Mutations in the HIV-1 envelope glycoprotein canbroadly rescue blocks at multiple steps in thevirus replication cycleRachel Van Duynea, Lillian S. Kuoa,1, Phuong Phama, Ken Fujiia,2, and Eric O. Freeda,3

aVirus–Cell Interaction Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702

Edited by Joseph G. Sodroski, Dana-Farber Cancer Institute, Boston, MA, and accepted by Editorial Board Member Stephen P. Goff March 19, 2019 (receivedfor review November 29, 2018)

The p6 domain of HIV-1 Gag contains highly conserved peptidemotifs that recruit host machinery to sites of virus assembly, therebypromoting particle release from the infected cell. We previouslyreported that mutations in the YPXnL motif of p6, which binds thehost protein Alix, severely impair HIV-1 replication. Propagation ofthe p6–Alix binding site mutants in the Jurkat T cell line led to theemergence of viral revertants containing compensatory mutationsnot in Gag but in Vpu and the envelope (Env) glycoprotein subunitsgp120 and gp41. The Env compensatory mutants replicate in JurkatT cells and primary human peripheral blood mononuclear cells, de-spite exhibiting severe defects in cell-free particle infectivity and Env-mediated fusogenicity. Remarkably, the Env compensatory mutantscan also rescue a replication-delayed integrase (IN) mutant, and ex-hibit reduced sensitivity to the IN inhibitor Dolutegravir (DTG), dem-onstrating that they confer a global replication advantage. Inaddition, confirming the ability of Env mutants to confer escape fromDTG, we performed de novo selection for DTG resistance and ob-served resistance mutations in Env. These results identify amino acidsubstitutions in Env that confer broad escape from defects in virusreplication imposed by either mutations in the HIV-1 genome or by anantiretroviral inhibitor. We attribute this phenotype to the ability ofthe Env mutants to mediate highly efficient cell-to-cell transmission,resulting in an increase in the multiplicity of infection. These findingshave broad implications for our understanding of Env function andthe evolution of HIV-1 drug resistance.

drug resistance | cell–cell transmission | Dolutegravir | virological synapse

The assembly of HIV type 1 (HIV-1) particles is driven by theexpression of the viral Gag polyprotein precursor, Pr55Gag,

which contains several major structural domains required forvirus-like particle production, including the p6 domain thatpromotes membrane scission to release budding virions (1–4).HIV-1 p6 encodes two highly conserved peptide motifs, known as“late domains,” that recruit components of the cellular endosomalsorting complexes required for transport (ESCRT) machinery tosites of virus assembly (5–7). The physiological function of theESCRT apparatus is to drive membrane-scission reactions thatoccur in a variety of cellular contexts, including the biogenesis ofmultivesicular bodies and cytokinesis (5–7). The Pro-Thr/Ser-Ala-Pro (PT/SAP) motif of p6 interacts directly with the ESCRT-Isubunit Tsg101 (8–14); the Tyr-Pro-Xn-Leu (YPXnL, where X isany residue and n = 1–3 amino acids) motif of p6 binds the ESCRT-associated protein Alix (15–21). While the requirement for the p6–Tsg101 interaction in HIV-1 release is well established, the physi-ological role of p6–Alix binding is less well defined.Expression of Gag alone is sufficient for the formation of

virus-like particles, but the incorporation of the HIV-1 envelope(Env) glycoprotein complex is required for the generation ofinfectious particles. Env expression on the membranes of bothfree virions and infected cells promotes viral spread. Productiveviral transmission from infected to uninfected cells can occur viatwo pathways: cell-free infection or cell-to-cell transmission (22–26). The latter pathway, which is thought to be a more rapid and

efficient mode of viral propagation than cell-free infection, isinitiated by interactions between Env expressed on the surface ofthe infected cell and CD4 on the surface of the target cell, in theabsence of cell–cell fusion, inducing the formation of a virologicalsynapse (VS) (27). Alternatively, when cell-surface HIV-1 Env en-gages CD4 on target cells, cell fusion can occur, resulting in theformation of multinucleated cells, or syncytia. Several studies havedemonstrated the importance of cell-to-cell transmission in vitro inovercoming barriers to cell-free infection, including target cellinfectability, virus stability, and defects in virus production (28–30).Additionally, cell-to-cell transmission can allow HIV-1 spread in thepresence of broadly neutralizing antibodies (bNabs) (31). Finally,cell-to-cell transmission of HIV-1 has been shown to be less sensitiveto antiretrovirals (ARVs) compared with cell-free transmission (29,32–35). The ability of the virus to evade blocks to infection may inpart be attributed to a higher multiplicity of infection (MOI) duringcell-to-cell vs. cell-free infection, allowing for a higher percentage ofcells to be infected with more than one virus (36). These findingsraise the intriguing possibility that HIV-1 could potentially escapethe inhibitory activity of antiviral agents through the acquisition ofmutations in Env that promote highly efficient cell–cell transmission.We have previously shown that mutations in the Alix binding

site of p6 induce relatively minor defects in Gag processing, virusrelease, and cell-free particle infectivity, but impose significantdelays in replication kinetics in physiologically relevant cell types

Significance

HIV-1 adapts over time to bypass blocks imposed by geneticlesions in the viral genome, typically by acquiring compensa-tory mutations in the defective gene itself. Here we report thatHIV-1 can evade replication blocks by acquiring mutations inthe envelope (Env) glycoprotein that enhance cell-to-celltransmission. We identified mutations in Env that arose inthe presence of the antiretroviral inhibitor Dolutegravir,thereby circumventing restriction. These data, which demon-strate that mutations in Env can provide escape from an anti–HIV-1 drug in vitro, could have broad implications for HIV-1 drugresistance and viral transmission.

Author contributions: R.V.D., L.S.K., K.F., and E.O.F. designed research; R.V.D., L.S.K., P.P.,and K.F. performed research; R.V.D., L.S.K., P.P., and K.F. analyzed data; and R.V.D. andE.O.F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.G.S. is a guest editor invited by theEditorial Board.

Published under the PNAS license.1Present address: Division of AIDS, National Institute of Allergy and Infectious Diseases,Bethesda, MD 20892.

2Present address: Neurovirology Project, Tokyo Metropolitan Institute of Medical Science,156-8506 Tokyo, Japan.

3To whom correspondence should be addressed. Email: efreed@nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1820333116/-/DCSupplemental.

Published online April 11, 2019.

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(37). To further characterize the significance of p6–Alix interac-tions, we selected for viral revertants that alleviate the replicationdefects imposed by a panel of mutations in the p6 YPXnL motif.We identified second-site compensatory changes in both Vpu andEnv that rescue replication defects imposed by the mutations in p6.The three Env compensatory mutations that arose can rescue virusreplication despite exhibiting severe defects in cell-free particle in-fectivity. Strikingly, these Env mutations also provide a replicationadvantage in the context of an integrase (IN) mutant and in thepresence of the IN strand-transfer inhibitor (INSTI) Dolutegravir(DTG). De novo selection in the presence of DTG led to theacquisition of at least one additional Env mutation that conferscell-line–independent resistance to DTG in vitro. We attributethe decreased DTG sensititivity of the Env mutants to their abilityto efficiently transmit viral material in a cell-associated manner,resulting in an increased MOI during spreading infections.

Resultsp6–Alix-Binding Site Mutants Acquire Second-Site Mutations in Vpuand Env. To further characterize the role of the p6–Alix in-teraction in HIV-1 replication, we propagated the p6 mutants (Fig.1A, Left) in culture to select for viral revertants. The Jurkat T cellline was transfected with pNL4-3 WT and p6 mutant proviral clonesand virus replication was monitored over time. Consistent with ourprevious results (37), we observed delayed replication kinetics withall five p6 mutants; the delays relative to the WT ranged from ∼1–2wk. Virus was collected at days of peak replication and seriallypassaged in Jurkat cells. By passage three, near-WT replicationkinetics were observed for all of the p6 mutants (Fig. 1B). Thesedata suggest that the p6 mutants reverted in culture, perhaps byacquiring second-site compensatory mutations. Viral DNA wasisolated from the third passage, amplified by PCR, and sequenced.Unexpectedly, we observed second-site mutations not in Gag but inthe Vpu and Env ORFs (Fig. 1A, Right). Two of the p6 mutants, p6-Y36S/L44R and p6-L41A, acquired inactivating mutations in Vpu,M1I, and K31stop, respectively. All five of the p6 mutants acquiredsubstitutions in Env as follows: Env-P81S, Env-A327T, and Env-A556T for p6-Y36A; Env-A556T for p6-Y36S/L44H; Env-I744Vand Env-R786K for p6-Y36S/L44R; Env-Y61H and Env-R166Ifor p6-L41A; and Env-A556T for p6-L41R (Fig. 1A, Right, and SIAppendix, Fig. S1A, Right).

Env Mutants Y61H, P81S, and A556T Rescue Replication-Defective p6–Alix Binding Site Mutants in Jurkat Cells. To determine whether theselected Vpu and Env substitutions can rescue the replicationdefects imposed by the p6–Alix binding-site mutations, we con-

structed pNL4-3 p6/Env and p6/Vpu mutant clones and evalu-ated their replication kinetics, in parallel with WT and theoriginal p6-mutant clones, in Jurkat cells. The Vpu-inactivatingmutations partially rescued the replication-defective p6-Y36S/L44Rand p6-L41A mutants (SI Appendix, Fig. S1 C and D). The p6-Y36Areplication defect was largely rescued by both Env-P81S and Env-A556T (Fig. 2A). Similarly, the replication defects of both p6-Y36S/L44H and p6-L41R were rescued by Env-A556T, with reverse-transcriptase (RT) peaks occurring at or near the day of peak RTfor the WT (Fig. 2 B and D, respectively). In contrast, the replicationdefective p6-Y36A substitution was not rescued by Env-A327T (SIAppendix, Fig. S1B) and the p6-Y36S/L44R mutations were notrescued by Env-I744V or Env-R786K (SI Appendix, Fig. S1C). Thedelay in replication exhibited by p6-L41A was rescued by Env-Y61H,but not by Env-R166I (Fig. 2C and SI Appendix, Fig. S1D). Becausethe Env mutations R166I, A327T, I744V, and R786K did not con-tribute to rescue of the p6 mutants, they were not analyzed further.Similarly, considering that Vpu mutations often arise during propa-gation of replication-defective HIV-1 mutants in Jurkat cells, weelected to focus on the Env compensatory mutations.

Env Compensatory Mutants Display Highly Efficient ReplicationKinetics in Jurkat T Cells and Peripheral Blood Mononuclear CellsDespite Severe Defects in Single-Cycle Infectivity and Fusogenicity.We next determined the replicative fitness of the Env compen-satory mutants in the context of WT Gag. We transfected Jurkatcells with pNL4-3 Env mutant proviral clones and observed thatthe Env compensatory mutants exhibited WT or faster-than-WTreplication kinetics (Fig. 3A). The 293T-derived virus-containingsupernatants were normalized for RT activity and used to infectthe reporter cell line TZM-bl, which contains an integrated lu-ciferase gene under transcriptional control of the HIV-1 longterminal repeat (LTR) (38, 39). The Env mutants displayedapproximately two- to sixfold defects in single-cycle infectivitycompared with WT (Fig. 3B), a phenotype that is highly dis-cordant with the robust replication fitness of these viruses. At anMOI greater than 1, we also observe defects in infectivity of Env-A556T compared with WT (SI Appendix, Fig. S2A). In contrastto their phenotype in Jurkat cells, in CEM12D7 cells the Envmutants exhibited replication defects relative to WT, consistentwith their defects in single-cycle infectivity. To determinewhether the infectivity defects of the Env mutants are dependenton the producer or target cell, we used Jurkat-derived virus toinfect TZM-bl cells and again observed severe defects in single-cycle infectivity of the three Env compensatory mutants (SIAppendix, Fig. S2B). Finally, we also inoculated Jurkat cells at

A B

Fig. 1. Identification of second-site compensatory changes obtained during propagation of p6–Alix binding site mutants. (A) Schematic of the HIV-1 genomeindicating the location of the Gag p6–Alix binding site mutations and the Vpu and Env substitutions. Mutations in p6, Vpu, and Env are indicated byunderlined residues and amino acid position (NL4-3 numbering). Location of the mutations within the genome are indicated within dashed regions. Labeled domainsare defined as follows: CA, capsid; C1–C5, constant region 1–5; FP, fusion peptide; CT, cytoplasmic tail; HR1/HR2, heptad repeat 1/2; MA, matrix; MSD, membrane-spanning domain; NC, nucleocapsid; V1–V5, variable region 1–5. (B) Replication kinetics of the p6–Alix binding site mutants at the third passage. Jurkat T cells weretransfected with the indicated pNL4-3 p6 mutant proviral clones and assayed for replication kinetics by measuring RT activity. Virus-containing supernatants werecollected at days of peak replication and used to infect new Jurkat cultures. After two rounds of reinfection, cells were collected at days of peak replication, viralgenomic DNA was extracted, amplified, and sequenced. Data shown are from one representative selection experiment.

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high viral inputs and observed very inefficient cell-free infection (SIAppendix, Fig. S2C). With spinoculation, a high MOI was achieved,and we again observed defects in single-cycle infectivity of Env-A556Tcompared with WT (SI Appendix, Fig. S2C). These results establishthat the single-cycle infectivity defects conferred by the Env mutantsare independent of producer or target cell type and viral input. Wealso observed that the Env mutants markedly reduced particle in-fectivity in the context of the original p6 mutations (Fig. 3C), againdemonstrating that the Env compensatory mutations rescue repli-cation despite exhibiting defects in cell-free particle infectivity.An additional interesting feature of the rescuing Env mutants

is that they did not form syncytia during a spreading infection in

Jurkat cells. To quantify the fusogenic activity of the Env mu-tants, we cocultured 293T cells coexpressing Env and Tat withTZM-bl or Jurkat-1G5 reporter cell lines. Fusion of the Env-expressing 293T cells with the CD4/CXCR4-expressing TZM-blor Jurkat-1G5 cells leads to Tat-mediated transactivation of theLTR-luciferase in the reporter cell and subsequent luciferaseexpression. The relative fusogenicity of the Env mutants paral-leled their single-cycle infectivity; Env-Y61H, P81S/A327T, andA556T were all significantly defective in cell–cell fusion relative toWT (Fig. 3D). In this experiment, the Env A327T substitution isincluded with the P81S mutation; however, we have shown that theA327T mutation does not contribute to phenotypic differences in

BA C D

Fig. 2. Rescue of replication-defective p6–Alix binding site mutants by compensatory changes in Env. Jurkat T cells were transfected with the indicated pNL4-3p6 and p6/Env mutant proviral clones and assayed for replication kinetics by measuring RT activity. Individual panels/graphs represent one p6 mutant with itscorresponding compensatory changes: (A) p6-Y36A, (B) p6-Y36S/L44H, (C) p6-L41A, (D) p6-L41R. Replication kinetics of WT NL4-3 are indicated by a solid blackline, p6 mutants by solid colored lines, and p6/Env mutants by colored line markers of varying shapes. B–D are from one experiment and the WT data are sharedacross these panels. Data are representative of at least two independent experiments.

A B C

D E

Fig. 3. Enhanced replication kinetics of Env mutants in Jurkat cells are discordant with defective cell-free particle infectivity and impaired fusogenicity. (A)Jurkat T cells were transfected with the indicated proviral clones and replication kinetics were monitored by measuring RT activity. (B) 293T-derived Envmutant viruses were collected 48 h posttransfection, RT normalized, and used to infect TZM-bl cells. Luciferase activity was measured ∼36 h postinfection;data are normalized to WT. Data from at least three independent experiments are shown as means ± SD. (C) Infectivity of the indicated mutants was analyzedas in B. Data from at least three independent experiments are shown as means ± SD. (D) 293T cells were cotransfected with the indicated Env mutant ex-pression vectors and an HIV-1 Tat expression vector at a ratio of 10:1. Twenty-four hours posttransfection, 293T cells were removed and overlaid onto TZM-blor Jurkat-1G5 cells with serial dilutions in duplicate. Twenty-four hours postoverlay, luciferase was measured as above and normalized relative to WT Env-expressing cells. Data from three independent experiments per reporter cell line are shown as means ± SD. (E) Cell-to-cell transmission of the indicatedmutants was measured by infecting Jurkat donor cells with VSV-G–pseudotyped pBR43IeG-Env mutant viruses, normalizing for GFP+ cells, and inoculatingtarget Jurkat cells. The accumulation of GFP+ cells during a 48-h coculture was measured. Data from three independent experiments were normalized to WTand plotted as means ± SD; ns, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001.

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replication kinetics or single-cycle infectivity (SI Appendix, Fig. S3 Aand B). Thus, the reduced fusogenicity of the Env mutants in thesequantitative fusion assays correlates well with their inability to formsyncytia in spreading infections.Finally, given that the Env compensatory mutants do not enhance

cell-free infectivity of the p6–Alix binding site mutants, we asked ifthey might affect cell-to-cell transmission. We infected Jurkat cellswith vesicular stomatitis virus-G glycoprotein (VSV-G)–pseudotypedpBR43IeG Env mutant viruses; this NL4-3–based constructexpresses GFP from an internal ribosome entry site (IRES)downstream of Nef. The infected donor Jurkat cells were normal-ized for GFP expression and cocultured with target Jurkat cells.Efficiency of Jurkat-to-Jurkat cell-to-cell transmission was measuredas the increase in GFP+ cells above input 48 h postcoculture. Forthis assay, we focused on the Env-A556T mutant, which displays thegreatest defect in fusion capacity (Fig. 3D). We found that not onlydid p6-Y36A alone exhibit a statistically significant decrease in cell-to-cell transmission compared with WT, but the Env-A556T mutantrescued this defect (Fig. 3E). These data indicate that the Envcompensatory mutants are able to overcome poor cell-free in-fectivity by enhancing cell-to-cell transmission.To investigate the replication fitness of the Env compensatory

mutants in physiologically relevant cells, we infected peripheralblood mononuclear cells (PBMCs) from three different donors induplicate (Fig. 4 and SI Appendix, Fig. S4) and monitored replica-tion kinetics as above (Fig. 4 A–C and SI Appendix, Fig. S4 A–C).Replication in Jurkat cells was analyzed in parallel (Fig. 4D and SIAppendix, Fig. S4D). To overcome the poor first-round infectivityinherent to the Env mutants, viruses were pseudotyped with theVSV-G glycoprotein. All replication downstream from the initialround of virus entry, reverse transcription, and integration wouldthen fully depend on HIV-1 Env. In general, the Env mutants arecapable of replicating with WT kinetics in PBMCs; this is particu-larly evident with mutants Y61H and P81S. However, we did ob-serve donor-to-donor variability in the ability of Env-A556T toreplicate in PBMCs (Fig. 4 A–C and SI Appendix, Fig. S4 A–C).“Donor 2” PBMCs supported near-WT levels of Env-A556T rep-lication (Fig. 4B and SI Appendix, Fig. S4B), whereas in PBMCsfrom “donor 1” and “donor 3,” replication of Env-A556T was im-paired (Fig. 4 A and C and SI Appendix, Fig. S4 A and C). Thus, inPBMCs, we observed donor-dependent variability in the capacity ofthe Env mutants to replicate, recapitulating the phenotypes ob-served in both Jurkat and CEM12D7 T cell lines. Consistent withour previous results, when we infected PBMCs with 293T-derived,luciferase-encoding Env-mutant viruses, we again observed defectsin single-cycle infectivity (Fig. 4E), although the reductions were notstatistically significant for the Env-P81S mutant. Thus, in T cells, theEnv mutants are capable of robust replication despite their gener-ally low particle infectivity.

The Compensatory Env Mutants Do Not Enhance Virus Release Efficiency,Env Expression, or Incorporation of Env or Pol Products into Virions. Tocharacterize the ability of the compensatory Env mutants to replicatedespite exhibiting low cell-free particle infectivity, we investigated theproperties of these mutants through biochemical analyses in Jurkatcells. We infected Jurkat cells with 293T-derived, VSV-G pseu-dotyped Env-mutant viruses and measured the expression ofmetabolically labeled viral proteins in cellular and viral lysates byradioimmunoprecipitation (SI Appendix, Fig. S5A). As expected,the Env compensatory mutants did not exhibit deficiencies in virusrelease efficiency compared with WT or Env (−) clones (SI Ap-pendix, Fig. S5 A and B). There was also no significant defect inmutant cellular Env expression or Env processing (gp120/gp160) orvirion Env, RT (p66/p51), or IN (p32) incorporation compared withWT (SI Appendix, Fig. S5). These results are in contrast to a recentstudy in which Env-mediated HIV-1 escape from APOBEC3G re-striction was associated with increased incorporation of RT in vi-rions (40). Taken together, these results indicate that the phenotypeof the Env mutants cannot be explained by effects on virus assemblyand release, viral protein expression, or the incorporation of Env orPol products into virions.

Mutagenesis of Env Residues Y61, P81, and A556 Reinforces thePhenotypes of the Original Env Mutants. To understand in moredetail the effects of mutations at Env residues Y61, P81, andA556 on HIV-1 replication and infectivity, we introduced bothconservative and nonconservative changes at these positions. Weobserved that nearly all of the mutants replicated efficiently inJurkat cells (Fig. 5A) yet exhibited severe defects in cell-free particleinfectivity (Fig. 5B). One exception is the conservative Env-Y61Fmutant, which displays WT levels of infectivity and forms syncytia inJurkat cultures. These data corroborate our observations with theoriginal three Env mutants that are highly defective for cell-freeinfectivity yet can replicate efficiently in Jurkat cells, in somecases with kinetics faster than those of the WT.

Mutations in Env Can Confer Drug Resistance. Given the overallrobust replication observed with the Env compensatory mutants inJurkat cells and in some PBMC donors, we next asked if the mu-tants could rescue a replication defect unrelated to Gag or Env. Wetransfected Jurkat cells with pNL4-3 proviral clones containing anonactive-site IN mutation, N155E (41), in the presence or absenceof Env compensatory mutations. We observed that all three of thecompensatory mutations largely rescue the replication defect im-posed by IN-N155E (Fig. 6A), demonstrating that these mutationscan broadly rescue replication-deficient viruses.Given the ability of the Env mutations to enhance the repli-

cation of an IN mutant, we asked whether they could alsoovercome inhibition mediated by the second-generation INSTI

A B C D E

Fig. 4. Replication kinetics of Env mutants in primary cells recapitulate their phenotypes in cell lines. (A–C) 293T-derived, VSV-G–pseudotyped, Env mutantswere used to infect PBMCs from three independent donors (donors 1–3) in duplicate (SI Appendix, Fig. S4). Jurkat cells (D) were included for comparison;replication kinetics were monitored by measuring RT activity. (E) PBMCs from three independent donors were infected with 293T-derived Env-pseudotypedpNLuc reporter viruses. Data from donor 1 are from two independent experiments, donor 2 from three independent experiments, and donor 3 from oneexperiment shown as means ± SD; ns, not significant. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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DTG, an ARV that is difficult for HIV-1 to evade both in vitroand in vivo (42). We transfected Jurkat cells with the WT or Env-mutant pNL4-3 proviral clones in the presence of three concen-trations of DTG and monitored replication kinetics. At the lowestconcentration of DTG tested, 0.3 nM, all viruses replicated like theno-drug control; however, at higher concentrations of inhibitor,1.5 nM and 3 nM, we observed replication of the three Env com-pensatory mutants but no, or severely delayed, replication of theWT (Fig. 6B). These results demonstrate that the Env mutationsare able to confer escape fromDTG. In the presence of 3 nMDTG,WT-transfected cultures showed evidence of virus replication at∼30 d posttransfection (Fig. 6B, Right), suggesting the acquisition ofDTG-resistance mutations. We collected virus-containing super-natants at the day of peak replication, infected new cultures ofJurkat cells, and found that the repassaged virus exhibited partialDTG resistance compared with naïve virus in the presence of 3 nMDTG. We collected cells from this repassaged, partially DTG-resistant virus, extracted the viral DNA, PCR amplified, and se-quenced. We did not identify any mutations in IN, but ratheridentified three mutations in Env: E209K, A539V, and H641Y (Fig.6C). These results were confirmed by the observation that the Env-A539V mutation arose under the same conditions in two in-dependent selections in Jurkat cells.To determine whether the Env mutations that arose during

virus propagation in DTG were able to confer resistance toDTG, we engineered these mutations into pNL4-3 and evaluatedtheir effects on replication kinetics in Jurkat cells in the presenceor absence of DTG. The Env-A539V mutant replicated with apeak on day 15 in the presence of 3 nM DTG, a concentration atwhich WT did not replicate (Fig. 6D). Additionally, the Env-A539V mutant replicated with WT or faster-than-WT kineticsin the absence of DTG (Fig. 6D), a phenotype similar to that ofthe Env compensatory mutants described above. Similarly, the

Env-E209K and H641Y mutants replicated with WT or faster-than-WT kinetics in the absence of DTG, but conferred onlypartial resistance to DTG (SI Appendix, Fig. S6A). Combining Env-A539V with either Env-E209K or Env-H641Y resulted in doublemutants that replicated in the presence and absence of 3 nM DTGwith kinetics similar to those of the WT without DTG (SI Appendix,Fig. S6B). In the presence of 3 nM DTG, the Env-A539V mutantalso conferred a replication advantage over WT in CEM12D7 cells(Fig. 6E). Further corroborating these findings, we also identifiedEnv-A539V independently during de novo selection for DTG re-sistance in CEM12D7 cells. To extend our observations to a clini-cally relevant HIV-1 strain, we propagated the clade C transmitted/founder virus, K3016 (CH185_TF), in the presence of 3 nM DTG.Consistent with our findings with NL4-3, the virus acquired a mu-tation in Env, specifically Env-T529I. Remarkably, residue T529 ofK3016 corresponds to Env-A539 of NL4-3, the position of theA539V mutation.

Env Mutants Exhibit Decreased Sensitivity to DTG by Enhancing VirusSpread. To further characterize the mechanism of DTG-resistantEnv mutants, we focused on the two gp41 mutants, Env-A556Tand Env-A539V. We calculated the DTG IC50 values of Env-A539V and A556T by performing a spreading infection in JurkatT cells in the presence of serial dilutions of DTG (SI Appendix, Fig.S7). The Env-A556T and A539V mutants displayed a fold-changein resistance compared with WT of ∼4.6 and 5.3, respectively (SIAppendix, Fig. S7); these values are comparable to currently char-acterized DTG drug-resistance mutations in IN [Stanford HIVDrug Resistance Database (43)]. Interestingly, and in contrast tothe Env compensatory mutants obtained during propagation of thep6-mutant viruses, Env-A539V exhibits near-WT levels of cell-freeparticle infectivity (SI Appendix, Fig. S8). The robust replicativefitness exhibited by Env-A556T in Jurkat cells and in some PBMCdonors, despite severe defects in cell-free infectivity, strongly sug-gests that this mutant is proficient in cell-to-cell infectivity. Indeed,we observed that the Env-A556T mutant exhibits a statisticallysignificant increase in cell-to-cell transmission efficiency comparedwith WT in the presence and absence of DTG (Fig. 7). The Env-A539V mutant also exhibits a statistically significant increase in cell-to-cell transmission efficiency in the presence and absence of DTG(Fig. 7). Taken together, these results demonstrate that the Envmutants that exhibit reduced sensitivity to DTG at concentrationsthat inhibit WT are proficient in cell-to-cell transmission.

The gp41 Env Mutants A556T and A539V Increase the MOI During aSpreading Infection. To determine the mechanism by which Env-A556T and Env-A539V confer DTG resistance, we again utilizedpBR43IeG, which allows us to quantify replication kinetics as afunction of viral gene expression. We inoculated Jurkat T cellswith virus-producing 293T cells, allowing for cell-to-cell transfer,and measured GFP expression over time (Fig. 8A and SI Ap-pendix, Fig. S9 A and B). In this system, the Env mutants repli-cate with accelerated kinetics compared with WT. We alsoobserved that at days of peak replication, we measured a higherpercentage of GFP+ cells with the Env mutants compared withWT (Fig. 8A). To further characterize the replication propertiesof the mutant viruses, we calculated the geometric mean fluo-rescence intensity (MFI) of the GFP+ cells at days of peakreplication and found that cells infected with Env mutant virusesexhibit dramatically brighter GFP fluorescence compared withWT (Fig. 8B and SI Appendix, Fig. S9C). Interestingly, we alsoobserve an increase in MFI with WT and Env-A539V when weinfect Jurkat cells by spinoculation at a high MOI (SI Appendix,Fig. S10 A and B). The increase in virally encoded GFP ex-pression, as measured by MFI, in the cells infected with the Envmutants relative to WT (Fig. 8B) suggests that, in the context of aspreading infection, the Env mutants may be overcoming blocksto viral replication by increasing the effective MOI, resulting inan increase in the number of productive infection events pertarget cell.

A

B

Fig. 5. Mutagenesis of Env residues Y61, P81, and A556 confirms the phe-notypes of the original Env mutants. (A) Jurkat cells were transfected withthe indicated proviral clones and replication kinetics were monitored bymeasuring RT activity. Data are representative of at least two independentexperiments. (B) Single-cycle infectivity of the indicated mutants was mea-sured in TZM-bl cells as in Fig. 3B. Data from at least three independentexperiments are shown as means ± SD.

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DiscussionIn this study, we describe mutations within HIV-1 Env thatrescue replication-defective p6–Alix binding site mutants. Wefurther characterize the Env compensatory mutants—Y61H,P81S, and A556T—and find that, despite exhibiting robust replica-tion in the Jurkat T cell line and PBMCs, they display severe defectsin fusogenicity and cell-free particle infectivity. This panel of Envsubstitution mutants not only rescues the replication defects con-ferred by disruption of p6–Alix binding but also enhances the rep-lication of an IN mutant and confers resistance to the INSTI DTG.We attribute the ability of these Env mutants to rescue replicationdefects to their efficient transmission via a cell-to-cell route. De novoselections for DTG resistance in both Jurkat and CEM12D7 T celllines led to the identification of an additional Env mutation, A539V,which confers DTG resistance in both T cell lines. Unlike the Y61H,P81S, and A556T mutations, A539V is highly fit in its ability to infectvia both cell-free and cell-to-cell routes. We also provide evidencethat the gp41 mutants A556T and A539V exhibit decreased sensi-tivity to DTG by increasing the MOI by cell-to-cell transmission.Finally, selection for DTG resistance with a subtype C transmitted/founder virus led to the selection of an Env mutation at the sameposition in gp41 as the A539V mutation, demonstrating that thephenomenon of Env-mediated DTG resistance is not confined tothe subtype B clone NL4-3.The selection of the original three Env escape mutations in

this study is influenced by several factors, including cell type,route of virus transmission, and Alix function. Our initial selec-tion experiments (37) were performed in Jurkat cells, which areinfected inefficiently by cell-free HIV-1 (28), and in which p6–Alix binding site mutants replicate with a severe delay. InPBMCs, the p6–Alix binding site mutants display variable phe-notypes, depending on the donor (37). While it is well estab-lished that the interaction between the PT/SAP motif of p6 andthe ESCRT-I subunit Tsg101 plays a key role in HIV-1 budding,Alix appears to play a more auxiliary and cell-type–dependentrole in HIV-1 replication. The observed reduction in cell-to-cell

transmission efficiency that we observed with a p6–Alix-bindingsite mutant suggests that recruitment of Alix to the VS maypromote virus spread.We demonstrate that the high levels of replicative fitness of

the Env mutants described here in Jurkat cells and in somePBMC donors is a result of their competence to mediate cell-to-cell transmission. The three Env mutants that were acquiredduring propagation of the p6-mutant viruses in Jurkat cells (Env-Y61H, P81S, and A556T) are impaired in their ability to induce

A

C

B

D E

Fig. 6. Env mutations partially rescue a replication-delayed IN mutant and provide a replicative advantage in the presence of DTG. (A) Jurkat cells weretransfected with the indicated proviral clones and replication kinetics were monitored by measuring RT activity. (B) Jurkat cells were transfected with theindicated proviral clones in the presence of 1.5 or 3 nM DTG and replication kinetics were monitored by measuring RT activity. Cells and virus from the WTescape mutant in the presence of 3 nM DTG were collected at the day of peak replication (gray line, day 28 posttransfection). (C) Schematic of HIV-1 Envindicating the location of the mutations identified by sequencing of DTG-resistant viruses. Mutations in Env are indicated by underlined residues and aminoacid position (NL4-3 numbering). Location of the mutations within the genome are indicated within dashed regions. Domains are defined as in Fig. 1A. Jurkat(D) and CEM12D7 (E) cells were transfected with the WT or Env-A539V pNL4-3 proviral clones in the presence or absence of 3 nM DTG and replication kineticswere monitored by measuring RT activity.

Fig. 7. DTG-resistant gp41 mutants Env-A556T and Env-A539V exhibit en-hanced cell-to-cell transmission relative to WT. Cell-to-cell transmission of theindicated mutants was measured by infecting Jurkat donor cells with VSV-G–pseudotyped pBR43IeG-Env mutant viruses, normalizing for GFP+ cells, andinoculating target Jurkat cells in the presence or absence of DTG. The accumu-lation of GFP+ cells during a 48-h coculture was measured. Data from fourindependent experiments were normalized to WT in the absence of drug, andplotted as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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syncytia and thus may decrease bystander cell killing and syn-cytial apoptosis (44). The selection of fusion-defective mutantshas been observed previously (45); mutations in Env arose inHIV-1–infected SupT1 cells that improved replication in theabsence of syncytia (46, 47), and Env mutations that abrogatedsyncytium formation were reported to arise during propagationof a Vif-defective virus (40). The lack of cell–cell fusion poten-tially increases the production of progeny virus before cell death(46, 47). The conservative and nonconservative substitutions thatwe introduced at positions Y61, P81, and A556 confirm ourinitial finding that poorly infectious mutants can replicate effi-ciently in Jurkat cells. The mutants that replicate efficiently inJurkats and PBMCs despite low cell-free particle infectivity arelikely functionally and structurally adapted to promote cell-to-cell transmission. Indeed, this idea is in agreement with the

suggestion that cell–cell fusion is inhibited during VS formationperhaps because Env is retained in a prefusogenic state (27).The positions of the Env compensatory mutations are highly

conserved within subtype B viruses. Among the >6,000 subtype BEnv amino acid sequences in the Los Alamos National Labo-ratory (LANL) database, Y61 is 93.7% conserved, P81 is 99.78%conserved, and A556 (A558 in HXB2; gp41 A47) is 99.91%conserved. A similar degree of conservation is found withinsubtype C viruses. Env-Y61H is present in 708 sequences in theLANL database, 76 of which are subtype B and 34 of which aresubtype C. Env-P81S occurs in only two subtype B sequences andEnv-A556T is present in only two nonsubtype B sequences(circulating recombinant form strains AE and BF). Env-Y61Hwas previously identified as an escape mutation that arose duringtreatment of HIV-1LAI–infected cells with the HIV-1 attachmentinhibitor BMS-378806 (48). To our knowledge, neither Env-P81Snor any other mutation at that position has been previously char-acterized in the literature. Finally, Env-A556 has been mutagenizedextensively in the context of characterizing intra- and interheptadrepeat (HR) interactions of the gp41 six-helix bundle (49, 50), whichis consistent with our findings with A556T. Recently, Env-A556T(A558T in HXB2) was also selected, along with several substitutionsin the C1 domain of gp120, as a resistance mutation to the peptidicinhibitor VIR165 (51). This study suggested that the C1 andHR1 substitutions may alter the kinetics of Env conformationalchanges, providing resistance by limiting substrate accessibility (51).In our study, the Env compensatory mutations are also locatedwithin the C1 domain of gp120 and the HR1 domain of gp41. Al-though gp120 and gp41 interact noncovalently, C1 and HR1 havebeen shown through mutagenesis and structural studies to be criticalfor the stability of gp120–gp41 association in the unliganded state(50, 52–54). These observations suggest that the Env mutationsdescribed here may alter the stability of gp120–gp41 interactions.We mapped the location of the Env compensatory mutations

onto a monomeric Env prefusion structure and a trimeric EnvCD4-bound structure (SI Appendix, Fig. S11 A and B). We foundthat the amino acid positions of the three Env compensatorychanges are closely clustered in the monomeric structure, withY61 and A556 only 4.5 Å apart (55) (SI Appendix, Fig. S11A).Interestingly, the trimeric, CD4-bound Env structure highlights arearrangement of Y61 to a solvent-exposed location central to allthree gp120 monomers (56) (SI Appendix, Fig. S11B). Our mu-tagenesis studies show that conservative mutation Y61F is welltolerated in terms of cell-free particle infectivity, syncytium for-mation, and replication relative to WT (Fig. 5). P81 is located ina loop region just C-terminal to the ⍺0 helix in the CD4-boundstructure; however, the gp41 residues surrounding A556 are notannotated in this structure (56). The Env mutants described herewill provide useful tools for further studies of Env structure andfunction, particularly in understanding how Env regulates cell-free vs. cell-to-cell modes of virus transmission.In addition to changes in Env, we also found that mutations in

Vpu arose in response to the substitutions in the p6–Alix binding

B

A

Fig. 8. Env-A539V confers resistance to DTG by accumulating a high per-centage of infected cells and by increasing the effective MOI. (A) Jurkat cellswere inoculated with virus-producing 293T cells expressing the indicatedproviral clones and replication kinetics were monitored by measuring %GFP+

cells. (B) The geometric MFI of GFP+ cells from A was calculated at days ofpeak infection. Data from four independent experiments are shown asmeans ± SD. *P < 0.05, **P < 0.01.

Fig. 9. Model for enhanced cell-to-cell transmission with mutant Envs. A model for cell-free and cell-to-cell infection for viruses encoding either WT (Upper)or mutant Env (Lower) proteins in the absence (Left) or presence (Right) of DTG. Producer cells are outlined in blue, target cells in black. Infected cells areindicated by the presence of a provirus (black) within the nucleus (gray). Inhibition with DTG is shown in red solid or dashed lines, indicating complete orpartial inhibition, respectively.

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site (Fig. 1A and SI Appendix, Fig. S1 C and D). We commonlyobserve Vpu-inactivating mutations during the propagation ofreplication-delayed HIV-1 in Jurkat cells. The basis for theseloss-of-function mutations in Vpu remains to be explored, butcould be associated with a beneficial effect of low-level tetherinexpression in cell-to-cell viral transmission (57, 58).Our characterization of the Env compensatory mutants continued

with the observation that they confer a global replication advantagein the presence of a replication-delayed IN mutant. We believe this isnot due to bypassing integration, but rather, as discussed above, tothe ability of the Env mutants to overcome blocks in the replicationcycle by mediating effective cell-to-cell transmission. We also foundthe Env compensatory mutants can replicate in the presence of DTGat concentrations that inhibit WT virus. De novo selection experi-ments led to the emergence of a DTG-resistant virus that lackedmutations in IN, but instead contained substitutions in Env. One ofthese Env mutations, Env-A539V, confers DTG resistance in bothJurkat and CEM12D7 cells, a phenotype that correlates with near-WT cell-free particle infectivity.The INSTIs—Raltegravir (RAL), Elvitegravir (EVG), DTG,

and Bictegravir (BIC)—are the most recently Food and DrugAdministration-approved class of anti-HIV inhibitor (59). HIV-1readily develops resistance to RAL and EVG both in vitro andin vivo as a result of mutations in IN; however, DTG is more dif-ficult for the virus to escape (42, 60, 61). Recently, IN-R263K hasemerged in ARV-experienced, INSTI-naïve patients experiencingvirological failure on a DTG regimen and in a patient during DTGmonotherapy (62–64). IN-R263K confers weak DTG resistance but,interestingly, can spread in culture in the context of cell-to-celltransmission (32, 65). Additional IN mutants in patients experi-encing virological failure during DTG monotherapy includeQ148H/R, N155H, G118R, and S230R (64). The fold-change inDTG resistance of reported IN mutants is significantly lower thanthat observed with IN mutants resistant to RAL and EVG; ourcalculated fold-change for Env-A556T and Env-A539V is compa-rable to several of these DTG-resistant mutants (43). Interestingly,we observed no differences in DTG IC50 values in the context of acell-free, single-cycle assay, supporting the hypothesis that reducedDTG sensitivity conferred by the Env mutations is manifestedduring cell-to-cell transmission.Several studies have investigated the ability of ARVs to inhibit

both cell-free and cell–cell infectivity (29, 33, 35, 66–68). Amodel proposed by Baltimore and coworkers (29) suggests that areduction in sensitivity to ARVs due to multiple infections percell caused by cell-to-cell spread can be a source of ongoingreplication in the presence of ARVs. Similarly, Mothes and co-workers (33) reported a correlation between the ability of ARVsto inhibit cell-to-cell transmission and effectiveness against highlocal MOI at cell–cell contacts. These models and observationswould lead to the prediction that mutations that enhance cell-to-cell transmission may lead to drug resistance and that the con-centrations of ARVs required to inhibit a cell-free infectionevent may be insufficient to inhibit cell-to-cell transmission, aswe observed in this study (Fig. 9). The hypothesis that cell–celltransfer of the Env mutants results in more proviruses per cell,relative to WT, is supported by the increased MFI observed inmutant vs. WT-infected cultures (Fig. 8B). While it is challengingto define the significance of cell-to-cell transmission in vivo, it isclear that ARVs need to remain effective against the high MOIoccurring during cell-to-cell transmission (27).Similar to Y61, P81, and A556, A539 (A541 in HXB2; gp41

A30) is 97.99% conserved across clade B viruses, occurring inonly 4 subtype B and 14 subtype C sequences of a total of 175.Mutations at this position have been described previously inseveral different contexts, but largely as fusion inhibitor escapemutations (shown mapped onto a monomeric Env prefusionstructure and a trimeric Env CD4-bound structure; see SI Ap-pendix, Fig. S11 C and D, respectively). Env-A539V was alsoselected during escape from a deleted form of the antiviral factorIFN-induced transmembrane protein 1 (IFITM1) (69) and inresponse to the MxB restriction factor (70). These studies also

observed the appearance of inactivating mutations in Vpu (69,70). Mutations in Env and Vpu also provided escape fromIFITM1 restriction in SupT1 cells; one of the Env mutations,Env-G367R, was described as being defective for cell-free in-fectivity, but able to spread via cell-to-cell transmission (71–73).These data provide further support for the concept that muta-tions in Vpu and Env can overcome barriers to virus replicationby promoting cell-to-cell transmission.The identification of ARV-resistance mutations outside of the

target gene is rare, although not unprecedented (74). For example,several studies have observed that patients on protease inhibitor(PI)-containing regimens experience virological failure in the ab-sence of drug-resistance mutations in protease (PR) (75–78).Siliciano and colleagues proposed that env sequences from thesepatients contain mutations, specifically in the Env CT, that conferPI resistance (79). A connection between the gp41 CT and PI re-sistance may be linked to the role of virus maturation, triggered byPR, in activating Env fusion activity (80, 81). Several clinical reportshave observed failure of DTG-containing therapy in the absence ofIN mutations, suggesting that mutations outside IN may conferresistance in these patients (82, 83). A recent study observed mu-tations in the nef gene that conferred INSTI resistance (84, 85).However, to our knowledge, we present a unique instance of denovo selection of Env mutations that confer resistance to DTG invitro. Ongoing studies should determine whether the Env mutationsdescribed here confer resistance to other classes of ARVs, andwhether mutations in Env can contribute to escape from ARVtherapy in infected individuals (86). The results of this work willprovide fundamental insights into mechanisms of drug resistanceand viral spread in vivo.

MethodsCell Culture. The 293T [obtained from American Type Culture Collection(ATCC)] and TZM-bl [obtained from J. C. Kappes, X. Wu, and Tranzyme, Inc.through the NIH AIDS Reagent Program (ARP), Germantown, MD] cells weremaintained in DMEM containing 5% or 10% (vol/vol) FBS, 2 mM glutamine,100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37 °C with 5%CO2. Jurkat (87), CEM12D7 (88), and Jurkat-1G5 (89) (obtained from E.Aguilar-Cordova and J. Belmont through the NIH ARP, Germantown, MD)T cell lines were maintained in RPMI-1640 medium containing 10% FBS,2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at37 °C with 5% CO2. PBMCs were stimulated with 2 μg/mL PHA-P for 3–5 dbefore infection, then cultured in 50 U/mL IL-2.

Preparation of Virus Stocks. The 293T cells were transfected with HIV-1proviral DNA using Lipofectamine 2000 (Invitrogen) according to the man-ufacturer’s instructions. Virus-containing supernatants were filtered througha 0.45-μm membrane 48 h posttransfection and virus was quantified bymeasuring RT activity. VSV-G–pseudotyped virus stocks were generated fromcells cotransfected with proviral DNA and the VSV-G expression vectorpHCMV-G (90), at a DNA ratio of 10:1. Env-pseudotyped reporter virus stockswere generated from cells cotransfected with pNLuc (91) and the Env ex-pression vector pIIINL4-Env (87), at a DNA ratio of 10:1.

Cloning and Plasmids. The full-length HIV-1 clade B molecular clone pNL4-3(pNL4-3 WT) was used for this study (92). The pNL4-3/KFS clone, referredto here as pNL4-3 Env (−) was described previously (93). pNL4-3 clonesbearing p6 mutations p6-Y36A, Y36S/L44H, Y36S/L44R, L41R, and L41Awere described previously (10, 16, 37). pNL4-3 clones bearing Vpu, Env,p6/Vpu, or p6/Env mutations were constructed with the QuikChange Site-Directed Mutagenesis kit (Stratagene) into subclones of pNL4-3 accordingto the manufacturer’s instructions, and were then recloned into pNL4-3.pIIINL4-Env clones bearing Env mutations were generated as above. The INmutant pNL4-3/IN-N155E was a kind gift from Alan Engelman, Dana FarberCancer Institute, Boston, MA (41) and IN/Env double mutants were gener-ated as above. pBR-NL43-IRES-eGFP-nef+ (pBR43IeG) is a proviral vector thatcoexpresses Nef and eGFP from a single bicistronic RNA (obtained fromF. Kirchhoff through the NIH ARP, Germantown, MD) (94, 95). pBR43IeG clonescontaining Env mutations were constructed as above. The full-length HIV-1 clade C molecular clone pK3016 (CH185_TF) was reported previously (96).DNA for transfections was purified in large-scale quantities using MaxiPrepKits (Qiagen) and mutations were verified by sequencing (Macrogen).

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Virus Replication Assays. Virus replication was assayed in a T cell line model ofspreading infection, as previously described (97). Briefly, T cells were trans-fected with proviral clones (1 μg DNA/1 × 106 cells) in the presence of 700 μg/mLDEAE-dextran or were infected with RT-normalized virus-containingsupernatants. Virus replication was quantified by measuring RT activity in col-lected supernatants over time. Where indicated, the assay was initiated by in-oculation of T cells with GFP virus-producing 293T cells at a ratio of 103 293T:106

Jurkat, a density that has been optimized to recapitulate WT kinetics; here,virus replication was quantified by measuring GFP+ cells by flow cytometry overtime. Cells were fixed in 4% PFA and analyzed by flow cytometry using aFACSCalibur (BD); data were collected via CellQuest and processed via FlowJo.When indicated, genomic DNA was extracted from infected cells using theQIAamp Genomic DNA Extraction Kit (Qiagen); viral DNA was amplified by PCR,and sequenced (Macrogen) (SI Appendix, Table S1). Frequency of residues ateach mutant position was determined by the AnalyzeAlign tool from LANL(https://www.hiv.lanl.gov/content/sequence/ANALYZEALIGN/analyze_align.html).DTG was a kind gift from S. Hughes, National Cancer Institute/NIH, Bethesda,MD. Data were plotted (transformed and normalized) and IC50 values werecalculated using GraphPad PRISM. Curves were fit using nonlinear regression aslog(inhibitor) vs. normalized response, variable slope using a least squares(ordinary) fit. Structural modeling were performed in MacPyMOL.

Single-Cycle Infectivity Assays. TZM-bl is a HeLa-derived reporter cell line thatcontains a stably integrated HIV–LTR–luciferase construct (38, 39). TZM-bl cellswere infected with serial dilutions of RT-normalized virus stocks in the presenceof 10 μg/mL DEAE-dextran. Approximately 36 h postinfection, cells were lysedwith BriteLite luciferase reagent (Perkin-Elmer) and luciferase was measured in aWallac BetaMax plate reader. Technical duplicates were normalized to pNL4-3WT and averaged; data represent the average of independent, normalized ex-periments. To measure single-cycle infectivity in PBMCs, cells were infected induplicate with the indicated RT-normalized Env mutant pseudotyped pNLucviruses. Luciferase was measured 48 h postinfection as above. Technical dupli-cates were normalized to pNL4-3 WT and averaged; data represent the averageof independent, normalized experiments.

Fusion Assay. The 293T cells were cotransfected with the indicated pIIINL4-Env expression vectors and the HIV-1 Tat expression vector pSV-Tat (98) at aratio of 10:1. Twenty-four hours posttransfection, 293T cells were removedand overlaid onto TZM-bl or Jurkat-1G5 cells (Jurkat-derived reportercell line containing a stably integrated HIV-1–LTR–luciferase construct)with serial dilutions in duplicate. Twenty-four hours postoverlay, luciferasewas measured as above. Technical duplicates were normalized to pNL4-3WT and averaged; data represent the average of independent, normalizedexperiments.

Cell-To-Cell Transmission Assay. Donor Jurkat cells were infected with 293T-derived VSV-G–pseudotyped pBR43IeG Env mutant viruses. Forty-eightto 72 h postinfection, the percent of GFP+ donor cells was measured byflow cytometry. Infected donor Jurkat cells were cocultured with un-infected target Jurkat cells at a ratio that normalized the GFP+ input cellsto ∼10% per coculture in the presence or absence of 1.5 nM DTG. Forty-eight hours postcoculture, cells were fixed in 4% PFA and analyzedby flow cytometry. Data were collected via CellQuest and processedvia FlowJo.

Statistics. Statistics were calculated using GraphPad PRISM. Unpaired t testswere performed and two-tailed *P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001 were considered statistically significant.

Ethics Statement. PBMCs were obtained from anonymous, deidentified blooddonors to the NIH Department of Transfusion Medicine Blood ProductsProgram (NIH CC-DTM).

ACKNOWLEDGMENTS. We thank members of the E.O.F. laboratory forhelpful discussion and critical review of the manuscript. Work in the E.O.F.laboratory is supported by the Intramural Research Program of the Centerfor Cancer Research, National Cancer Institute, NIH, the Intramural AIDSTargeted Antiviral Program.

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