre/loxPand Tet system (6, 60) with the receptor–envelope system allowsmodifiers of neural activity—such as channelrhodopsin-2 (61, 62),tetanus toxin (63–65), allatostatin (66), and immunotoxin (67–69)—to be selectively introduced and expressed in a more limited pop-ulation of cell types. In addition, the combination of new opto-genetic tools activated by different light wavelengths (70, 71) withthe receptor–envelope system may allow the manipulation of eachconnection separately and simultaneously. Therefore, when com-bined with other emerging technologies, the system we describehere should make a powerful contribution to the functional analysisof multiple neural populations in vivo.

Materials and MethodsAll experiments were approved by the Institutional Review Committee ofthe University of Tokyo School of Medicine. Lentiviral vectors were producedby cotransfection of HEK293T cells with four plasmids (Table S3) and titratedas described (25, 26). In in vitro experiments, HEK293T cells were seriallyinfected by the FuGB2 vector and the Env vector (3 d after FuGB2 vectorinfection). Three days later, infected HEK293T cells were collected and fixedwith 4% (wt/vol) paraformaldehyde, and analyzed by using fluorescencemicroscopy or flow cytometer. In in vivo experiments, ten-week-old ratswere serially injected with the FuGB2 vector (somatosensory cortex) and theEnv vectors (thalamus, 3 wk after FuGB2 vector injections). Injected rats werethen perfused with 4% (wt/vol) paraformaldehyde and their brains wereprocessed for histological analyses as described (26). All data are presentedas mean ± SE (SEM). We considered P < 0.01 as significant statistical dif-ferences. Additional materials and methods, including procedures for invitro experiments, are provided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Ms. Kaori Mamada, Ms. Ayumi Fukuda,and Mr. Takeru Sekine (Department of Physiology, University of Tokyo) forexcellent technical support; Dr. Brian Lewis (Program in Gene Function andExpression, University of Massachusetts Medical School) for the pCB6-WTA-VCT plasmid; Dr. John A. T. Young (The Salk Institute for Biological Studies)for the pCI-neo-TVBS1, pTEF24ΔDD, pAB6, and pAB9 plasmids; and Dr. MarkJ. Federspiel (Department of Molecular Medicine, Mayo Clinic) for thepTVA950(H6) and pTVC-F plasmids. The St. Jude lentiviral vector system waskindly provided by St. Jude Children’s Research Hospital (Dr. Arthur W.Nienhuis) and George Washington University. This work was supportedin part by Ministry of Education, Culture, Sports, Science, and Technology(MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI Grants19002010 and 24220008 (to Y.M.); Core Research for Evolutional Science and

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Technology from the Japan Science and Technology Agency (Y.M.); theTakeda Science Foundation (Y.M.); MEXT Grants-in-Aid for Young Scientists

23700489 and 26830004 (to Y.O.); Uehara Memorial Fund (Y.O.); and JSPSResearch Fellowships for Young Scientists 235569 (to T.T.) and 256060 (to M.M.).

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Fig. 8. Fluorescent dissociation of three thalamic neuronal populations, each projecting to different cortical regions, visualized simultaneously with or-thogonal receptor–envelope pairs. (A) Schematic representation of the lentiviral vector constructs used in this test. (B) Stereotaxic coordinates of two-stepviral injection. First, each retrograde TVA950/TVBS3/DR-46TVB–expressing vector was injected into the different primary somatosensory cortices. Three weeksafter the first injection, a mixture of Env vectors was injected into the thalamus. The locations of the serial sections shown in C–E are depicted as nos. 1–5.(C–E) Representative images of sagittal sections stained with antibodies against the three fluorescent proteins (BFP, cyan; EGFP, green; tdTomato, red) andcounterstained with NeuN antibody (white). (C) The sagittal section near the injection sites of the FuGB2 vectors shows each fluorescence-positive axondifferentially innervating the S1 regions. (D and E) Serial sagittal sections show the distribution of neurons positive for each type of fluorescence in thethalamus. (E) The boxed area near the second injection site is magnified (E, Inset) to show that the three types of projection neurons are intermingled in thethalamus. (Scale bars: C; D, Upper Left; D, Middle; D, Lower; and E, 1,000 μm; D, Upper Right, and E, Inset, 200 μm.) (F) Confocal images of the boxed area in E,Lower Right. Neurons expressed the three fluorescent proteins in a mutually exclusive manner. (Scale bar: 20 μm.)

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