Mutations in the HIV-1 Envelope Glycoprotein Can Broadly Rescue Blocks at Multiple Steps in the Virus Replication Cycle

Home , Env (gene), Nef (protein), Tat (HIV)

Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle

Rachel 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 (received for review November 29, 2018)

The p6 domain of HIV-1 Gag contains highly conserved peptide efficient mode of viral propagation than cell-free infection, is motifs that recruit host machinery to sites of virus assembly, thereby initiated by interactions between Env expressed on the surface of promoting particle release from the infected cell. We previously the infected cell and CD4 on the surface of the target cell, in the – reported that mutations in the YPXnL motif of p6, which binds the absence of cell cell fusion, inducing the formation of a virological host protein Alix, severely impair HIV-1 replication. Propagation of synapse (VS) (27). Alternatively, when cell-surface HIV-1 Env en- the p6–Alix binding site mutants in the Jurkat T cell line led to the gages CD4 on target cells, cell fusion can occur, resulting in the emergence of viral revertants containing compensatory mutations formation of multinucleated cells, or syncytia. Several studies have not in Gag but in Vpu and the envelope (Env) glycoprotein subunits demonstrated the importance of cell-to-cell transmission in vitro in gp120 and gp41. The Env compensatory mutants replicate in Jurkat overcoming barriers to cell-free infection, including target cell T cells and primary human peripheral blood mononuclear cells, de- infectability, virus stability, and defects in virus production (28–30). spite exhibiting severe defects in cell-free particle infectivity and Env- Additionally, cell-to-cell transmission can allow HIV-1 spread in the mediated fusogenicity. Remarkably, the Env compensatory mutants presence of broadly neutralizing antibodies (bNabs) (31). Finally, can also rescue a replication-delayed integrase (IN) mutant, and ex- cell-to-cell transmission of HIV-1 has been shown to be less sensitive hibit reduced sensitivity to the IN inhibitor Dolutegravir (DTG), dem- to antiretrovirals (ARVs) compared with cell-free transmission (29, onstrating that they confer a global replication advantage. In 32–35). The ability of the virus to evade blocks to infection may in addition, confirming the ability of Env mutants to confer escape from part be attributed to a higher multiplicity of infection (MOI) during DTG, we performed de novo selection for DTG resistance and ob- cell-to-cell vs. cell-free infection, allowing for a higher percentage of served resistance mutations in Env. These results identify amino acid cells to be infected with more than one virus (36). These findings substitutions in Env that confer broad escape from defects in virus raise the intriguing possibility that HIV-1 could potentially escape replication imposed by either mutations in the HIV-1 genome or by an the inhibitory activity of antiviral agents through the acquisition of – antiretroviral inhibitor. We attribute this phenotype to the ability of mutations in Env that promote highly efficient cell cell transmission. the Env mutants to mediate highly efficient cell-to-cell transmission, We have previously shown that mutations in the Alix binding resulting in an increase in the multiplicity of infection. These findings site of p6 induce relatively minor defects in Gag processing, virus have broad implications for our understanding of Env function and release, and cell-free particle infectivity, but impose significant the evolution of HIV-1 drug resistance. delays in replication kinetics in physiologically relevant cell types

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

he assembly of HIV type 1 (HIV-1) particles is driven by the HIV-1 adapts over time to bypass blocks imposed by genetic Texpression of the viral Gag polyprotein precursor, Pr55Gag, lesions in the viral genome, typically by acquiring compensa- which contains several major structural domains required for tory mutations in the defective gene itself. Here we report that virus-like particle production, including the p6 domain that HIV-1 can evade replication blocks by acquiring mutations in promotes membrane scission to release budding virions (1–4). the envelope (Env) glycoprotein that enhance cell-to-cell HIV-1 p6 encodes two highly conserved peptide motifs, known as transmission. We identified mutations in Env that arose in “late domains,” that recruit components of the cellular endosomal the presence of the antiretroviral inhibitor Dolutegravir, thereby circumventing restriction. These data, which demon- sorting complexes required for transport (ESCRT) machinery to – sites of virus assembly (5–7). The physiological function of the strate that mutations in Env can provide escape from an anti HIV-1 drug in vitro, could have broad implications for HIV-1 drug ESCRT apparatus is to drive membrane-scission reactions that resistance and viral transmission. occur in a variety of cellular contexts, including the biogenesis of – multivesicular bodies and cytokinesis (5 7). The Pro-Thr/Ser-Ala- Author contributions: R.V.D., L.S.K., K.F., and E.O.F. designed research; R.V.D., L.S.K., P.P., Pro (PT/SAP) motif of p6 interacts directly with the ESCRT-I and K.F. performed research; R.V.D., L.S.K., P.P., and K.F. analyzed data; and R.V.D. and subunit Tsg101 (8–14); the Tyr-Pro-Xn-Leu (YPXnL, where X is E.O.F. wrote the paper. any residue and n = 1–3 amino acids) motif of p6 binds the ESCRT- The authors declare no conflict of interest. associated protein Alix (15–21). While the requirement for the p6– This article is a PNAS Direct Submission. J.G.S. is a guest editor invited by the Tsg101 interaction in HIV-1 release is well established, the physi- Editorial Board. ological role of p6–Alix binding is less well defined. Published under the PNAS license. Expression of Gag alone is sufficient for the formation of 1Present address: Division of AIDS, National Institute of Allergy and Infectious Diseases, virus-like particles, but the incorporation of the HIV-1 envelope Bethesda, MD 20892. (Env) glycoprotein complex is required for the generation of 2Present address: Neurovirology Project, Tokyo Metropolitan Institute of Medical Science, infectious particles. Env expression on the membranes of both 156-8506 Tokyo, Japan. free virions and infected cells promotes viral spread. Productive 3To whom correspondence should be addressed. Email: [email protected]. viral transmission from infected to uninfected cells can occur via This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. two pathways: cell-free infection or cell-to-cell transmission (22– 1073/pnas.1820333116/-/DCSupplemental. 26). The latter pathway, which is thought to be a more rapid and Published online April 11, 2019.

9040–9049 | PNAS | April 30, 2019 | vol. 116 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1820333116 Downloaded by guest on October 1, 2021 (37). To further characterize the significance of p6–Alix interac- structed pNL4-3 p6/Env and p6/Vpu mutant clones and evalu- tions, we selected for viral revertants that alleviate the replication ated their replication kinetics, in parallel with WT and the defects imposed by a panel of mutations in the p6 YPXnLmotif. original p6-mutant clones, in Jurkat cells. The Vpu-inactivating We identified second-site compensatory changes in both Vpu and mutations partially rescued the replication-defective p6-Y36S/L44R Env that rescue replication defects imposed by the mutations in p6. and p6-L41A mutants (SI Appendix,Fig.S1C and D). The p6-Y36A The three Env compensatory mutations that arose can rescue virus replication defect was largely rescued by both Env-P81S and Env- replication despite exhibiting severe defects in cell-free particle in- A556T (Fig. 2A). Similarly, the replication defects of both p6-Y36S/ fectivity. Strikingly, these Env mutations also provide a replication L44H and p6-L41R were rescued by Env-A556T, with reverse- advantage in the context of an integrase (IN) mutant and in the transcriptase (RT) peaks occurringatornearthedayofpeakRT presence of the IN strand-transfer inhibitor (INSTI) Dolutegravir for the WT (Fig. 2 B and D, respectively). In contrast, the replication (DTG). De novo selection in the presence of DTG led to the defective p6-Y36A substitution was not rescued by Env-A327T (SI acquisition of at least one additional Env mutation that confers Appendix,Fig.S1B) and the p6-Y36S/L44R mutations were not cell-line–independent resistance to DTG in vitro. We attribute rescued by Env-I744V or Env-R786K (SI Appendix,Fig.S1C). The the decreased DTG sensititivity of the Env mutants to their ability delay in replication exhibited by p6-L41A was rescued by Env-Y61H, to efficiently transmit viral material in a cell-associated manner, but not by Env-R166I (Fig. 2C and SI Appendix,Fig.S1D). Because resulting in an increased MOI during spreading infections. the Env mutations R166I, A327T, I744V, and R786K did not contribute to rescue of the p6 mutants, they were not analyzed further. Results Similarly, considering that Vpu mutations often arise during propa- p6–Alix-Binding Site Mutants Acquire Second-Site Mutations in Vpu gation of replication-defective HIV-1 mutants in Jurkat cells, we and Env. To further characterize the role of the p6–Alix in- elected to focus on the Env compensatory mutations. teraction in HIV-1 replication, we propagated the p6 mutants (Fig. 1A, Left) in culture to select for viral revertants. The Jurkat T cell Env Compensatory Mutants Display Highly Efficient Replication line was transfected with pNL4-3 WT and p6 mutant proviral clones Kinetics in Jurkat T Cells and Peripheral Blood Mononuclear Cells and virus replication was monitored over time. Consistent with our Despite Severe Defects in Single-Cycle Infectivity and Fusogenicity. previous results (37), we observed delayed replication kinetics with We next determined the replicative fitness of the Env compen- all five p6 mutants; the delays relative to the WT ranged from ∼1–2 satory mutants in the context of WT Gag. We transfected Jurkat wk. Virus was collected at days of peak replication and serially cells with pNL4-3 Env mutant proviral clones and observed that passaged in Jurkat cells. By passage three, near-WT replication the Env compensatory mutants exhibited WT or faster-than-WT

kinetics were observed for all of the p6 mutants (Fig. 1B). These replication kinetics (Fig. 3A). The 293T-derived virus-containing MICROBIOLOGY data suggest that the p6 mutants reverted in culture, perhaps by supernatants were normalized for RT activity and used to infect acquiring second-site compensatory mutations. Viral DNA was the reporter cell line TZM-bl, which contains an integrated lu- isolated from the third passage, amplified by PCR, and sequenced. ciferase gene under transcriptional control of the HIV-1 long Unexpectedly, we observed second-site mutations not in Gag but in terminal repeat (LTR) (38, 39). The Env mutants displayed the Vpu and Env ORFs (Fig. 1A, Right). Two of the p6 mutants, p6- approximately two- to sixfold defects in single-cycle infectivity Y36S/L44R and p6-L41A, acquired inactivating mutations in Vpu, compared with WT (Fig. 3B), a phenotype that is highly dis- M1I, and K31stop, respectively. All five of the p6 mutants acquired cordant with the robust replication fitness of these viruses. At an substitutions in Env as follows: Env-P81S, Env-A327T, and Env- MOI greater than 1, we also observe defects in infectivity of Env- A556T for p6-Y36A; Env-A556T for p6-Y36S/L44H; Env-I744V A556T compared with WT (SI Appendix, Fig. S2A). In contrast and Env-R786K for p6-Y36S/L44R; Env-Y61H and Env-R166I to their phenotype in Jurkat cells, in CEM12D7 cells the Env for p6-L41A; and Env-A556T for p6-L41R (Fig. 1A, Right,andSI mutants exhibited replication defects relative to WT, consistent Appendix,Fig.S1A, Right). with their defects in single-cycle infectivity. To determine whether the infectivity defects of the Env mutants are dependent Env Mutants Y61H, P81S, and A556T Rescue Replication-Defective p6– on the producer or target cell, we used Jurkat-derived virus to Alix Binding Site Mutants in Jurkat Cells. To determine whether the infect TZM-bl cells and again observed severe defects in single- selected Vpu and Env substitutions can rescue the replication cycle infectivity of the three Env compensatory mutants (SI defects imposed by the p6–Alix binding-site mutations, we con- Appendix, Fig. S2B). Finally, we also inoculated Jurkat cells at

Fig. 1. Identification of second-site compensatory changes obtained during propagation of p6–Alix binding site mutants. (A) Schematic of the HIV-1 genome indicating the location of the Gag p6–Alix binding site mutations and the Vpu and Env substitutions. Mutations in p6, Vpu, and Env are indicated by underlined residues and amino acid position (NL4-3 numbering). Location of the mutations within the genome are indicated within dashed regions. Labeled domains are 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 were transfected with the indicated pNL4-3 p6 mutant proviral clones and assayed for replication kinetics by measuring RT activity. Virus-containing supernatants were collected 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, viral genomic DNA was extracted, amplified, and sequenced. Data shown are from one representative selection experiment.

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Fig. 2. Rescue of replication-defective p6–Alix binding site mutants by compensatory changes in Env. Jurkat T cells were transfected with the indicated pNL4-3 p6 and p6/Env mutant proviral clones and assayed for replication kinetics by measuring RT activity. Individual panels/graphs represent one p6 mutantwithits corresponding 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 black line, 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 shared across these panels. Data are representative of at least two independent experiments.

high viral inputs and observed very inefficient cell-free infection (SI Jurkat cells. To quantify the fusogenic activity of the Env mu- Appendix,Fig.S2C). With spinoculation, a high MOI was achieved, tants, we cocultured 293T cells coexpressing Env and Tat with and we again observed defects in single-cycle infectivity of Env-A556T TZM-bl or Jurkat-1G5 reporter cell lines. Fusion of the Env- compared with WT (SI Appendix,Fig.S2C). These results establish expressing 293T cells with the CD4/CXCR4-expressing TZM-bl that the single-cycle infectivity defects conferred by the Env mutants or Jurkat-1G5 cells leads to Tat-mediated transactivation of the are independent of producer or target cell type and viral input. We LTR-luciferase in the reporter cell and subsequent luciferase also observed that the Env mutants markedly reduced particle in- expression. The relative fusogenicity of the Env mutants paral- fectivity in the context of the original p6 mutations (Fig. 3C), again leled their single-cycle infectivity; Env-Y61H, P81S/A327T, and demonstrating that the Env compensatory mutations rescue repli- A556T were all significantly defective in cell–cell fusion relative to cation despite exhibiting defects in cell-free particle infectivity. WT (Fig. 3D). In this experiment, the Env A327T substitution is An additional interesting feature of the rescuing Env mutants included with the P81S mutation; however, we have shown that the is that they did not form syncytia during a spreading infection in A327T mutation does not contribute to phenotypic differences in

ABC

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 Env mutant 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 analyzed as in B. Data from at least three independent experiments are shown as means ± SD. (D) 293T cells were cotransfected with the indicated Env mutant expression 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-bl or 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 indicated + mutants was measured by infecting Jurkat donor cells with VSV-G–pseudotyped pBR43IeG-Env mutant viruses, normalizing for GFP cells, and inoculating + target Jurkat cells. The accumulation of GFP cells during a 48-h coculture was measured. Data from three independent experiments were normalized to WT and plotted as means ± SD; ns, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001.

9042 | www.pnas.org/cgi/doi/10.1073/pnas.1820333116 Van Duyne et al. Downloaded by guest on October 1, 2021 replication kinetics or single-cycle infectivity (SI Appendix,Fig.S3A The Compensatory Env Mutants Do Not Enhance Virus Release Efficiency, and B). Thus, the reduced fusogenicity of the Env mutants in these Env Expression, or Incorporation of Env or Pol Products into Virions. To quantitative fusion assays correlates well with their inability to form characterize the ability of the compensatory Env mutants to replicate syncytia in spreading infections. despite exhibiting low cell-free particle infectivity, we investigated the Finally, given that the Env compensatory mutants do not enhance properties of these mutants through biochemical analyses in Jurkat cell-free infectivity of the p6–Alix binding site mutants, we asked if cells. We infected Jurkat cells with 293T-derived, VSV-G pseu- they might affect cell-to-cell transmission. We infected Jurkat cells dotyped Env-mutant viruses and measured the expression of with vesicular stomatitis virus-G glycoprotein (VSV-G)–pseudotyped metabolically labeled viral proteins in cellular and viral lysates by pBR43IeG Env mutant viruses; this NL4-3–based construct radioimmunoprecipitation (SI Appendix, Fig. S5A). As expected, expresses GFP from an internal ribosome entry site (IRES) the Env compensatory mutants did not exhibit deficiencies in virus downstream of Nef. The infected donor Jurkat cells were normal- release efficiency compared with WT or Env (−) clones (SI Ap- ized for GFP expression and cocultured with target Jurkat cells. pendix,Fig.S5A and B). There was also no significant defect in Efficiency of Jurkat-to-Jurkat cell-to-cell transmission was measured mutant cellular Env expression or Env processing (gp120/gp160) or + as the increase in GFP cells above input 48 h postcoculture. For virion Env, RT (p66/p51), or IN (p32) incorporation compared with this assay, we focused on the Env-A556T mutant, which displays the WT (SI Appendix,Fig.S5). These results are in contrast to a recent greatest defect in fusion capacity (Fig. 3D). We found that not only study in which Env-mediated HIV-1 escape from APOBEC3G re- did p6-Y36A alone exhibit a statistically significant decrease in cell- striction was associated with increased incorporation of RT in vi- to-celltransmissioncomparedwithWT,buttheEnv-A556Tmutant rions (40). Taken together, these results indicate that the phenotype rescued this defect (Fig. 3E). These data indicate that the Env of the Env mutants cannot be explained by effects on virus assembly compensatory mutants are able to overcome poor cell-free in- and release, viral protein expression, or the incorporation of Env or fectivity by enhancing cell-to-cell transmission. Pol products into virions. To investigate the replication fitness of the Env compensatory mutants in physiologically relevant cells, we infected peripheral Mutagenesis of Env Residues Y61, P81, and A556 Reinforces the blood mononuclear cells (PBMCs) from three different donors in Phenotypes of the Original Env Mutants. To understand in more duplicate (Fig. 4 and SI Appendix,Fig.S4)andmonitoredreplica- detail the effects of mutations at Env residues Y61, P81, and tion kinetics as above (Fig. 4 A–C and SI Appendix,Fig.S4A–C). A556 on HIV-1 replication and infectivity, we introduced both Replication in Jurkat cells was analyzed in parallel (Fig. 4D and SI conservative and nonconservative changes at these positions. We Appendix,Fig.S4D). To overcome the poor first-round infectivity observed that nearly all of the mutants replicated efficiently in inherent to the Env mutants, viruses were pseudotyped with the Jurkat cells (Fig. 5A) yet exhibited severe defects in cell-free particle VSV-G glycoprotein. All replication downstream from the initial infectivity (Fig. 5B). One exception is the conservative Env-Y61F MICROBIOLOGY round of virus entry, reverse transcription, and integration would mutant, which displays WT levels of infectivity and forms syncytia in then fully depend on HIV-1 Env. In general, the Env mutants are Jurkat cultures. These data corroborate our observations with the capable of replicating with WT kinetics in PBMCs; this is particu- original three Env mutants that are highly defective for cell-free larly evident with mutants Y61H and P81S. However, we did ob- infectivity yet can replicate efficiently in Jurkat cells, in some serve donor-to-donor variability in the ability of Env-A556T to cases with kinetics faster than those of the WT. replicate in PBMCs (Fig. 4 A–C and SI Appendix,Fig.S4A–C). “Donor 2” PBMCs supported near-WT levels of Env-A556T rep- Mutations in Env Can Confer Drug Resistance. Given the overall lication (Fig. 4B and SI Appendix,Fig.S4B), whereas in PBMCs robust replication observed with the Env compensatory mutants in from “donor 1” and “donor 3,” replication of Env-A556T was im- Jurkat cells and in some PBMC donors, we next asked if the mu- paired (Fig. 4 A and C and SI Appendix,Fig.S4A and C). Thus, in tants could rescue a replication defect unrelated to Gag or Env. We PBMCs, we observed donor-dependent variability in the capacity of transfected Jurkat cells with pNL4-3 proviral clones containing a the Env mutants to replicate, recapitulating the phenotypes ob- nonactive-site IN mutation, N155E (41), in the presence or absence served in both Jurkat and CEM12D7 T cell lines. Consistent with of Env compensatory mutations. We observed that all three of the our previous results, when we infected PBMCs with 293T-derived, compensatory mutations largely rescue the replication defect im- luciferase-encoding Env-mutant viruses, we again observed defects posed by IN-N155E (Fig. 6A), demonstrating that these mutations in single-cycle infectivity (Fig. 4E), although the reductions were not can broadly rescue replication-deficient viruses. statistically significant for the Env-P81S mutant. Thus, in T cells, the Given the ability of the Env mutations to enhance the repli- Env mutants are capable of robust replication despite their gener- cation of an IN mutant, we asked whether they could also ally low particle infectivity. overcome inhibition mediated by the second-generation INSTI

ABCD 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 mutants were 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-pseudotyped pNLuc reporter viruses. Data from donor 1 are from two independent experiments, donor 2 from three independent experiments, and donor 3 from one experiment shown as means ± SD; ns, not significant. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Van Duyne et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 9043 Downloaded by guest on October 1, 2021 A Env-E209K and H641Y mutants replicated with WT or faster- than-WT kinetics in the absence of DTG, but conferred only partial resistance to DTG (SI Appendix,Fig.S6A). Combining Env- A539V with either Env-E209K or Env-H641Y resulted in double mutants that replicated in the presence and absence of 3 nM DTG with kinetics similar to those of the WT without DTG (SI Appendix, Fig. S6B). In the presence of 3 nM DTG, the Env-A539V mutant also conferred a replication advantage over WT in CEM12D7 cells (Fig. 6E). Further corroborating these findings, we also identified Env-A539V independently during de novo selection for DTG resistance 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- B tation in Env, specifically Env-T529I. Remarkably, residue T529 of K3016 corresponds to Env-A539 of NL4-3, the position of the A539V mutation.

Env Mutants Exhibit Decreased Sensitivity to DTG by Enhancing Virus Spread. To further characterize the mechanism of DTG-resistant Env mutants, we focused on the two gp41 mutants, Env-A556T and Env-A539V. We calculated the DTG IC50 values of Env- A539V and A556T by performing a spreading infection in Jurkat T cells in the presence of serial dilutions of DTG (SI Appendix,Fig. S7). The Env-A556T and A539V mutants displayed a fold-change in resistance compared with WT of ∼4.6 and 5.3, respectively (SI Appendix,Fig.S7); these values are comparable to currently char- acterized DTG drug-resistance mutations in IN [Stanford HIV Drug Resistance Database (43)]. Interestingly, and in contrast to Fig. 5. Mutagenesis of Env residues Y61, P81, and A556 confirms the phe- the Env compensatory mutants obtained during propagation of the notypes of the original Env mutants. (A) Jurkat cells were transfected with p6-mutant viruses, Env-A539V exhibits near-WT levels of cell-free the indicated proviral clones and replication kinetics were monitored by particle infectivity (SI Appendix,Fig.S8). The robust replicative measuring RT activity. Data are representative of at least two independent fitness exhibited by Env-A556T in Jurkat cells and in some PBMC experiments. (B) Single-cycle infectivity of the indicated mutants was mea- donors, despite severe defects in cell-free infectivity, strongly sug- sured in TZM-bl cells as in Fig. 3B. Data from at least three independent gests that this mutant is proficient in cell-to-cell infectivity. Indeed, experiments are shown as means ± SD. we observed that the Env-A556T mutant exhibits a statistically significant increase in cell-to-cell transmission efficiency compared with WT in the presence and absence of DTG (Fig. 7). The Env- DTG, an ARV that is difficult for HIV-1 to evade both in vitro A539V mutant also exhibits a statistically significant increase in cell- and in vivo (42). We transfected Jurkat cells with the WT or Env- to-cell transmission efficiency in the presence and absence of DTG mutant pNL4-3 proviral clones in the presence of three concen- (Fig. 7). Taken together, these results demonstrate that the Env trations of DTG and monitored replication kinetics. At the lowest mutants that exhibit reduced sensitivity to DTG at concentrations concentration of DTG tested, 0.3 nM, all viruses replicated like the that inhibit WT are proficient in cell-to-cell transmission. no-drug control; however, at higher concentrations of inhibitor, 1.5 nM and 3 nM, we observed replication of the three Env com- The gp41 Env Mutants A556T and A539V Increase the MOI During a pensatory mutants but no, or severely delayed, replication of the Spreading Infection. To determine the mechanism by which Env- WT (Fig. 6B). These results demonstrate that the Env mutations are able to confer escape from DTG. In the presence of 3 nM DTG, A556T and Env-A539V confer DTG resistance, we again utilized WT-transfected cultures showed evidence of virus replication at pBR43IeG, which allows us to quantify replication kinetics as a ∼30 d posttransfection (Fig. 6B, Right), suggesting the acquisition of function of viral gene expression. We inoculated Jurkat T cells DTG-resistance mutations. We collected virus-containing super- with virus-producing 293T cells, allowing for cell-to-cell transfer, natants at the day of peak replication, infected new cultures of and measured GFP expression over time (Fig. 8A and SI Ap- Jurkat cells, and found that the repassaged virus exhibited partial pendix, Fig. S9 A and B). In this system, the Env mutants repli- DTG resistance compared with naïve virus in the presence of 3 nM cate with accelerated kinetics compared with WT. We also observed that at days of peak replication, we measured a higher DTG. We collected cells from this repassaged, partially DTG- + resistant virus, extracted the viral DNA, PCR amplified, and se- percentage of GFP cells with the Env mutants compared with quenced. We did not identify any mutations in IN, but rather WT (Fig. 8A). To further characterize the replication properties of the mutant viruses, we calculated the geometric mean fluo- identified three mutations in Env: E209K, A539V, and H641Y (Fig. + 6C). These results were confirmed by the observation that the Env- rescence intensity (MFI) of the GFP cells at days of peak A539V mutation arose under the same conditions in two in- replication and found that cells infected with Env mutant viruses dependent selections in Jurkat cells. exhibit dramatically brighter GFP fluorescence compared with To determine whether the Env mutations that arose during WT (Fig. 8B and SI Appendix, Fig. S9C). Interestingly, we also virus propagation in DTG were able to confer resistance to observe an increase in MFI with WT and Env-A539V when we DTG, we engineered these mutations into pNL4-3 and evaluated infect Jurkat cells by spinoculation at a high MOI (SI Appendix, their effects on replication kinetics in Jurkat cells in the presence Fig. S10 A and B). The increase in virally encoded GFP ex- or absence of DTG. The Env-A539V mutant replicated with a pression, as measured by MFI, in the cells infected with the Env peak on day 15 in the presence of 3 nM DTG, a concentration at mutants relative to WT (Fig. 8B) suggests that, in the context of a which WT did not replicate (Fig. 6D). Additionally, the Env- spreading infection, the Env mutants may be overcoming blocks A539V mutant replicated with WT or faster-than-WT kinetics to viral replication by increasing the effective MOI, resulting in in the absence of DTG (Fig. 6D), a phenotype similar to that of an increase in the number of productive infection events per the Env compensatory mutants described above. Similarly, the target cell.

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C DE

Fig. 6. Env mutations partially rescue a replication-delayed IN mutant and provide a replicative advantage in the presence of DTG. (A) Jurkat cells were transfected with the indicated proviral clones and replication kinetics were monitored by measuring RT activity. (B) Jurkat cells were transfected with the indicated 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 WT

escape 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 Env MICROBIOLOGY indicating the location of the mutations identified by sequencing of DTG-resistant viruses. Mutations in Env are indicated by underlined residues and amino acid 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 kinetics were monitored by measuring RT activity.

Discussion transmission efficiency that we observed with a p6–Alix-binding In this study, we describe mutations within HIV-1 Env that site mutant suggests that recruitment of Alix to the VS may rescue replication-defective p6–Alix binding site mutants. We promote virus spread. further characterize the Env compensatory mutants—Y61H, We demonstrate that the high levels of replicative fitness of P81S, and A556T—and find that, despite exhibiting robust replica- the Env mutants described here in Jurkat cells and in some tion in the Jurkat T cell line and PBMCs, they display severe defects PBMC donors is a result of their competence to mediate cell-to- in fusogenicity and cell-free particle infectivity. This panel of Env cell transmission. The three Env mutants that were acquired substitution mutants not only rescues the replication defects con- during propagation of the p6-mutant viruses in Jurkat cells (Env- – ferred by disruption of p6 Alix binding but also enhances the rep- Y61H, P81S, and A556T) are impaired in their ability to induce lication of an IN mutant and confers resistance to the INSTI DTG. We attribute the ability of these Env mutants to rescue replication defects to their efficient transmission via a cell-to-cell route. De novo selections for DTG resistance in both Jurkat and CEM12D7 T cell lines 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 infect via both cell-free and cell-to-cell routes. We also provide evidence that the gp41 mutants A556T and A539V exhibit decreased sensitivity 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 same position in gp41 as the A539V mutation, demonstrating that the phenomenon of Env-mediated DTG resistance is not confined to the 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 selection experiments (37) were performed in Jurkat cells, which are infected inefficiently by cell-free HIV-1 (28), and in which p6– Fig. 7. DTG-resistant gp41 mutants Env-A556T and Env-A539V exhibit en- Alix binding site mutants replicate with a severe delay. In – hanced cell-to-cell transmission relative to WT. Cell-to-cell transmission of the PBMCs, the p6 Alix binding site mutants display variable phe- indicated mutants was measured by infecting Jurkat donor cells with VSV-G– + notypes, depending on the donor (37). While it is well estab- pseudotyped pBR43IeG-Env mutant viruses, normalizing for GFP cells, and lished that the interaction between the PT/SAP motif of p6 and inoculating target Jurkat cells in the presence or absence of DTG. The accumu- + the ESCRT-I subunit Tsg101 plays a key role in HIV-1 budding, lation of GFP cells during a 48-h coculture was measured. Data from four Alix appears to play a more auxiliary and cell-type–dependent independent experiments were normalized to WT in the absence of drug, and role in HIV-1 replication. The observed reduction in cell-to-cell plotted as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Van Duyne et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 9045 Downloaded by guest on October 1, 2021 A suggestion that cell–cell fusion is inhibited during VS formation perhaps 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 B Env 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 within subtype C viruses. Env-Y61H is present in 708 sequences in the LANL database, 76 of which are subtype B and 34 of which are subtype C. Env-P81S occurs in only two subtype B sequences and Env-A556T is present in only two nonsubtype B sequences (circulating recombinant form strains AE and BF). Env-Y61H was previously identified as an escape mutation that arose during treatment of HIV-1LAI–infected cells with the HIV-1 attachment B inhibitor BMS-378806 (48). To our knowledge, neither Env-P81S nor any other mutation at that position has been previously char- acterizedintheliterature.Finally, Env-A556 has been mutagenized extensively in the context of characterizing intra- and interheptad repeat (HR) interactions of the gp41 six-helix bundle (49, 50), which is consistent with our findings with A556T. Recently, Env-A556T (A558T in HXB2) was also selected, along with several substitutions in the C1 domain of gp120, as a resistance mutation to the peptidic inhibitor VIR165 (51). This study suggested that the C1 and HR1 substitutions may alter the kinetics of Env conformational changes, providing resistance by limiting substrate accessibility (51). In our study, the Env compensatory mutations are also located within the C1 domain of gp120 and the HR1 domain of gp41. Al- though gp120 and gp41 interact noncovalently, C1 and HR1 have Fig. 8. Env-A539V confers resistance to DTG by accumulating a high per- been shown through mutagenesis and structural studies to be critical for the stability of gp120–gp41 association in the unliganded state centage of infected cells and by increasing the effective MOI. (A) Jurkat cells – were inoculated with virus-producing 293T cells expressing the indicated (50, 52 54). These observations suggest that the Env mutations + proviral clones and replication kinetics were monitored by measuring %GFP described here may alter the stability of gp120–gp41 interactions. cells. (B) The geometric MFI of GFP+ cells from A was calculated at days of We mapped the location of the Env compensatory mutations peak infection. Data from four independent experiments are shown as onto a monomeric Env prefusion structure and a trimeric Env means ± SD. *P < 0.05, **P < 0.01. CD4-bound structure (SI Appendix, Fig. S11 A and B). We found that the amino acid positions of the three Env compensatory changes are closely clustered in the monomeric structure, with syncytia and thus may decrease bystander cell killing and syn- Y61 and A556 only 4.5 Å apart (55) (SI Appendix, Fig. S11A). cytial apoptosis (44). The selection of fusion-defective mutants Interestingly, the trimeric, CD4-bound Env structure highlights a has been observed previously (45); mutations in Env arose in rearrangement of Y61 to a solvent-exposed location central to all HIV-1–infected SupT1 cells that improved replication in the three gp120 monomers (56) (SI Appendix, Fig. S11B). Our mu- absence of syncytia (46, 47), and Env mutations that abrogated tagenesis studies show that conservative mutation Y61F is well syncytium formation were reported to arise during propagation tolerated in terms of cell-free particle infectivity, syncytium for- of a Vif-defective virus (40). The lack of cell–cell fusion poten- mation, and replication relative to WT (Fig. 5). P81 is located in tially increases the production of progeny virus before cell death a loop region just C-terminal to the ⍺0 helix in the CD4-bound (46, 47). The conservative and nonconservative substitutions that structure; however, the gp41 residues surrounding A556 are not we introduced at positions Y61, P81, and A556 confirm our annotated in this structure (56). The Env mutants described here initial finding that poorly infectious mutants can replicate effi- will provide useful tools for further studies of Env structure and ciently in Jurkat cells. The mutants that replicate efficiently in function, particularly in understanding how Env regulates cell- Jurkats and PBMCs despite low cell-free particle infectivity are free vs. cell-to-cell modes of virus transmission. likely functionally and structurally adapted to promote cell-to- In addition to changes in Env, we also found that mutations in cell transmission. Indeed, this idea is in agreement with the Vpu arose in response to the substitutions in the p6–Alix binding

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 are indicated by the presence of a provirus (black) within the nucleus (gray). Inhibition with DTG is shown in red solid or dashed lines, indicating complete or partial inhibition, respectively.

9046 | www.pnas.org/cgi/doi/10.1073/pnas.1820333116 Van Duyne et al. Downloaded by guest on October 1, 2021 site (Fig. 1A and SI Appendix, Fig. S1 C and D). We commonly observed the appearance of inactivating mutations in Vpu (69, observe Vpu-inactivating mutations during the propagation of 70). Mutations in Env and Vpu also provided escape from replication-delayed HIV-1 in Jurkat cells. The basis for these IFITM1 restriction in SupT1 cells; one of the Env mutations, loss-of-function mutations in Vpu remains to be explored, but Env-G367R, was described as being defective for cell-free in- could be associated with a beneficial effect of low-level tetherin fectivity, but able to spread via cell-to-cell transmission (71–73). expression in cell-to-cell viral transmission (57, 58). These data provide further support for the concept that muta- Our characterization of the Env compensatory mutants continued tions in Vpu and Env can overcome barriers to virus replication with the observation that they confer a global replication advantage by promoting cell-to-cell transmission. in the presence of a replication-delayed IN mutant. We believe this is The identification of ARV-resistance mutations outside of the not due to bypassing integration, but rather, as discussed above, to target gene is rare, although not unprecedented (74). For example, the ability of the Env mutants to overcome blocks in the replication several studies have observed that patients on protease inhibitor cycle by mediating effective cell-to-cell transmission. We also found (PI)-containing regimens experience virological failure in the ab- the Env compensatory mutants can replicate in the presence of DTG sence of drug-resistance mutations in protease (PR) (75–78). at concentrations that inhibit WT virus. De novo selection experi- Siliciano and colleagues proposed that env sequences from these ments led to the emergence of a DTG-resistant virus that lacked patients contain mutations, specifically in the Env CT, that confer mutations in IN, but instead contained substitutions in Env. One of PI resistance (79). A connection between the gp41 CT and PI re- these Env mutations, Env-A539V, confers DTG resistance in both sistance may be linked to the role of virus maturation, triggered by Jurkat and CEM12D7 cells, a phenotype that correlates with near- PR, in activating Env fusion activity (80, 81). Several clinical reports WT cell-free particle infectivity. have observed failure of DTG-containing therapy in the absence of The INSTIs—Raltegravir (RAL), Elvitegravir (EVG), DTG, IN mutations, suggesting that mutations outside IN may confer — and Bictegravir (BIC) are the most recently Food and Drug resistance in these patients (82, 83). A recent study observed mu- Administration-approved class of anti-HIV inhibitor (59). HIV-1 tations in the nef gene that conferred INSTI resistance (84, 85). readily develops resistance to RAL and EVG both in vitro and However, to our knowledge, we present a unique instance of de in vivo as a result of mutations in IN; however, DTG is more dif- novo selection of Env mutations that confer resistance to DTG in ficult for the virus to escape (42, 60, 61). Recently, IN-R263K has vitro. Ongoing studies should determine whether the Env mutations emerged in ARV-experienced, INSTI-naïve patients experiencing described here confer resistance to other classes of ARVs, and virological failure on a DTG regimen and in a patient during DTG – whether mutations in Env can contribute to escape from ARV monotherapy (62 64). IN-R263K confers weak DTG resistance but, therapy in infected individuals (86). The results of this work will

interestingly, can spread in culture in the context of cell-to-cell provide fundamental insights into mechanisms of drug resistance MICROBIOLOGY transmission (32, 65). Additional IN mutants in patients experi- and viral spread in vivo. encing virological failure during DTG monotherapy include Q148H/R, N155H, G118R, and S230R (64). The fold-change in Methods DTG resistance of reported IN mutants is significantly lower than Cell Culture. The 293T [obtained from American Type Culture Collection that observed with IN mutants resistant to RAL and EVG; our (ATCC)] and TZM-bl [obtained from J. C. Kappes, X. Wu, and Tranzyme, Inc. calculated fold-change for Env-A556T and Env-A539V is compa- through the NIH AIDS Reagent Program (ARP), Germantown, MD] cells were rable to several of these DTG-resistant mutants (43). Interestingly, maintained in DMEM containing 5% or 10% (vol/vol) FBS, 2 mM glutamine, we observed no differences in DTG IC50 values in the context of a 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37 °C with 5%

cell-free, single-cycle assay, supporting the hypothesis that reduced CO2. Jurkat (87), CEM12D7 (88), and Jurkat-1G5 (89) (obtained from E. DTG sensitivity conferred by the Env mutations is manifested Aguilar-Cordova and J. Belmont through the NIH ARP, Germantown, MD) during cell-to-cell transmission. T cell lines were maintained in RPMI-1640 medium containing 10% FBS, Several studies have investigated the ability of ARVs to inhibit 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at – – both cell-free and cell cell infectivity (29, 33, 35, 66 68). A 37 °C with 5% CO2. PBMCs were stimulated with 2 μg/mL PHA-P for 3–5d model proposed by Baltimore and coworkers (29) suggests that a before infection, then cultured in 50 U/mL IL-2. reduction in sensitivity to ARVs due to multiple infections per cell caused by cell-to-cell spread can be a source of ongoing Preparation of Virus Stocks. The 293T cells were transfected with HIV-1 replication in the presence of ARVs. Similarly, Mothes and co- proviral DNA using Lipofectamine 2000 (Invitrogen) according to the man- workers (33) reported a correlation between the ability of ARVs ufacturer’s instructions. Virus-containing supernatants were filtered through to inhibit cell-to-cell transmission and effectiveness against high a 0.45-μm membrane 48 h posttransfection and virus was quantified by local MOI at cell–cell contacts. These models and observations measuring RT activity. VSV-G–pseudotyped virus stocks were generated from would lead to the prediction that mutations that enhance cell-to- cells cotransfected with proviral DNA and the VSV-G expression vector cell transmission may lead to drug resistance and that the con- pHCMV-G (90), at a DNA ratio of 10:1. Env-pseudotyped reporter virus stocks centrations of ARVs required to inhibit a cell-free infection were generated from cells cotransfected with pNLuc (91) and the Env ex- event may be insufficient to inhibit cell-to-cell transmission, as pression vector pIIINL4-Env (87), at a DNA ratio of 10:1. we observed in this study (Fig. 9). The hypothesis that cell–cell transfer of the Env mutants results in more proviruses per cell, Cloning and Plasmids. The full-length HIV-1 clade B molecular clone pNL4-3 relative to WT, is supported by the increased MFI observed in (pNL4-3 WT) was used for this study (92). The pNL4-3/KFS clone, referred − mutant vs. WT-infected cultures (Fig. 8B). While it is challenging to here as pNL4-3 Env ( ) was described previously (93). pNL4-3 clones to define the significance of cell-to-cell transmission in vivo, it is bearing p6 mutations p6-Y36A, Y36S/L44H, Y36S/L44R, L41R, and L41A clear that ARVs need to remain effective against the high MOI were described previously (10, 16, 37). pNL4-3 clones bearing Vpu, Env, p6/Vpu, or p6/Env mutations were constructed with the QuikChange Site- occurring during cell-to-cell transmission (27). Directed Mutagenesis kit (Stratagene) into subclones of pNL4-3 according Similar to Y61, P81, and A556, A539 (A541 in HXB2; gp41 to the manufacturer’s instructions, and were then recloned into pNL4-3. A30) is 97.99% conserved across clade B viruses, occurring in pIIINL4-Env clones bearing Env mutations were generated as above. The IN only 4 subtype B and 14 subtype C sequences of a total of 175. mutant pNL4-3/IN-N155E was a kind gift from Alan Engelman, Dana Farber Mutations at this position have been described previously in Cancer Institute, Boston, MA (41) and IN/Env double mutants were gener- several different contexts, but largely as fusion inhibitor escape ated as above. pBR-NL43-IRES-eGFP-nef+ (pBR43IeG) is a proviral vector that mutations (shown mapped onto a monomeric Env prefusion coexpresses Nef and eGFP from a single bicistronic RNA (obtained from structure and a trimeric Env CD4-bound structure; see SI Ap- F. Kirchhoff through the NIH ARP, Germantown, MD) (94, 95). pBR43IeG clones pendix, Fig. S11 C and D, respectively). Env-A539V was also containing Env mutations were constructed as above. The full-length HIV- selected during escape from a deleted form of the antiviral factor 1 clade C molecular clone pK3016 (CH185_TF) was reported previously (96). IFN-induced transmembrane protein 1 (IFITM1) (69) and in DNA for transfections was purified in large-scale quantities using MaxiPrep response to the MxB restriction factor (70). These studies also Kits (Qiagen) and mutations were verified by sequencing (Macrogen).

Van Duyne et al. PNAS | April 30, 2019 | vol. 116 | no. 18 | 9047 Downloaded by guest on October 1, 2021 Virus Replication Assays. Virus replication was assayed in a T cell line model of Fusion Assay. The 293T cells were cotransfected with the indicated pIIINL4- spreading infection, as previously described (97). Briefly, T cells were trans- Env expression vectors and the HIV-1 Tat expression vector pSV-Tat (98) at a fected with proviral clones (1 μg DNA/1 × 106 cells) in the presence of 700 μg/mL ratio of 10:1. Twenty-four hours posttransfection, 293T cells were removed DEAE-dextran or were infected with RT-normalized virus-containing and overlaid onto TZM-bl or Jurkat-1G5 cells (Jurkat-derived reporter supernatants. Virus replication was quantified by measuring RT activity in col- cell line containing a stably integrated HIV-1–LTR–luciferase construct) lected supernatants over time. Where indicated, the assay was initiated by in- with serial dilutions in duplicate. Twenty-four hours postoverlay, luciferase 3 6 oculation of T cells with GFP virus-producing 293T cells at a ratio of 10 293T:10 was measured as above. Technical duplicates were normalized to pNL4-3 Jurkat, a density that has been optimized to recapitulate WT kinetics; here, + WT and averaged; data represent the average of independent, normalized virus replication was quantified by measuring GFP cells by flow cytometry over experiments. time. Cells were fixed in 4% PFA and analyzed by flow cytometry using a FACSCalibur (BD); data were collected via CellQuest and processed via FlowJo. Cell-To-Cell Transmission Assay. Donor Jurkat cells were infected with 293T- When indicated, genomic DNA was extracted from infected cells using the derived VSV-G–pseudotyped pBR43IeG Env mutant viruses. Forty-eight QIAamp Genomic DNA Extraction Kit (Qiagen); viral DNA was amplified by PCR, + to 72 h postinfection, the percent of GFP donor cells was measured by and sequenced (Macrogen) (SI Appendix,TableS1). Frequency of residues at flow cytometry. Infected donor Jurkat cells were cocultured with un- each mutant position was determined by the AnalyzeAlign tool from LANL + (https://www.hiv.lanl.gov/content/sequence/ANALYZEALIGN/analyze_align.html). infected target Jurkat cells at a ratio that normalized the GFP input cells ∼ DTG was a kind gift from S. Hughes, National Cancer Institute/NIH, Bethesda, to 10% per coculture in the presence or absence of 1.5 nM DTG. Forty- eight hours postcoculture, cells were fixed in 4% PFA and analyzed MD. Data were plotted (transformed and normalized) and IC50 values were calculated using GraphPad PRISM. Curves were fit using nonlinear regression as by flow cytometry. Data were collected via CellQuest and processed log(inhibitor) vs. normalized response, variable slope using a least squares via FlowJo. (ordinary) fit. Structural modeling were performed in MacPyMOL. Statistics. Statistics were calculated using GraphPad PRISM. Unpaired t tests Single-Cycle Infectivity Assays. TZM-bl is a HeLa-derived reporter cell line that were performed and two-tailed *P < 0.05, **P < 0.01, ***P < 0.001, and contains a stably integrated HIV–LTR–luciferase construct (38, 39). TZM-bl cells ****P < 0.0001 were considered statistically significant. were infected with serial dilutions of RT-normalized virus stocks in the presence of 10 μg/mL DEAE-dextran. Approximately 36 h postinfection, cells were lysed Ethics Statement. PBMCs were obtained from anonymous, deidentified blood with BriteLite luciferase reagent (Perkin-Elmer) and luciferase was measured in a donors to the NIH Department of Transfusion Medicine Blood Products Wallac BetaMax plate reader. Technical duplicates were normalized to pNL4-3 Program (NIH CC-DTM). WT and averaged; data represent the average of independent, normalized experiments. To measure single-cycle infectivity in PBMCs, cells were infected in ACKNOWLEDGMENTS. We thank members of the E.O.F. laboratory for duplicate with the indicated RT-normalized Env mutant pseudotyped pNLuc helpful discussion and critical review of the manuscript. Work in the E.O.F. viruses. Luciferase was measured 48 h postinfection as above. Technical dupli- laboratory is supported by the Intramural Research Program of the Center cates were normalized to pNL4-3 WT and averaged; data represent the average for Cancer Research, National Cancer Institute, NIH, the Intramural AIDS of independent, normalized experiments. Targeted Antiviral Program.

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