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1 Title: 2 Elucidating the basis for permissivity of the MT-4 T-cell line to replication of an HIV-1 3 mutant lacking the gp41 cytoplasmic tail 4 5 6 7 8 Authors and affiliations: 9 Melissa V. Fernandez1, Huxley K. Hoffman2, Nairi Pezeshkian2, Philip R. Tedbury1, 10 Schuyler B. van Engelenburg2, and Eric O. Freed1* 11 12 1 HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer 13 Institute, Frederick, MD 21702, USA 14 2 Molecular and Cellular Biophysics Program, Department of Biological Sciences, 15 University of Denver, Denver, CO 80210, USA 16 17 *Author for correspondence ([email protected]) 18 19 Institutional addresses: 20 National Cancer Institute 21 HIV Dynamics and Replication Program 22 1050 Boyles St. 23 Building 535, Room 110 24 Frederick, MD 21702 25 26 Department of Biological Sciences 27 Seeley G. Mudd Building 28 2101 E. Wesley Ave. 29 Denver, CO 80210 30 31

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32 Abstract 33 34 HIV-1 encodes an envelope glycoprotein (Env) that contains a long cytoplasmic tail (CT) 35 harboring trafficking motifs implicated in Env incorporation into virus particles and viral 36 transmission. In most physiologically relevant cell types, the gp41 CT is required for 37 HIV-1 replication, but in the MT-4 T-cell line the gp41 CT is not required for a spreading 38 infection. To help elucidate the role of the gp41 CT in HIV-1 transmission, in this study 39 we investigated the viral and cellular factors that contribute to the permissivity of MT-4 40 to gp41 CT truncation. We found that the kinetics of HIV-1 production are faster in MT-4 41 than in the other T-cell lines tested, but MT-4 express equivalent amounts of HIV-1 42 proteins on a per-cell basis relative to cells not permissive to CT truncation. MT-4 43 express higher levels of plasma-membrane-associated Env than non-permissive cells 44 and Env internalization from the plasma membrane is slower compared to another T- 45 cell line, SupT1. Paradoxically, despite the high levels of Env on the surface of MT-4, 46 two-fold less Env is incorporated into virus particles in MT-4 compared to SupT1. Cell- 47 to-cell transmission between co-cultured 293T and MT-4 is higher than in co-cultures of 48 293T with most other T-cell lines tested, indicating that MT-4 are highly susceptible to 49 this mode of infection. These data help to clarify the long-standing question of how MT- 50 4 cells overcome the requirement for the HIV-1 gp41 CT and support a role for gp41 51 CT-dependent trafficking in Env incorporation and cell-to-cell transmission in 52 physiologically relevant cell lines. 53 54 Importance 55 56 The HIV-1 Env cytoplasmic tail (CT) is required for efficient Env incorporation into 57 nascent particles and viral transmission in primary CD4+ T cells. The MT-4 T-cell line 58 has been reported to support multiple rounds of infection of HIV-1 encoding a gp41 CT 59 truncation. Uncovering the underlying mechanism of MT-4 T-cell line permissivity to 60 gp41 CT truncation would provide key insights into the role of the gp41 CT in HIV-1 61 transmission. This study reveals that multiple factors contribute to the unique ability of a 62 gp41 CT truncation mutant to spread in cultures of MT-4 cells. The lack of a 63 requirement for the gp41 CT in MT-4 is associated with the combined effects of rapid 64 HIV-1 protein production, high levels of cell-surface Env expression, and increased 65 susceptibility to cell-to-cell transmission compared to non-permissive cells. 66 67

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68 Keywords: HIV-1, Env, gp41, cytoplasmic tail, virological synapse, transmission, HTLV- 69 I, Tax

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70 Introduction 71 72 HIV-1 Env is initially synthesized in the endoplasmic reticulum (ER) as a polyprotein 73 precursor, gp160, which is cleaved during trafficking through the Golgi apparatus to 74 generate the mature surface Env subunit gp120 and the transmembrane subunit gp41 75 (1, 2). The two subunits are non-covalently linked to form a trimeric gp120:gp41 76 heterodimer in the functional Env glycoprotein complex. The mature, trimeric Env 77 complex traffics via the secretory pathway to the plasma membrane (PM), the site of 78 viral assembly and budding. Env is expressed on the surface of infected cells and is 79 incorporated into virus particles where it is embedded in the viral envelope. 80 81 The two subunits of Env are responsible for different functions of the glycoprotein 82 complex. The gp120 subunit promotes particle attachment and entry by binding to 83 receptor (CD4) and co-receptor (CXCR4 or CCR5). The gp41 subunit comprises three 84 domains: an ectodomain that associates with gp120 and contains the determinants 85 critical for membrane fusion, a transmembrane domain that anchors Env in the lipid 86 bilayer, and a cytoplasmic tail (CT) that regulates a number of aspects of Env function. 87 While the principal functions of the Env complex are well characterized, and it is clear 88 that the gp41 CT regulates Env incorporation into virions, the precise role of the CT in 89 Env biology remains poorly understood. 90 91 HIV-1 is transmitted to target cells in vitro and in vivo via either cell-free or cell-to-cell 92 (C-C) infection (3, 4). Cell-free infection occurs when virions that are not associated with 93 the virus-producing cell bind and enter uninfected target cells. C-C infection is defined 94 as direct transmission of nascent particles at points of contact, known as virological 95 synapses (VSs), between infected and uninfected cells. Studies have established that, 96 in vitro, viral dissemination by C-C transmission is a highly efficient mode of viral 97 transfer relative to cell-free infection (5-9). However, the relative contribution of cell-free 98 vs C-C transmission to viral spread in vivo is less clear. In most cell types, viral 99 transmission requires CT-dependent localization of Env to viral assembly sites (10-13) 100 and Env binding to CD4 and co-receptor. A hallmark of C-C spread is the accumulation 101 of viral proteins, in particular Gag and Env, at the VS (5, 6, 11, 14). How Env is directed 102 to the VS is not well understood; further elucidation of this process is fundamental to our 103 ability to design therapies capable of blocking C-C transmission. 104 105 The lentiviral gp41 CT is very long compared to those of other retroviruses; it contains 106 150 amino acids in the case of HIV-1 and harbors trafficking motifs implicated in Env 107 recycling, incorporation, and viral transmission. The gp41 CT contains a highly 108 conserved YxxΦ motif (with Φ representing a hydrophobic amino acid) known to interact 109 with host cell clathrin adaptor protein complex 2 (AP-2) and mediate fast internalization 110 of Env via clathrin-mediated endocytosis (2). The gp41 CT contains several other well- 111 conserved tyrosine and dileucine motifs that may also play a role in Env trafficking and 112 subcellular localization (2). The high degree of conservation in both the length of the 113 gp41 CT and the YxxΦ motif suggests that these features play key roles in viral 114 transmission. It is currently unclear whether Env recycling from the PM prior to 115 incorporation into the assembling Gag lattice is a requisite step in Env incorporation.

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116 Recent evidence suggests a role for recycling in Env incorporation (15-17) and many 117 studies have explored the role of trafficking motifs in the gp41 CT in promoting the 118 proper spatio-temporal localization of Env during assembly (1, 2, 18, 19), but the role of 119 Env recycling in Env incorporation is not well-defined. 120 121 Wild-type (WT) HIV-1 has an average of ~10 Env trimers per virion (20) and truncation 122 of the gp41 CT generally results in a 10-fold decrease in Env incorporation in 123 physiologically relevant cell types (which we refer to as being non-permissive to gp41 124 CT truncation) (21). The sparsity of Env on HIV-1 particles suggests that Env 125 incorporation is tightly regulated. The degree of regulation seems to be cell-type and CT 126 dependent. For example, in the non-permissive T-cell line CEM-A, WT Env is localized 127 at the neck of the budding particle while truncation of the gp41 CT results in a more 128 uniform Env distribution around the virus particle (19). In the permissive COS7 129 fibroblast-like cell line, both WT and CT-truncated Env are evenly distributed around the 130 virus particle. CT-dependent endocytosis of WT Env from the PM is more active in 131 CEM-A than in COS7, suggesting that Env recycling regulates Env distribution on the 132 virus particle. Importantly, in both cell lines, incorporation of the gp41 CT-truncated 133 mutant was reduced compared to the WT, consistent with the essential role for the CT 134 in trapping Env in the assembling Gag lattice (22). These data highlight the multifaceted 135 role of the gp41 CT in regulating Env incorporation and the cell-type dependent 136 utilization of the CT in viral assembly. 137 138 A functional interaction between the gp41 CT and the matrix (MA) domain of the Gag 139 polyprotein has been postulated to play an important role in capturing Env during viral 140 assembly (23, 24). Compelling evidence for the trapping of the gp41 CT by the Gag 141 lattice has been previously demonstrated by various studies showing the clustering of 142 Env trimers at sites of virus assembly (10, 12, 19, 22) and retention of full-length Env, 143 but not CT-truncated Env, in detergent-stripped Gag virus-like particles (25). Further 144 evidence for the trapping of the gp41 CT by the Gag lattice is provided by the ability of 145 single amino acid changes in MA to block WT Env incorporation (26-33). Truncation of 146 the gp41 CT reverses the Env incorporation block imposed by these point mutations in 147 MA (26, 29, 31, 33). Similarly, Env incorporation is inhibited by small intragenic 148 deletions in MA or deletion of the globular domain of MA, and this inhibition is relieved 149 by truncation of the gp41 CT (31, 34). Highlighting the importance of MA- gp41 CT 150 interactions, a small deletion in the gp41 CT that inhibits Env incorporation is rescued 151 by a single amino acid change in MA (35). Further underscoring the intimate 152 relationship between MA and the gp41 CT, truncation of the gp41 CT abrogates Gag’s 153 ability to repress premature fusion of Env with the target cell membrane (12, 36, 37). 154 Recent studies have demonstrated an important role for trimerization of the MA domain 155 of Gag in the formation of a Gag lattice that accommodates the long gp41 CT during 156 Env incorporation (27, 28, 38). While evidence of the relationship between the gp41 CT 157 and MA is well appreciated, the precise mechanism of how Env is incorporated into the 158 assembling Gag lattice is not well understood. 159 160 Our understanding of the function of the gp41 CT and MA in Env incorporation suggests 161 four general models of Env incorporation: 1) passive incorporation (Env incorporation

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162 does not require its concentration at assembly sites), 2) Gag-Env co-targeting (Gag and 163 Env are both targeted to an assembly platform, such as a membrane microdomain), 3) 164 direct Gag-Env interaction (Gag directly binds the gp41 CT, thereby capturing it into 165 assembling virions), and 4) indirect Gag-Env interaction (a host factor serves as a 166 bridge between Gag and Env for the capture of Env by the assembling Gag lattice) (1, 167 23). These models are not mutually exclusive; for example, Env could colocalize with 168 Gag at sites of virus assembly and then be captured by the Gag lattice via direct 169 interactions between MA and the gp41 CT, and other combinations of these models can 170 be readily envisioned. Interestingly, even foreign viral glycoproteins have been shown to 171 cluster at HIV-1 assembly sites in the context of pseudotype particle formation (39, 40). 172 173 The unusual ability of certain T-cell lines (notably MT-4, C8166, and M8166) to be 174 highly susceptible to HIV-1 infection and permissive to loss of certain viral protein 175 functions is incompletely understood. In particular, MT-4 are known to be permissive, 176 relative to other T-cell lines, to deletion of the gp41 CT (14, 21, 29, 31, 33, 38, 41-45), 177 loss of integrase (IN) function (46, 47), disruption of proper capsid (CA) multimerization 178 (48), deletion of Nef (49), and a large MA deletion (34). A functional IN protein is 179 required for productive HIV-1 replication in a majority of cell lines and human peripheral 180 blood mononuclear cells (hPBMCs) (47). MT-4 and C8166 are known to be permissive 181 to type I IN mutations (mutations in IN that specifically block viral DNA integration into 182 the host cell genome) while Jurkat E6.1 and hPBMCs are non-permissive to defects in 183 integration (47). It was recently reported that MT-4 and C8166 are likely permissive to 184 type I IN mutations due to HTLV-I Tax expression inducing NF-B protein recruitment to 185 the HIV-1 LTR on unintegrated HIV-1 DNA (46). Furthermore, it was previously 186 suggested by Emerson et al. (14) that HTLV-I Tax expression may contribute to MT-4 187 permissivity by causing potent and chronic activation of NF-B signaling (50), resulting 188 in transactivation of the HIV-1 LTR (51). The ability of MT-4 to support rapid replication 189 of HIV-1 and gain second-site compensatory mutations unlikely to be acquired in less- 190 permissive cells has led to the use of this cell line for selection experiments with a wide 191 variety of defective MA and CA mutants (29, 48, 52-54). Uncovering the underlying 192 mechanisms of MT-4 cell line permissivity is important because of the frequent use of 193 this cell line in HIV-1 replication studies (13, 55-57). 194 195 In this study, we examined a number of factors involved in viral replication and spread in 196 cells both permissive (i.e., MT-4) and non-permissive (i.e., all other T-cell lines tested) 197 for gp41 CT truncation. Our results show that HTLV-I Tax expression is not the sole 198 determinant of permissivity to gp41 CT truncation. Rather, high surface Env expression, 199 rapid kinetics of HIV protein production, and efficient C-C transmission likely contribute 200 to the MT-4 permissivity to CT-truncation. 201

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202 Results 203 204 Lineage and validation of cell lines used to interrogate T-cell line permissivity to 205 replication of an HIV-1 mutant lacking the gp41 CT. 206 To investigate the mechanistic basis for the permissivity of MT-4 cells to replication of a 207 gp41 CT-truncation mutant, a panel of five T-cell lines were selected based on their 208 origins (Fig. 1) and previously reported permissivity, or lack thereof, to gp41 CT 209 truncation. Previous studies have reported that MT-4 (14, 21, 29, 31, 33, 38, 41-45) and 210 M8166 (58) are permissive to truncation of the gp41 CT, and it is established that 211 hPBMCs, SupT1, and Jurkat E6.1 are not permissive (14, 21). Therefore, two HTLV-1– 212 lymphoma-derived cell lines, SupT1 (59) and Jurkat E6.1 (60) (Fig. 1A-B); and three 213 HTLV-1-transformed lines, MT-4 (61), C8166 (62), and M8166 (Fig. 1C-D) were 214 selected for this study. The cell line lineage and HTLV-I particle and RNA production 215 status of each cell line are shown in Fig 1. C8166, M8166, and MT-4 all express HTLV-I 216 RNA but do not produce viral particles. Due to the presence of HTLV-I RNA, they do 217 express the HTLV-I Tax protein (43, 63, 64). SupT1 and Jurkat E6.1 do not express 218 HTLV-I RNA or proteins (59, 60). 219 220 Short tandem repeat (STR) profiling was utilized to validate the identity of all the cell 221 lines in our T-cell panel (Table 1). The MT-4 STR profile, recently validated by functional 222 assays, morphological analysis, and assessment of HTLV-I Tax protein expression (43), 223 was an exact match to the Cellosaurus reference profile (Table 1). Jurkat E6.1 and 224 SupT1 STR profiles were both close matches to the published STR profiles. The STR 225 profile revealed some genetic instability in the SupT1 line compared to SupT1-CCR5, 226 indicated by the presence of extra, lower-intensity alleles at several gene loci (data not 227 shown). Overall, the STR profile of our SupT1 cell line was a 95% match to the SupT1- 228 CCR5 line, confirming the identity of our laboratory SupT1 line. The Jurkat E6.1 line 229 also displayed some genetic instability (data not shown), which accounts for its 230 imperfect match to the Cellosaurus reference profile. 231 232 M8166 is a subclone of C8166 (Fig. 1D), and therefore these two lines share the same 233 STR profile (Table 1), which was a ~98% match to the cell line profile published in the 234 Cellosaurus database and in a separate report (65). To further confirm the identity of the 235 M8166 and C8166 cell lines, they were assessed for their capacity to host replication of 236 WT SIVmac239 (Fig. 2). The C8166 line has been reported to exhibit a block to efficient 237 SIV replication (66), whereas M8166 is reportedly capable of hosting multiple rounds of 238 SIVmac239 replication (67). Consistent with these reports, SIVmac239 did not establish 239 a spreading infection in C8166 (Fig. 2a) while it did in M8166 (Fig. 2B). Together, the 240 STR profile and capacity of M8166, but not C8166, to support multiple rounds of 241 SIVmac239 replication confirm the identity of the C8166 and M8166 cell lines used in 242 this study. 243 244 MT-4 is the only T-cell line tested in this study that is permissive to the gp41 CT 245 truncation mutant CTdel144. 246 HIV-1 replication in most T-cell lines is abrogated by truncation of the gp41 CT (14, 21), 247 but several studies have demonstrated that the MT-4 cell line is able to propagate a

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248 gp41 CT-deleted mutant (14, 21, 29, 31, 33, 38, 41-45). To evaluate the ability of the 249 CTdel144 mutant, which lacks 144 amino acids from the gp41 CT (26), to replicate in 250 HTLV-I-transformed T-cell lines, MT-4, C8166 and M8166 cells were transfected with 251 the WT pNL4-3 molecular clone or the CTdel144 derivative, to initiate a spreading 252 infection (Fig. 3A). MT-4, C8166, and M8166 (Fig. 3B-D) supported rapid WT HIV-1 253 replication with supernatant HIV-1 reverse transcriptase (RT) activity detectable within 254 3-5 days post-transfection (Fig. 3B). Replication of HIV-1 in T-cell lines not permissive 255 to truncation of the gp41 CT - or in phytohemagglutinin (PHA)-activated PBMCs isolated 256 from healthy hPBMCs - was markedly slower than in MT-4 cells (Fig. 3E-G compared to 257 Fig. 3B). Consistent with previous reports (14, 21, 29, 31, 33, 38, 41-45), the MT-4 cell 258 line also supported replication of the CTdel144 mutant. In contrast, neither of the other 259 two HTLV-1-transformed T-cell lines (C8166 and M8166), the two HTLV-1– lymphoma- 260 derived cell lines (SupT1 and Jurkat E6.1), or hPBMCs supported replication of 261 CTdel144 (Fig. 3B compared to Fig. 3C-G). Therefore, under these standard conditions, 262 MT-4 is unique among the T-cell lines tested here in their capacity to support multiple 263 rounds of CTdel144 replication. 264 265 NF-B-target gene expression in Tax-expressing T-cell lines is higher than in SupT1. 266 To determine the relative levels of Tax expression in MT-4, C8166 and M8166, western 267 blot analysis of cell lysates was performed (Fig. 4A). To control for antibody specificity, 268 two sets of controls were included: 1) a panel of adult T-cell leukemia (ATL) cells 269 previously reported to have lost Tax expression (68), and 2) 293T cells transfected with 270 a Tax expression vector (Fig. 4A). Western blot analysis indicated that C8166 271 expressed substantially more Tax protein than MT-4 or M8166, while a small amount of 272 Tax expression in the ATL-55T cell line was detectable. 273 274 Tax is a known oncogene whose expression induces rapid senescence through potent 275 and persistent NF-B hyperactivation and upregulation of NF-B-regulated genes (68, 276 69). To ascertain whether Tax expression in these cells has resulted in hyperactivation 277 of NF-B signaling, Illumina RNA-seq was employed to measure the RNA expression of 278 NF-B-dependent and -independent genes in untreated cells. A panel of NF-B target 279 genes was selected from a database of genes activated by NF-B; these included 280 immunoreceptor genes and genes involved in proliferation, apoptosis, stress response, 281 and cytokine-stimulation (70). A control panel of NF-B-independent genes targeted by 282 the IRF3 transcription factor was derived from the ENCODE Transcription Factors 283 database (available on the Harmonizome search engine) for IRF3-target genes 284 identified by ChIP-seq (71, 72). IRF3-target genes also targeted by NF-B, identified by 285 their presence in the NF-B-target genes panel (70), were not used in the IRF3- 286 dependent target gene analysis. Overall, NF-B-target gene transcripts were 287 upregulated in all three HTLV-I-transformed lines relative to SupT1 (Fig. 4B-D - left). 288 IRF3-target genes were not consistently up- or down-regulated (Fig. 4B-D - right), 289 consistent with a Tax-dependent chronic hyperactivation of NF-B, but not IRF3, 290 signaling in Tax-expressing cell lines. These data establish that Tax transactivation of 291 the HIV-1 LTR is not solely responsible for MT-4 permissivity to gp41 CT truncation. 292

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293 Viral entry mediated by full-length and CT-truncated Env is equivalent among HTLV- 294 transformed T-cell lines. 295 Binding of virion-associated Env to the CD4 receptor and CXCR4 or CCR5 co-receptor 296 is an essential step for infection of human T cells by most strains of HIV-1. T-cell line 297 tropic isolates, like NL4-3, use CXCR4 as their coreceptor. To determine whether high- 298 level expression of CD4 or CXCR4 on the surface of MT-4 cells could contribute to the 299 permissivity of this cell line to CT-truncated HIV-1, surface CD4 and CXCR4 was 300 measured on the panel of T-cell lines (Fig. 5A-B). While MT-4 cells did express more 301 CD4 and CXCR4 than C8166 or M8166, they expressed less CD4 than SupT1. 302 Therefore, receptor and coreceptor expression levels do not account for the permissive 303 phenotype exhibited by MT-4. 304 305 To determine whether increased efficiency of viral entry contributes to MT-4 306 permissivity, the β-lactamase (BlaM)-based viral entry assay was performed using virus 307 bearing either VSV-G, WT HIV-1 Env, or the CTdel144 Env truncation mutant (Fig. 5C- 308 F). Differences in viral entry mediated by VSV-G were not statistically significant 309 between the cell lines (Fig. 5D). Entry mediated by WT HIV-1 Env was significantly 310 lower in SupT1 than in the other T-cell lines, with MT-4 cells intermediate between 311 Jurkat E6.1 and the other HTLV-1-transformed T-cell lines. Entry mediated by WT Env 312 was not statistically different between MT-4 and Jurkat E6.1, C8166, and M8166 (Fig. 313 5E). Entry mediated by CTdel144 was equivalent among all cell lines tested, with the 314 exception of SupT1, which displayed restricted entry consistent with low susceptibility to 315 cell-free HIV-1 infection (6). These results demonstrate that the permissivity of MT-4 316 cells to the gp41 CT truncation mutant is not explained at the level of cell-free virus 317 entry. 318 319 Kinetics of virus release in MT-4 are faster than in other T-cell lines tested. 320 Because the kinetics of HIV-1 replication in MT-4 cells are more rapid than in the other 321 T-cell lines tested, we hypothesized that the kinetics of HIV-1 gene expression might 322 also be faster in MT-4 cells. An equal number of cells were transduced with equivalent 323 amounts of VSV-G-pseudotyped Env– NL4-3 encoding eGFP in place of Nef (Fig. 6A- 324 C). In this reporter virus, eGFP expression is under transcriptional control of the viral 325 LTR; eGFP thus serves as a surrogate for LTR-mediated gene expression. Cells were 326 analyzed for eGFP expression by flow cytometry at multiple time points post- 327 transduction. We observed that a higher percentage of MT-4 cells expressed eGFP 24 328 hours post-transduction relative to the other T-cell lines (Fig. 6A). The percentage of 329 MT-4 expressing eGFP increased ~3-fold 48 hours post-transduction, whereas the other 330 cell lines tested exhibited a ~2-fold increase in the number of eGFP+ cells, with the 331 exception of M8166, which exhibited only a minor increase in eGFP expression 332 between 24 and 48 hours post-transduction. Therefore, the kinetics of HIV-1 protein 333 production in MT-4 are faster relative to the other T-cell lines examined here. 334 335 Enhanced virus production and release on a per-cell basis could contribute to the gp41 336 CT-truncation permissive phenotype of MT-4 cells. To determine whether MT-4 express 337 more HIV proteins than non-permissive cells, the eGFP median fluorescence intensity 338 (MFI) was measured at different time points (Fig. 6B-C). At 24 hours post-transduction,

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339 the MFI of MT-4 was less than that of SupT1, C8166, or M8166. At 48 hours post- 340 transduction, the MT-4 MFI increased to that of C8166 and M8166, indicating that the 341 HIV gene expression on a per-cell basis between MT-4 and C8166 is equivalent, 342 consistent with the data of Emerson et al (14). The finding that SupT1, a Tax– non- 343 permissive cell line, expressed more eGFP than MT-4 is inconsistent with the 344 hypothesis that MT-4 are permissive to CT-truncation due to overall enhanced protein 345 production per cell relative to non-permissive cells. 346 347 To explore the role of viral protein production kinetics in virus output, virus release in the 348 T-cell panel was measured. Cells were transduced with Env– VSV-G-pseudotyped NL4- 349 3 and HIV-1 RT released into the supernatant was measured 42 hours post- 350 transduction (Fig. 6D). Consistent with increased kinetics of HIV-1 protein expression 351 compared to the other T-cell lines in the panel, HIV-1 RT production was highest for 352 MT-4 compared to other cell lines. Taken together, these data indicate that on a per-cell 353 basis, individual MT-4 cells do not exhibit higher steady-state cell-associated viral gene 354 expression than the non-permissive cell lines tested here. However, MT-4 cultures 355 release nearly three-fold more HIV-1 RT in a 42-hour period, indicating that the kinetics 356 of HIV protein production in MT-4 cells are faster than in the other cell lines tested. 357 358 Env incorporation in MT-4 is inefficient. 359 Because Env incorporation into virus particles is essential for viral infectivity, we next 360 investigated the role of the gp41 CT in Env incorporation in SupT1 and MT-4. Env 361 incorporation was determined by two methods, radio-immunoprecipitation (radio-IP) and 362 western blot (Fig. 7A-C). Equivalent numbers of both MT-4 and SupT1 cells were 363 transduced overnight with VSV-G-pseudotyped HIV-1 expressing either WT or 364 CTdel144 Env and washed extensively the following morning to remove unabsorbed 365 virus. Cell- and virus-containing supernatants were collected, lysed, and prepared for 366 analysis as described in the Methods. Consistent with our previous report (21), 367 truncation of the gp41 CT in a non-permissive T-cell line, SupT1, resulted in an 368 approximately 10-fold decrease in Env incorporation, as measured by gp120 and gp41 369 levels in virions, compared to WT (Fig. 7A). In contrast, in MT-4 cells, truncation of the 370 gp41 CT resulted in an approximately 2-fold reduction in Env incorporation (Fig. 7B). 371 We next tested whether higher levels of WT Env are incorporated into virions produced 372 in MT-4 compared to SupT1 cells. Virus lysates were similarly prepared as in Fig. 7A-B, 373 RT-normalized, subjected to SDS-PAGE, and Env band intensities were measured (Fig. 374 7C). Surprisingly, MT-4 were found to incorporate less WT Env than SupT1. To gain 375 more insight into why MT-4 incorporate less Env than the non-permissive SupT1 cell 376 line, various parameters of viral assembly were determined (Fig. 7D-F). Env processing 377 in MT-4 was lower than in SupT1 (Fig. 7D). The ratio of cell-associated gp120 to total 378 Gag was also lower for MT-4 than SupT1 (Fig. 7E). Consistent with a previous report 379 that examined other non-permissive T-cell lines (14), we observed that Gag processing 380 in MT-4 is more efficient than in the non-permissive SupT1 (Fig. 7F). More rapid Gag 381 processing is likely an indication of more rapid Gag trafficking, membrane association, 382 and/or assembly, perhaps resulting in assembly of the Gag lattice before Env is 383 recruited (19). 384

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385 High infectivity of virions produced from MT-4 does not explain their permissivity to gp41 386 CT truncation. 387 The surprising result that Env incorporation in MT-4 is inefficient relative to SupT1 388 suggested that there is something inherently more efficient about viral transmission in 389 the MT-4 cell line. To determine whether cell-free virions produced from MT-4 cells are 390 more infectious than virions produced from the other cell lines in our panel, TZM-bl 391 infectivity assays were performed (Fig. 8A-C). Cells were transduced with VSV-G- 392 pseudotyped HIV-1 encoding either WT or CTdel144 Env and collected 42 hours post- 393 transduction. RT-normalized virus supernatants from the T-cell panel were used to 394 infect TZM-bl cells in parallel. Consistent with our findings that Env incorporation of the 395 CT-truncated Env is reduced ~10-fold and ~2-fold in SupT1 and MT-4, respectively, 396 cell-free infectivity was also reduced to a similar extent by the CTdel144 mutation (Fig. 397 8A). Thus, under these conditions and with these cell lines, cell-free infectivity closely 398 correlates with levels of virion-associated Env. 399 400 To directly compare the infectivity between WT and CTdel144 virus produced from all 401 five T-cell lines, TZM-bl cells were infected in parallel with the same RT-normalized 402 virus produced as in Fig. 7A. SupT1 were used as the standard of comparison. We 403 observed the infectivity of WT virus produced by all cells, with the exception of M8166, 404 to be significantly lower than WT virus produced by SupT1 (Fig. 8B). Interestingly, 405 CTdel144 virions produced in the non-permissive cell line M8166 exhibited ~3-fold 406 higher levels of infectivity compared to MT-4 and C8166 (Fig. 8C), suggesting that cell- 407 free infectivity is, at most, a minor contributing factor to the CT-permissive phenotype of 408 MT-4. 409 410 MT-4 express higher levels of cell surface CT-truncated Env than other T-cell lines 411 tested. Because Env incorporation in MT-4 is reduced compared to the other T-cell lines 412 tested, we next investigated whether Env is efficiently expressed on the cell surface of 413 MT-4. To measure cell surface-associated Env, the T-cell panel was transduced with 414 VSV-G pseudotyped WT or CTdel144 NL4-3 and fixed 42 hours later, a time point at 415 which the cells would be expected to express HIV-1 proteins, but not display extensive 416 syncytium formation. Fixed cells were then fluorescently labeled with anti-gp120 417 antibodies and Env expression was measured by flow cytometry as a shift in the 418 histogram relative to an Env– control (Fig. 9A). WT Env expression on MT-4 cells was 419 higher than on the other cell lines tested (Fig. 9B). Consistent with the role of the gp41 420 CT in regulating endocytosis (73-76), truncation of the gp41 CT enhanced cell-surface 421 Env expression in all cell lines including MT-4, although in M8166 the increase was not 422 statistically significant (Fig. 9B). 423 424 The enhanced surface expression of Env observed in MT-4 cells could be explained by 425 either defects in Env internalization or high levels of viral protein production relative to 426 the other cell lines, resulting in high levels of cell-associated Env. Having established 427 that MT-4 do not exhibit higher levels of LTR-mediated gene expression than some 428 other cell lines in the panel, we sought to determine whether Env internalization is 429 defective in MT-4. To confirm the higher levels of cell-associated Env on MT-4 relative 430 to the nonpermissive SupT1 cell line, MT-4 and SupT1 cells were transduced with VSV-

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431 G-pseudotyped HIV-1, pulse-chased with a fluorescently labeled anti-gp120 monovalent 432 Fab probe and analyzed by confocal microscopy (Fig. 10A) (19). A monovalent Fab 433 fragment was used to probe for Env levels to avoid bivalent antibody crosslinking of Env 434 trimers, which has been shown to induce Env internalization (77). The anti-gp120 probe 435 used in the pulse-chase assay labeled both the internalized pool of Env and surface 436 Env. The total (non-biosynthetic) Env was quantified by combining both the internalized 437 pool and surface Env values. As a control to establish whether AP-2-dependent 438 internalization machinery was functional, the cell media were supplemented during the 439 short anti-gp120 pulse with fluorescent transferrin (Tfn) to label the endosomal 440 compartments (78, 79) (Fig. 10A – uninfected conditions). Tfn was internalized in both 441 SupT1 and MT-4, confirming that both cell lines have an intact endocytosis machinery. 442 443 To determine whether differences exist in Env internalization in SupT1 and MT-4, the 444 ratio of surface to internalized Env was analyzed (Fig. 10B-C). Consistent with the flow 445 cytometric analysis (Fig. 9), MT-4 expressed higher levels of total and surface Env than 446 SupT1 (Fig. 10B). Surface Env expression on MT-4 was further increased by truncating 447 the gp41 CT. To gain further insight into the ability of MT-4 to internalize Env, we 448 determined the percent Env internalization during the pulse-chase (Fig. 10C). WT Env 449 was internalized more rapidly in SupT1 than in MT-4. As expected, truncation of the 450 gp41 CT resulted in less internalized Env for both MT-4 and SupT1. Altogether, the data 451 suggest that Env levels on the surface of MT-4 cells may be higher than on other lines 452 due to slower internalization. 453 454 MT-4 serve as better targets for C-C transmission than the other cell lines tested. 455 The results presented above suggest that levels of virion-associated Env and cell-free 456 particle infectivity do not account for the permissivity of MT-4 cells to CT-truncated Env. 457 We therefore explored the role of C-C transmission in the gp41 CT-truncation 458 permissive phenotype. To gain a better understanding of MT-4 cells as a target for HIV- 459 1 infection, we compared the relative ability of cell lines in the T-cell panel to be infected 460 via C-C transmission, using 293T cells as the donor. Non-lymphoid 293T cells do not 461 express the adhesion molecules LFA-1, ICAM-1, or ICAM-3 (80). Using 293T cells as 462 the virus-producing donor cell enabled the study of viral transmission independent of 463 differences in LFA-1:ICAM1/3 engagement, surface Env levels, and kinetics of HIV 464 release, as C-C transfer in this system is dependent primarily on Env-CD4 interactions. 465 Because ~50% of our Jurkat E6.1 cells do not express CD4 (Fig. 5A), this cell line was 466 excluded from the analysis as the CD4– cells would not become infected in the assay. 467 The vector used in this analysis encodes eGFP under control of the HIV-1 LTR; eGFP 468 therefore labels cells infected by either the cell-free or C-C route. 293T were transiently 469 transfected with an HIV-1 proviral clone encoding eGFP and 24 hours later either co- 470 cultured with dye-labelled T cells or overlaid with a transwell containing dye-labelled T 471 cells. Viral transfer in the co-culture thus represents the summation of both cell-free and 472 C-C infection events (Fig. 11A). Evaluation of the transwell data showed that cell-free 473 infection of MT-4 is less efficient relative to C8166 and M8166, but more efficient than 474 SupT1 (Fig. 11B). Subtracting the transwell from the co-culture values produced the C- 475 C contribution (Fig. 11C). The results indicated that MT-4 cells were more efficiently 476 infected by C-C transmission than SupT1 or C8166. High MT-4 and M8166

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477 susceptibility as target cells is not due to higher CD4 expression, since we found that 478 SupT1 express higher levels of CD4 compared to either MT-4 or M8166 (Fig. 5A) and 479 equivalent or greater levels of CXCR4 than MT-4 or M8166, respectively (Fig. 5B). The 480 differences in susceptibility to C-C transmission from 293T donor to MT-4 and M8166 481 targets were not statistically significant, suggesting that high susceptibility to C-C 482 transmission likely contributes to, but does not entirely account for, the CT-truncation 483 permissive phenotype of MT-4.

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484 Discussion 485 486 It has been previously reported in a number of studies that the CTdel144 gp41 487 truncation mutant is unable to establish a spreading infection in most T-cell lines or 488 primary hPBMCs. In this study, we sought to elucidate the basis for the unusual, and in 489 our analysis unique, permissivity of the MT-4 T-cell line to truncation of the gp41 CT. To 490 this end, a panel of validated T-cell lines known or reported to be permissive or non- 491 permissive to gp41 CT truncation was compared to MT-4. We confirmed that the MT-4 492 T-cell line is permissive to CT-truncation, but observed that other HTLV-transformed T- 493 cell lines previously reported as permissive (14, 58, 81) are not. Differences in 494 methodology or cell line origin likely explain the contrast in these findings. In part 495 because of these differences, we extensively validated the T-cell lines used in the study. 496 We found that HTLV-I Tax expression, viral entry efficiency, cellular levels of viral 497 protein expression, virion-associated Env content, and cell-free viral infectivity did not 498 solely account for permissivity to gp41 CT truncation. A combination of rapid HIV-1 499 gene expression, enhanced cell-surface Env expression, and high susceptibility to C-C 500 transmission compared to the non-permissive lines tested in this study likely explains 501 the ability of MT-4 to overcome the requirement for the gp41 CT. 502 503 It was previously suggested that MT-4 express higher levels of Gag proteins after HIV-1 504 infection relative to the non-permissive cells examined (14). We found that in MT-4 505 cultures, more cells were eGFP+ over time indicating faster HIV-1 protein production 506 kinetics compared to non-permissive cells. HTLV-I Tax expression and upregulated NF- 507 B activity were not associated with greater HIV-1 protein production; C8166 and 508 M8166 expressed comparable levels of eGFP per cell relative to Tax– SupT1 and Jurkat 509 E6.1. These contrasting results are likely explained by methodological differences; our 510 flow cytometry approach allowed us to quantify both eGFP expression per cell, and 511 number of cells expressing eGFP, at various time points, while the western blot 512 approach used by Emerson et al. (14) measured total HIV-1 Gag expression in the cell 513 cultures at a single time point. Both our study and that of Emerson et al. found that MT- 514 4 cells release approximately 3-fold more virus than non-permissive cells. We conclude 515 that this is due to faster kinetics of HIV-1 protein production in MT-4 cells rather than 516 more protein being expressed per infected cell, relative to non-permissive cells. 517 518 A recent report found that MT-4 and C8166 are likely permissive to type I IN mutations 519 due to HTLV-I Tax expression inducing NF-B protein recruitment to the HIV-1 LTR on 520 unintegrated HIV-1 DNA (46). If this phenomenon were the cause of T-cell line 521 permissivity to truncation of the gp41 CT, Tax+ C8166 and M8166 would also be 522 permissive to gp41 CT truncation. Furthermore, we found that MT-4, C8166, and M8166 523 all display higher NF-B activity compared to SupT1, indicating that they are all in a 524 hyper-NF-B activated state. Therefore, while Tax expression was found to overcome 525 type I IN defects and activate transcription of non-integrated HIV-1 DNA in C8166 and 526 MT-4 (46), this phenomenon does not account for the unique ability of MT-4 to host 527 multiple rounds of CTdel144 infection. 528

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529 Reduced Env incorporation in MT-4 was associated with a commensurate reduction in 530 cell-free infectivity, suggesting that viral particles produced by MT-4 are inherently less 531 infectious than those produced by non-permissive cells. We also observed that MT-4 532 cells exhibit a higher level of Gag processing relative to SupT1 and a lower level of Env 533 processing. More efficient Gag processing may be a consequence of increased 534 assembly kinetics. Previous work has led to the hypothesis that the timing of Gag 535 assembly versus Env expression on the surface can influence the efficiency of Env 536 incorporation; a delay in Env trafficking to the surface relative to completion of Gag 537 assembly reduces the efficiency of Env trapping by the nascent Gag lattice (19), a 538 phenomenon that could account for the relatively inefficient Env incorporation observed 539 in MT-4 relative to SupT1. 540 541 Consistent with the role of the gp41 CT in regulating Env internalization from the PM, 542 truncation of the gp41 CT resulted in enhanced surface Env expression on all T-cell 543 lines tested (with the exception of M8166 in which the increase was not statistically 544 significant) as observed in previous studies (17, 82, 83). Analysis of Env internalization, 545 using a pulse-chase assay, found that the rate of Env internalization was reduced in 546 MT-4 compared to SupT1. This reduced Env internalization may contribute to the 547 reduced Env incorporation seen in MT-4, consistent with a role for gp41 CT-dependent 548 internalization from the PM in Env incorporation during viral assembly (15-17). This 549 finding suggests a recycling-independent Env incorporation model in MT-4, wherein the 550 presence of a full-length tail allows for some enhancement of WT Env incorporation by 551 Gag lattice trapping during particle assembly (22), compared to the passive, less 552 efficient incorporation of the gp41 CT-truncated mutant which is unable to be trapped. 553 The higher surface expression of Env in MT-4 cells contributes to the smaller effect of 554 CT truncation in Env incorporation in MT-4 relative to non-permissive T-cell lines. This is 555 consistent with the observation that in HeLa cells the Env incorporation defect observed 556 with CTdel144 could be overcome by increasing Env expression (15); more Env on the 557 surface leads to an increase in non-specific (passive) incorporation that is less 558 dependent on the gp41 CT and its trapping by the Gag lattice (1). The higher surface 559 expression of Env in MT-4 relative to non-permissive T-cell lines may also contribute to 560 the formation of VSs and more productive transfer of CTdel144, which is more 561 fusogenic than Env with a full-length CT (18), by C-C transmission. 562 563 Our approach of using 293T as virus-donor cells allowed us to test the susceptibility of 564 the T-cell line panel to infection by either cell-free or C-C transmission independent of 565 the ability of the T-cell lines to serve as donor cells. We observed an approximately 3- 566 fold higher susceptibility of MT-4 and M8166 cells to C-C transmission from 293T 567 donors compared to SupT1 and C8166. These data indicate that a differential 568 susceptibility to C-C transmission between C8166 and M8166 (a subclone of C8166 569 more susceptible to formation of syncytia) arose during the generation of M8166. 570 Because the lipid composition of the target cell membrane has been shown to affect 571 viral fusion and entry (84, 85), it is possible that differences in PM lipid composition 572 between the cell lines studied here account for the high susceptibility of MT-4 and 573 M8166 to C-C transmission. Further studies will be required to evaluate this possibility. 574

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575 This study reveals that multiple factors are associated with the ability of the MT-4 T-cell 576 line to support the replication of a gp41 CT truncation mutant. The requirement for the 577 gp41 CT in MT-4 is likely overcome by the additive effects of rapid HIV-1 protein 578 production, high levels of cell-surface Env expression, and increased susceptibility to C- 579 C transmission compared to non-permissive cells. These results highlight that Env 580 incorporation, viral transfer, and ultimately the establishment of a spreading infection 581 are influenced by a number of factors including the kinetics of viral protein expression 582 and virus assembly, the levels of Env expressed on the cell surface, and the rate of Env 583 trafficking and internalization. 584

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585 Materials & Methods 586 587 Cell lines and culture 588 293T [obtained from American Type Culture Collection (ATCC)] and TZM-bl [obtained 589 from J. C. Kappes, X. Wu, and Tranzyme, Inc. through the NIH AIDS Reagent Program 590 (ARP), Germantown, MD] cell lines were maintained in DMEM containing 5% or 10% 591 (vol/vol) fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL 592 streptomycin (Gibco) at 37 °C with 5% CO2. Jurkat E6.1, MT-4, C8166-45 (referred to 593 as C8166), and M8166 were obtained from Arthur Weiss (Cat# 177), Douglas Richman 594 (Cat# 120), Robert Gallo (Cat# 404), and Paul Clapham (Cat# 11395), respectively, 595 through the NIH ARP. The source of SupT1 used in this study is unknown. The SupT1- 596 CCR5 (86) T-cell line was a generous gift from James Hoxie. The ATL Tax– cells lines 597 (ED, ATL-35T, and TL-Om1) were a generous gift from Chou-Zen Giam. T-cell lines 598 were maintained in RPMI-1640 medium containing 10% FBS, 2 mM glutamine, 100 599 U/mL penicillin, and 100 µg/mL streptomycin (Gibco) at 37 °C with 5% CO2. Whole 600 blood was obtained from healthy donors via the NIH Clinical Center. hPBMCs were 601 isolated using a ficoll gradient and stimulated with 2 μg/mL PHA-P for 3 – 5 days before 602 infection, then cultured in 50 U/mL IL-2. 603 604 Cloning and plasmids 605 The HIV-1 pNL43-nef–-eGFP reporter vector (also called pBR43IeGFP-nef–; Cat# 606 11351) [obtained through the NIH ARP from Frank Kirchhoff]. The Env– construct was 607 generated by restriction digest of the pNL4-3/KFS clone (87), referred to here as pNL4- 608 3 Env–, and target vector with StuI and XhoI restriction enzymes followed by ligation of 609 the Env fragment into pBR43IeGFP-nef– to generate pBR43IeGFP-nef–Env–. Unless 610 otherwise indicated, the full-length HIV-1 clade B molecular clone pNL4-3 was used 611 (88). The Env– (87) and CTdel144 clone (26) were described previously. Wild-type and 612 CTdel SIVmac239 were generated by Bruce Crise and Yuan Li and were a generous 613 gift from Jeff Lifson. Plasmid sequences were confirmed by restriction digest with HindIII 614 and Sanger sequencing. The HIV-1 YU2 Vpr β-lactamase expression vector (pMM310) 615 was [obtained from Michael Miller, Cat # 11444, through the NIH ARP]. The plasmids 616 pHR’CMV-GFP and pHR’CMV-Tax were a generous gift from Chou-Zen Giam. 617 618 For confocal microscopy, non-propagating, release-defective constructs were generated 619 (pSVNL4-3-ctfl-dPol-dVV-GFP-3'LTR and pSVNL4-3-ctfl-dPol-dVV-dCT-GFP-3'LTR 620 where ctfl = C-terminal FLAG tag on Gag, dPol = deletion of Pol, dVV = deletion of Vif 621 and Vpr, by removal of the Aflll fragment, GFP = green fluorescent protein in place of 622 Nef). Expression plasmids for Env and associated mutants in addition to Gag were 623 natively expressed from the reference HIV-1 clone NL4-3 with the following 624 modifications: the pNL4-3 vector was sub-cloned into an SV40 ori-containing backbone 625 (pN1 vector; Clontech; pSVNL4-3), deletion of pol by removal of the BclI-NsiI fragment, 626 mutation of the p6 PTAP motif (455PTAP458-LIRL) (89), addition of a coding C-terminal 627 FLAG tag to the Gag open reading frame to detect Gag expression (GSDPSSQ500- 628 SGDYKDDDDK) (90) and removal of the 5′ portion of the nef open reading frame and 629 replacement with a GFP coding sequence. 630

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631 Preparation of virus stocks. 632 293T cells were transfected with HIV-1 proviral DNA using Lipofectamine 2000 633 (Invitrogen) according to the manufacturer’s instructions. Virus-containing supernatants 634 were filtered through a 0.45-µm membrane 48 h post-transfection and virus was 635 quantified by measuring RT activity. VSV-G–pseudotyped virus stocks were generated 636 from 293T cells co-transfected with proviral DNA and the VSV-G expression vector 637 pHCMV-G (91) at a DNA ratio of 10:1. 638 639 For confocal microcopy, virus particles were made using the pSVNL4-3 plasmids. 640 Briefly, VSV-G-pseudotyped, single-round viruses were produced by transfecting 293T 641 cells with the pSVNL4-3 plasmids, the psPAX2 packaging plasmid (a gift from Didier 642 Trono, Addgene plasmid #12260), and pVSV-G expression plasmid using PEI 643 transfection reagent (Alfa Aesar/Thermo Fisher Scientific). The virus was harvested at 644 ~48 hours after transfection and stored at -80 °C. 645 646 STR profiling and cell line validation 647 The identity of cells in the T-cell line panel was confirmed by performing STR profiling 648 as described previously (43). Briefly, genomic DNA was extracted and sent to Genetica 649 (LabCorp) for profiling. The URL for this data set is: 650 https://amp.pharm.mssm.edu/Harmonizome/gene_set/IRF3/ENCODE+Transcription+Fa 651 ctor+Targets. The obtained STR profile was compared to the Cellosaurus reference 652 STR using the % Match formula (92). 653 654 Illumina RNA-Seq 655 RNA was extracted from T cells in their exponential growth phase using QIAshredder 656 (Qiagen, Cat# 79654) and RNeasy Plus Mini Kit (Qiagen, Cat# 74134). RNA sample 657 integrity was assessed by determining the RNA Integrity Number (RIN) with an Agilent 658 2100 Bioanalyzer instrument and applying the Eukaryote Total RNA Nano assay. RIN 659 values were between 9 and 10, indicating intact RNA. Samples were sequenced on a 660 Hiseq 2500, generating an average of 50 million raw reads per sample. Transcript 661 sequence reads were normalized against the total reads for each cell line to generate a 662 reads per kilobase per million mapped reads (RPKM) value. The RPKM is a relative 663 measure of transcript abundance. 664 665 A panel of NF-B-dependent genes was obtained by random selection of genes from a 666 database of NF-B target genes (70). An IRF3-dependent gene panel was obtained 667 from the Harmonizome search engine (71) by searching the ENCODE Transcription 668 Factors gene set titled “IRF3” (72). This gene set contains 4159 IRF3 transcription 669 factor target genes obtained from DNA-binding by ChIP-seq datasets. Genes dually 670 targeted by NF-B and IRF3 were removed from the IRF3-dependent gene panel used 671 in our analysis. 672 673 To analyze expression of NF-B and IRF3 target genes, the average RPKM for each 674 parent gene transcript was mined from the total acquired RNA-seq data set. RPKM 675 values of zero were set to 1 to allow for fold-change analysis. HTLV-transformed cell

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676 lines were compared to the lymphoma-derived SupT1 line to generate the fold-change 677 value. This value was then converted by Log2 transformation. 678 679 Virus Replication Assays 680 Virus replication kinetics were determined in Jurkat E6.1 and MT-4 cell lines as 681 previously described (26). Briefly, T cells were transfected with proviral clones (1 μg 682 DNA/106 cells) in the presence of 700 μg/ml DEAE-dextran. MT-4, C8166, and M8166 683 cells were split 1:2 every 2 days, and SupT1 and Jurkat E6.1 were split 1:3 every 2-3 684 days with fresh media. VSV-G-pseudotyped virus was used to inoculate stimulated 685 hPBMCs. Virus stocks were normalized by 32P RT activity and used to initiate spreading 686 infection. After a 2-hour incubation with VSV-G-pseudotyped virus, cells were washed 687 and resuspended in fresh RPMI-10% FBS. Every other day half the medium was 688 replaced without disturbing the cells. Virus replication was monitored by measuring the 689 RT activity in collected supernatants over time. RT activity values were plotted using 690 GraphPad Prism to generate replication curves. 691 692 HIV-1 infection of T cells 693 293T cells were plated and co-transfected via lipofectamine the next day with the pNL4- 694 3 proviral clone and the VSV-G expression vector, pHCMV-VSV-G, at a 10:1 ratio. 48 695 hours post-transfection, supernatants were passed through a 0.45 µm filter and RT 696 activity was measured by 32P RT activity assay as described (93). T cells were plated 697 the night before infection in fresh media at a density of 5x106 cells / 2 mL. The following 698 day, cells were infected overnight with RT-normalized virus. 699 700 Western blotting for viral proteins 701 The morning after transduction, cells were washed extensively to remove any 702 unabsorbed virus, and plated in 1.5 mL of RPMI-10. 42 hours post-infection, cells were 703 pelleted and lysed. Virus-containing supernatants were passed through a 0.45µm filter. 704 10 µL were set aside for RT assay and 200 µL were set aside for TZM-bl infectivity 705 assay. The remaining filtered virus-containing supernatant was layered on a 20% w/v 706 sucrose/phosphate buffered saline (PBS) solution and spun for 1.25 h at 41,500 x g at 707 4°C in a Sorvall S55-A2 fixed angle rotor (ThermoFisher Scientific). Cell and virus 708 fractions were lysed in lysis buffer (30 mM NaCl, 50 mM Tris-HCL pH 7.5, 0.5 % Triton 709 X-100, 10mM Iodoacetamide, complete protease inhibitor (Roche)). Lysates boiled with 710 6x loading buffer (7 mL 0.5 M Tris-HCL/0.4 % SDS, 3.8 g glycerol, 1 g SDS, 0.93 g 711 DTT, 1.2 mg bromophenol blue) were subjected to SDS-PAGE on 12% 1.5 mm gels 712 and processed using standard western blotting techniques. All antibodies were diluted 713 in 10 mL of 5% milk in TBS blocking buffer. HIV proteins were detected with 10 µg/mL 714 polyclonal HIV immunoglobulin (HIV-Ig) obtained from the NIH ARP. Anti-human IgG 715 conjugated to horseradish peroxidase (HRP) was obtained from SigmaAldrich (Cat# 716 GENA933) and used at a 1:5000 dilution. gp41 was detected with 2 µg/mL10E8 717 monoclonal antibody obtained from the NIH ARP (Cat# 12294) followed by anti-human 718 IgG-HRP as above. 719 720 To detect HTLV-I Tax protein, 2x106 cells were lysed in 100 µL of lysis buffer and 30 µL 721 of 6x loading buffer and boiled for 10 min. 20 µL of cell lysates were loaded on a 12%

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722 1.5mm Tris-glycine gel and processed using standard western blotting techniques. 723 HTLV-I Tax was detected with an anti-Tax-1 mouse monoclonal (Abcam, Cat# 724 ab26997) at a 2 µg/mL concentration followed by goat-anti-mouse-HRP antibody 725 (Thermo Fisher, Cat # 32230) at a 0.5 µg/mL concentration. β-actin was used as a 726 loading control and detected using anti-β-actin conjugated directly to HRP (Abcam, Cat# 727 ab49900). Protein bands were visualized using chemiluminescence with a Bio-Rad 728 Universal Hood II ChemiDoc and then analyzed with ImageLab v5.1 software or the 729 Azure Spot Analysis Software. 730 731 Metabolic labeling and radioimmunoprecipitation were performed as previously 732 described (21). Phosphor screens were imaged with a Bio-Rad Personal Molecular 733 Imager System and quantitative analysis of bands was performed with the Azure Spot 734 Analysis Software. 735 736 Viral entry assay 737 BlaM-Vpr-containing viruses were produced by transient co-transfection of 239T cells 738 with pNL4-3 constructs encoding either Env–, WT, or CTdel144 sequences, a second 739 plasmid (pMM310 – NIH ARP Cat# 11444) encoding the BlaM-Vpr fusion protein, and a 740 third plasmid (pAdvantage - Promega, Cat# E1711) to enhance transient protein 741 expression at a 6:2:1 ratio. For the VSV-G control, pHCMV-VSV-G was provided in 742 trans at a 10:1 ratio relative to pNL4-3. Virus-containing supernatant was filtered 48 743 hours post-transfection and concentrated using Lenti-X concentrator (TakaraBio, Cat# 744 631231) according to the manufacturer’s instructions. After concentration, the virus 745 particles were resuspended in CO2-independent media (ThermoFisher, Cat# 746 18045088), aliquoted, and stored at -80oC. Resuspended virus was diluted 10,000x and 747 virus concentration was measured using two methods: (1) p24gag concentration by 748 Lenti-X GoStix Plus (TakaraBio, Cat# 631280) and (2) 32P RT activity (93). 749 750 Entry of BlaM-Vpr-containing virus was detected using a fluorescent substrate, CCF2- 751 AM (Thermo Fisher, Cat# K1032) (94). To perform the BlaM-Vpr assay, a suspension of 752 20x106 cells/mL was made. 100 µL of this suspension was aliquoted per well of a U- 753 bottom plate. Cells were infected for 4 h at 37oC with 400 ng p24gag. After incubation 754 with virus, cells were washed 2x with CO2-independent media supplemented with 10% 755 FBS. Cells were then resuspended in 100 µL CO2 independent media with 10% FBS 756 and 20 µL of the CCF2-AM reagent (prepared according to the manufacturer’s 757 instructions). Cells were incubated for 1 hour with the CCF2-AM reagent in darkness at 758 room temperature. Cells were then washed 2x with PBS, resuspended in CO2- 759 indpendent media + 10 % FBS and left at room temperature in darkness overnight. 760 Sixteen hours later, cells were fixed in 100 µL 4% PFA and analyzed the same day. 761 Flow cytometric analysis utilized a Fortessa X-20 flow cytometer (BD Bioscience) and 762 FlowJo software (Tree Star Inc, Ashland, OR, USA). 763 764 Single-cycle infectivity assays 765 TZM-bl infectivity assays were performed as previously described (95). Briefly, 20,000 766 TZM-bl cells were plated in a flat-bottomed white-walled plate (Sigma Aldrich, Cat# 767 CLS3903-100EA). The following day, the cells were infected with serial dilutions of RT-

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768 normalized virus stocks in the presence of 10 µg/mL DEAE-dextran. Approximately 36 769 hours post-infection, cells were lysed with BriteLite luciferase reagent (Perkin-Elmer) 770 and luciferase was measured in a Wallac BetaMax plate reader. 771 772 Flow cytometry 773 Cells were resuspended in 8% BSA/PBS at a concentration of 107 cells/mL. 100 µL was 774 aliquoted into a 96-well V-bottom plate (SigmaAldrich, Cat# CLS3897). An antibody 775 solution was made using 20 µL of either isotype or target antibody per test, as 776 recommended by the manufacturer. The following antibodies from BD Pharmigen were 777 used: APC mouse IgG2a 훋 isotype control (Ca# 555576), APC mouse anti-CD184 778 (CXCR4) (Cat# 560936), PE mouse anti-CD4 (Cat # 555347). The isotype control for 779 CD4 was acquired from Biolegend (PE mouse IgG1, 훋 isotype control, Cat# 400113). 780 Cells were incubated with antibodies for 20 min at room temperature, then washed 3x 781 with PBS. Samples were analyzed via the BD FACSCalibur flow cytometer (BD 782 Bioscience) and FlowJo software (Tree Star Inc.). 783 784 To detect cell-surface Env, infected cells were washed with PBS and fixed with 4% PFA 785 overnight at 4oC. The following day, the PFA was quenched with 0.3 M glycine in PBS 786 using three rounds of 5 min incubations. Cells were then resuspended in 100 µL of 8% 787 BSA/PBS. An antibody cocktail was made by diluting b12 and 2G12 in 8% BSA/PBS 788 [obtained from Dr. Dennis Burton and Carlos Barbas, and Polymun Scientific, 789 respectively, via the NIH-ARP] and aliquoting 100 µL to each sample to obtain a final 790 concentration of 2 µg/mL of both antibodies. The cell:antibody mixture was incubated at 791 room temperature for 20 min. Unbound antibody was removed with three PBS washes. 792 Goat anti-human conjugated to Alexa Fluor 488 was used as a secondary antibody 793 (Invitrogen, Cat# A11013) at a concentration of 2 μg/mL. Cells were incubated with 794 secondary antibody for 20 min at room temperature and washed 3x with PBS before 795 analysis on the BD FACSCalibur. 796 797 C-C transmission assay 798 293T were transfected with pBR43IeGFP-nef+ (Cat# 11349; NIH ARP) or an Env– 799 derivative (described above). 24 hours post-transfection, dye-labelled T cells were 800 added to the transfected 293T cells at a 4:1 ratio in either a co-culture or to the top layer 801 of a transwell. 12 hours post-coculture, BMS-806 was supplemented into the media at a 802 concentration of 2000 nM to prevent the formation of syncytia and T cell to T cell 803 transmission. Forty-eight hours post-coculture, cells were fixed in 4% PFA and analyzed 804 by flow cytometry. Data were collected via CellQuest and processed with FlowJo 805 software (Tree Star Inc). 806 807 Production of anti-Env monovalent Fab b12-Atto565 probe 808 The anti-Env b12 Fab fragment recombinant expression vector was a kind gift from 809 Dennis Burton (96). A 4x lysine tag, to enhance dye conjugation efficiency (97), was 810 added to the C-terminus of the b12 light chain using site-directed mutagenesis. 811 Expression of b12 was carried out as previously described (19, 98). Briefly, clarified cell 812 lysates were purified by protein G affinity chromatography (Gold Biotechnology, Inc.). 813 The b12 Fab, typically 99% pure, was conjugated with Atto565 N-hydroxysuccinimidyl

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814 ester (Sigma-Aldrich). Typically, a labeling ratio of 1 Atto565 dye molecule per b12 Fab 815 molecule was achieved. 816 817 Pulse-chase staining 818 For each sample, 6-8x105 MT-4 or SupT1 cells were transduced with VSV-G- 819 pseudotyped virus encoding either WT or CTdel144 Env (described above). 820 Approximately 40 hours post-infection, the cells were blocked with 10% BSA in 821 complete media for 30 min at 37°C with 5% CO2, stained with custom Fab b12-Atto565 822 (25 nM) and Transferrin-AlexaFluor647 (Transferrin-AF647; Invitrogen; 25 μg/mL) in 823 media with 6% BSA, and washed thrice for 5 min with the same media. The cells were 824 then placed onto 18 mm coverslips pre-treated with poly-L-lysine (Ted Pella Inc.), fixed 825 with 4% and 0.2% paraformaldehyde and gluteraldehyde in PBS, respectively. 826 Coverslips were then mounted onto glass slides with Fluoromount-G (Southern 827 Biotech). 828 829 Microscopy 830 Imaging of the cellular specimens was performed with a spinning-disk confocal 831 microscope built on an inverted Nikon Ti-E base (Solamere Technology Group Inc., Salt 832 Lake City, UT) using a 60x, CFI Plan Apo Lambda 1.4 NA oil-immersion objective 833 (Nikon Instruments). Fiber-coupled lasers (OBIS, Coherent) were used with a CSU-X 834 A1 spinning disk unit (Yokogawa Electronics) to excite and collect confocal fluorescence 835 sections. Transferrin-AF647 was imaged using a 640 nm laser at 12 mW with 40 ms 836 exposure, Atto565 was imaged using a 561 nm laser at 24 mW with 100 ms exposure, 837 and GFP was imaged using a 488 nm laser at 12 mW with 40 ms exposure. Laser 838 power was measured at the specimen. Z-stacks for each field of view were collected at 839 0.3 μm spacing. 840 841 Image analysis 842 Quantitation of surface-exposed and internalized Env signal in the pulse-chase assay 843 images was performed using ImageJ software. First, for each z-stack, a maximum 844 intensity projection was generated for a 4.5 μm range through the middle of the cells. 845 The background was subtracted using ImageJ’s built-in “rolling ball” background 846 subtraction process with a radius of 150 pixels. Cells were excluded from analysis if 847 they did not express GFP (non-infected cells) or did not internalize transferrin (dead or 848 dysfunctional cells), or if the maximum intensity projection did not represent an 849 equatorial section of the whole cell. Integrated intensity was then estimated using two 850 regions of interest (ROIs) in the Atto565 channel: (1) an outer ROI was manually 851 segmented to enclose the entire cell, including any plasma-membrane-associated 852 signal, and (2) an interior ROI was manually segmented just inside the PM, excluding 853 any PM-associated signal. Signal for Env on the PM was thus enclosed between these 854 two ROIs, and signal for the internalized Env pool was enclosed within the interior ROI. 855 The integrated intensity of the outer ROI (1) minus that of the inner ROI (2) served as a 856 measure of surface-exposed Env, while the integrated intensity of the inner ROI (2) 857 served as a measure of internalized Env. 858 859 Statistics

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860 Statistics were calculated using GraphPad Prism version 8 for Mac OS (GraphPad 861 Software, La Jolla, CA). Unpaired Student’s t-tests were performed and two-tailed *P < 862 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically 863 significant. GraphPad Prism was also used to calculate standard error and to assess 864 statistical significance by one-way ANOVA. P values for Student’s t-test and one-way 865 ANOVA analysis are defined with the same cut-offs. 866 867 Ethics Statement 868 PBMCs were obtained from anonymous, de-identified blood donors to the NIH 869 Department of Transfusion Medicine Blood Products Program (NIH CC-DTM). 870

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871 List of abbreviations. 872 CT – cytoplasmic tail 873 VS – virological synapse 874 C-C – cell-to-cell 875 Env – envelope glycoprotein 876 STR – short tandem repeat 877 MFI – median fluorescence intensity 878 PM – plasma membrane 879 Radio-IP – radio-immunoprecipitation 880 hPBMC – human peripheral blood mononuclear cells 881 RT –reverse transcriptase 882 VSV-G – Vesicular stomatitis virus glycoprotein 883 PBS – phosphate buffered saline 884 FBS – fetal bovine serum 885 WT – wild type 886 HRP – horseradish peroxidase 887 ER – endoplasmic reticulum 888 MA – HIV-1 Matrix protein 889 EBV – Epstein-Barr virus 890 HTLV – Human T-cell leukemia virus 891 ALL – Acute lymphocytic leukemia 892 ATL – Adult T-cell lymphoma 893 ATCC – American Type Culture Collection 894 NIH ARP – NIH AIDS Reagent Program 895 n.s. – not statistically significant 896 IN – HIV-1 Integrase protein 897 CA – HIV-1 Capsid protein 898 RIN - RNA Integrity Number 899

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900 Declarations. 901 Competing interests. 902 The authors declare that they have no competing interests. 903 904 Authors’ contributions 905 MVF and EOF designed the study. MVF and HKH performed the experiments and 906 analysis, and NP generated the fluorescently labelled b12 monovalent Fab for the 907 microscopic analysis. PT performed initial western blots comparing Env content in 908 virions produced in SupT1 and MT-4. SBvE contributed valuable feedback and 909 guidance on the microscopy experiments. All authors made contributions to writing the 910 manuscript. 911 912 Acknowledgements 913 We thank members of the Freed and van Engelenberg labs for helpful discussion and 914 for critical review of the manuscript. We thank James Hoxie for providing the SupT1- 915 CCR5 cell line, Erin M. Hall at Genetica (Lab Corp) for assistance with STR profiling 916 and data interpretation, Douglas Richman for providing historical information on the 917 treatment of MT-4 cells prior to donation to the NIH ARP, and James A. Thomas for 918 assisting with the BlaM-Vpr flow cytometry acquisition. We thank Chou-Zen Giam for 919 helpful discussion and for providing the ATL Tax– T-cells (ED, ATL-55T, and TL-Om1) 920 and expression vectors for Tax and GFP expression. We also thank Yongmei Zhao at 921 the NCI-Center for Cancer Research Sequencing Facility for performing the Illumina 922 RNA-seq and assisting with the differential RNA analysis. This study was supported by 923 the Intramural Research Program of the Center for Cancer Research, National Cancer 924 Institute, NIH and by an Intramural AIDS Research Fellowship (for MVF). 925

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926 Figure Legends. 927 928 Figure 1. Lineage of cell lines used to interrogate T-cell line permissivity to replication of 929 an HIV-1 mutant lacking the gp41 CT. 930 A) The SupT1 T-cell line was derived from a pleural effusion of a male patient with non- 931 Hodgkin’s lymphoma. Cells were sub-cloned by limiting dilution to generate a cell line 932 capable of continual growth from a single-cell colony (59). B) The peripheral blood of a 933 14-year-old boy with relapsed acute lymphocytic leukemia (ALL) was used to generate 934 the EBV–, IL-2-dependent JM T-cell line (60). The JM line was then subcloned to 935 generate the Jurkat-FHCRC subclone for its ability to produce IL-2 upon stimulation with 936 phorbol esters or lectins (99). “-FHCRC” designation indicates the cell line originated at 937 the Fred Hutchinson Cancer Research Center. Jurkat-FHCRC was then subjected to 938 limiting dilution to generate an IL-2 -independent, mycoplasma-free cell line, Jurkat E6.1 939 (100). C) To generate the MT-4 cell line, cells from an adult male ATL patient were co- 940 cultured with male human infant cord leukocytes, therefore it is unknown whether these 941 cells are of cord leukocyte or ATL cell origin (61). Cells were gifted to the lab of Douglas 942 Richman by Harada et al. (101) and serially passaged by terminal dilution cloning of the 943 cells in the presence of ciprofloxacin until they were determined to be mycoplasma free. 944 These cells were then donated to the NIH ARP by Douglas Richman (D. Richman, 945 personal communication). D) To generate the C8166 T-cell line, cells were first acquired 946 from a 26-year-old male ATL patient’s inguinal lymph node (102) and maintained in IL-2 947 for several passages until they were deemed IL-2 independent (103). This T-cell line, 948 HUT 102, was determined to be EBV– and HTLV+. The CR-M2 subclone of HUT 102 949 was then isolated and further purified by percoll gradient isolation to generate a cell line 950 with increased HTLV production, designated CRII (104). CRII was then used to 951 transform human umbilical cord blood T leukocytes by cell hybridization to generate the 952 C63/CRII-2 cell line, also known as the C8166-45 (aka 81-66 or C8166) cell line (62). 953 The “-45” designation indicates the cell line has 45 chromosomes. “CR” indicates the 954 cells were transformed by the HTLV-ICR virus. Characterization of this line found that it 955 produces HTLV-I RNA but no virus particles (62). C8166 were then subjected to limiting 956 dilution to generate a clone more susceptible to formation of syncytia when cultures 957 were infected with HIV-1 (105). This new C8166-derived cell line was named M8166. 958 959 Figure 2. Spreading infection kinetics of SIVmac239 in C8166 and M8166. 960 A) C8166 and B) M8166 were transfected with SIVmac239 encoding the indicated Env 961 CT genotype. Supernatant was sampled every 2-3 days for analysis by HIV-1 RT assay 962 and cell cultures were split 1/2. Data are representative of 3 independent experiments. 963 964 Figure 3. MT-4 is the only T-cell line tested that is permissive to the gp41 CT truncation 965 mutant CTdel144. 966 A) Schematic representation of the NL4-3 gp41 CT and gp41 CT genotypes used in this 967 study. The lentiviral lytic peptide (LLP) domains are indicated in grey boxes and the 968 highly conserved tyrosine endocytosis motif is indicated with a shaded black rectangle. 969 The CTdel144 mutant was generated by introducing two stop codons in the highly 970 conserved tyrosine endocytosis motif, resulting in a CT of 4 amino acids (26). The 971 numbers above the gp41 CT schematic indicate the first and last amino acid positions of

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972 the gp41 CT. The number below the gp41 CT indicates the position of the QGYSPL 973 sequence. The tyrosine endocytosis motif, GYSPL, is underlined. (B-G) Replication 974 curves for spreading infection are shown. B-F) Cell lines were either mock transfected 975 or transfected with pNL4-3 encoding the indicated gp41 CT genotype. Cells were split 976 B-D) 1/2 or E-F) 1/3 every 2-3 days, and an aliquot of the supernatant was reserved for 977 analysis of HIV-1 RT activity at each time point. G) hPBMCs were transduced with VSV- 978 G-pseudotyped NL4-3 encoding the indicated gp41 CT genotype or mock infected. 979 Supernatant was sampled every 2-3 days for analysis of HIV-1 RT activity and cell 980 cultures were supplemented with fresh media. Data are representative of 3 independent 981 experiments (B-F) and 3 donors (G). 982 983 Figure 4. NF-B-target gene expression in Tax-expressing T-cell lines is higher than in 984 SupT1. 985 A) Cell lysates from the indicated cell lines were analyzed by western blot for HTLV-I 986 Tax expression. 293T were transfected with a GFP expression vector as a transfection 987 control, or a Tax expression vector as a control for antibody specificity. ATL Tax- 988 deficient cells were included as negative control for Tax expression in immortalized T- 989 cell lines. B-D) RNA levels of the indicated NF-B- and IRF3-target genes were 990 compared to SupT1. RNA levels were determined by Illumina RNA-seq. As described in 991 more detail in the methods, comparisons are reported as the Log2 of the fold-change 992 (FC) of the HTLV-transformed line (MT-4, C8166, or M8166) relative to the lymphoma- 993 derived T-cell line, SupT1. 994 995 Figure 5. Viral entry mediated by full-length and CT-truncated Env is equivalent among 996 HTLV-transformed T-cell lines. 997 A) Surface CD4 and B) CXCR4 was measured by flow cytometry. MFI was determined 998 by measuring the MFI value of CD4+ or CXCR4+ histograms and subtracting the isotype 999 control MFI to directly compare MFIs between samples. To account for fluctuations in 1000 flow cytometry data that occur when comparing data from different experiments, cells 1001 from different acquisition dates were stained with antibody, fixed, and analyzed in 1002 parallel by flow cytometry. The bar graphs show the mean MFI values of cell surface A) 1003 CD4 and B) CXCR4, ± SD from three independent experiments and the bar shading for 1004 ease of comparison between panels A) and B). C) BlaM-Vpr-based viral entry assays 1005 were performed using NL4-3 expressing either WT or CTdel144 Env. A mock treatment 1006 and Env– NL4-3 pseudotyped with VSV-G were used as a positive control and Env– was 1007 used as a negative control. Representative FACS dot plots are shown. Virus dose used 1008 for each condition was titrated to avoid saturation (in the case of VSV-G and WT) and to 1009 be above the Env– conditions (in the case of CTdel144). Therefore, values can only be 1010 compared between cell lines for each condition, not between conditions. The bar graphs 1011 show the fold change in viral entry relative to SupT1 (set at 1) between D) VSV-G- 1012 pseudotyped Env– NL4-3, E) WT NL4-3, and F) CTdel144 NL4-3 with SD representing 1013 comparison of values derived from three independent experiments. Statistical 1014 significance was assessed by one-way ANOVA and Tukey’s multiple comparison test. 1015 P-values are defined in the Materials and Methods. 1016 1017 Figure 6. Kinetics of virus release in MT-4 are faster than in other T-cell lines tested.

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1018 Cells were transduced with VSV-G-pseudotyped pBR43IeGFP-nef–Env– reporter vector 1019 and collected at various time points. A) The percent of cells expressing eGFP at each 1020 time point was plotted. Error bars indicating the standard deviation of duplicate 1021 infections are too small to be seen. B) Histogram of eGFP expression in the cell lines 1022 48-hour post-transduction indicating the percent of cells expressing eGFP and their MFI 1023 relative to an eGFP– control. C) eGFP levels at various time points post-transduction 1024 (p.t.) were determined by the eGFP MFI from which the background MFI from the 1025 eGFP– construct was subtracted. The bar graph shows the mean eGFP MFI, ± SD from 1026 three independent experiments. D) Cells lines were transduced with VSV-G- 1027 pseudotyped NL4-3 Env–, washed, and supernatant HIV-1 RT activity was measured 1028 42-hours post-transduction. The bar graph shows the mean fold-change relative to the 1029 SupT1 reference line, ± SD from three independent experiments. Statistical analysis 1030 was performed to compare cell lines relative to SupT1 at individual time points post- 1031 transduction or between two samples as indicated by the horizontal line between two 1032 bars. n.s. indicates no statistical difference between the indicated cell line and the 1033 SupT1 reference or between two samples as indicated by the horizontal line between 1034 two bars. Statistical significance was assessed by one-way ANOVA and Tukey’s 1035 multiple comparison test. 1036 1037 Figure 7. Env incorporation in MT-4 cells is inefficient. 1038 SupT1 (A) and MT-4 (B) cells were transduced with RT-normalized VSV-G- 1039 pseudotyped NL4-3 encoding either WT or CTdel144 Env. Cells were metabolically 1040 labeled with [35S]Cys and cell and virus lysates were immunoprecipitated to detect HIV 1041 proteins. The locations of p66(RT), the Gag precursor Pr55Gag, p32(IN), and p24(CA) 1042 are indicated. Western blotting was performed on the virus fraction to detect gp41 and 1043 p24(CA) using equal amounts of WT and CTdel144 viral lysates. gp41.t indicates the 1044 position of the truncated gp41, CTdel144. The fold-change in Env incorporation 1045 between WT and CTdel144 Env, calculated by determining the ratio of virion-associated 1046 gp41 to p24(CA) relative to the WT condition, is indicated below the western blots. C) 1047 SupT1 and MT-4 were transduced as in (A-B) and RT-normalized virus was used to 1048 compare gp41 content in the virus fraction. Samples were analyzed by [35S]Cys radio- 1049 immunoprecipitation (IP) and western blot (detected by chemiluminescence). The bar 1050 graph shows the mean values relative to the SupT1 reference line, ± SD from three 1051 independent experiments. D) Env processing efficiency was quantified by dividing cell- 1052 associated gp120 by the total cell-associated Env band intensity 1053 [gp120/(gp120+gp160)]. E) The cell-associated Env-to-Gag ratio was determined as 1054 [gp120/(Pr55Gag+p24)]. F) Gag processing was determined by dividing p24(CA) by 1055 total Gag band intensity. D-F) The bar graphs show the mean values, ± SD from three 1056 independent experiments. n.s. indicates no statistical difference between two samples 1057 as indicated by the horizontal line. C-E) Statistical significance was assessed by one- 1058 way ANOVA and Tukey’s multiple comparison test or F) paired Student’s t-test. 1059 1060 Figure 8. High infectivity of virions produced from MT-4 cells does not explain their 1061 permissivity to gp41 CT-truncation. 1062 Cells were transduced with VSV-G-pseudotyped NL4-3 encoding either WT or 1063 CTdel144 Env. Viral supernatant was collected 42 hr post-transduction. Supernatants

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1064 were RT normalized, and a serial dilution of virus was used to infect TZM-bl cells. 1065 Luciferase values were then used to determine the relative infectivity of virus produced 1066 from each T-cell line. A) The bar graph shows the mean values of CTdel144 relative to 1067 WT (set at 100), ± SD from three independent experiments. B-C) The same virus was 1068 used as in A) to compare WT and CTdel144 virus infectivity between cell lines. The bar 1069 graphs show the mean values of B) WT and C) CTdel144 relative to the SupT1 1070 reference line (set at 100), ± SD from three independent experiments. Shading of 1071 individual columns indicates values for the individual cell lines for ease of comparison 1072 between data sets in A-C. Statistics relative to the SupT1 reference line, or between two 1073 samples as indicated by the horizontal line between two bars, are shown. “n.s.” 1074 indicates no statistical difference between the indicated cell line to the SupT1 reference. 1075 Statistical significance was assessed by A) Student’s t-test and B) one-way ANOVA and 1076 Tukey’s multiple comparison test. 1077 1078 Figure 9. MT-4 cells express higher levels of surface CT-truncated Env than other T-cell 1079 lines tested as determined by flow cytometry. 1080 VSV-G-pseudotyped NL4-3 encoding either WT or Ctdel144 Env, or no Env (Env–) was 1081 used to transduce T cells. 40 hours post-transduction, cells were fixed and stained with 1082 anti-Env antibodies for analysis by flow cytometry. A) Histogram of surface Env for each 1083 cell line is shown. Env– cells were used as a control for background staining. B) Surface 1084 Env MFI was determined by measuring the MFI value of the Env+ histogram and 1085 subtracting the Env– MFI to directly compare MFIs between samples. To account for 1086 fluctuations in flow cytometry data that occur when comparing data from three 1087 independent experiments, cells were stained with antibody and analyzed in parallel by 1088 flow cytometry. The bar graph shows the mean values of WT and CTdel144 Env MFI, ± 1089 SD from three independent experiments. n.s. indicates no statistical difference between 1090 WT and CTdel144 for the M8166 cell line. Error bars ± SD from 3 independent 1091 experiments. Statistical significance was assessed by Student’s t-test. 1092 1093 Figure 10. MT-4 cells express higher levels of Env than SupT1, which is further 1094 enhanced by truncating the gp41 CT, as determined by confocal microscopy. 1095 MT-4 or SupT1 cells were infected with a replication-and-release-defective HIV-1 1096 mutant (described in methods) expressing an eGFP reporter (yellow), and either WT or 1097 CTdel144 Env. Approximately 40 hours post-infection, cells were simultaneously pulsed 1098 with anti-Env Fab b12-Atto565 (magenta) and Transferrin-AF647 (blue) for 12 min and 1099 then chased for 50 min. Cells were fixed and imaged by confocal fluorescence 1100 microscopy. A) Representative confocal slices of transduced MT-4 or Jurkat E6.1 cells 1101 after the pulse-chase labeling. Scale bars 15 µm, inset scale bars 3 µm. B) Total 1102 surface Env and total internal Env per cell were measured using the integrated 1103 fluorescence intensity for regions defining the PM of the cell (surface) and a region 1104 defining the interior of the cell (internal), using a maximum intensity projection through a 1105 4.5 μm confocal range centered in the approximate middle of a single cell. n = 50 1106 infected cells per sample. The bar graph shows the mean values of Surface and Internal 1107 Env levels. C) Percent Env internalization was calculated as the percent of internal Env 1108 above the total Env signal (internal and surface). The scatter plot shows the mean 1109 values of percent internalized Env. n = 50 infected cells per sample. Error bars ± SEM.

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

1110 Statistical significance was assessed by one-way ANOVA and Tukey’s multiple 1111 comparison test. 1112 1113 Figure 11. MT-4 serve as better targets for C-C transmission than the other cell lines 1114 tested. 1115 293T cells were transfected with pBR43IeGFP-nef– encoding either WT Env or Env–. 1116 Twenty-four hours post-transfection, dye-labelled T cells were either co-cultured or 1117 added to a transwell exposed to the 293T supernatant. Eighteen hours post-co-culture, 1118 BMS-806 was added to prevent multiple cycles of infection and the formation of 1119 syncytia. Forty-four hours post initial co-culture, cells were collected, fixed, and 1120 analyzed by flow cytometry. A-B) The percentage of cells expressing eGFP was 1121 determined and plotted. C) The transwell value was subtracted from the co-culture 1122 value to determine the contribution of cell-to-cell transmission. The bar graphs show the 1123 mean values of the percent of cells positive for eGFP expression for the A) co-culture, 1124 B) transwell (cell-free), and C) C-C transmission, ± SD from three independent 1125 experiments. Shading of individual columns indicates values for the cell line for ease of 1126 comparison between data sets. n.s. indicates no statistically significant difference 1127 between the indicated cell line and the SupT1 reference or between two samples as 1128 indicated by the horizontal line connecting two bars. Statistical significance was 1129 assessed by one-way ANOVA and Tukey’s multiple comparison test. 1130

30 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

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40 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 1. and is also made available for use under a CC0 license. A) D) Male T-cell Male ATL patient – – Non-Hodgkin’s Lymphoma (EBV , HTLV-I ) IL-2 dependent limiting dilution sub-clone

SupT1 IL -2 independent HUT 102 B) (EBV–, HTLV-I+) Male ALL patient (EBV–) subcloning CR-M2 IL-2 dependent (HTLV-I+)

Jurkat (JM) Percoll gradient isolation produces IL-2 upon stimulation Human umbilical cord CRII blood T lymphocytes (↑ HTLV production) Jurkat-FHCRC (mycoplasma+)

limiting dilution sub-clone IL-2 independent C63/CRII-2 aka C8166-45 (C8166) (HTLV-I RNA+, virus–) Jurkat E6.1 (mycoplasma–) limiting dilution sub-clone M8166 C) Human umbilical cord ATL cells blood T lymphocytes (EBV–, HTLV-I+)

co-culture MT-4 (mycoplasma+ HTLV-I RNA+, virus–)

Limiting dilution and serial passaging in ciprofloxacin (~1987) MT4 - (mycoplasma–) bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. TABLE 1. T-cell Line STR profiles Genetic Loci Jurkat E6.1 MT-4 C8166 & M8166 SupT1 D3S1358 15, 17 17 15,16 16, 17, 18, 19 TH01 6, 9.3 7 6, 9.3 9.3 D21S11 30.2, 31.2, 33.2 28, 30.3 27, 30 28, 31, 32, 33 D18S51 12, 13, 20, 21 13 14, 17 13, 14 Penta E 10, 12 5, 15 7, 11 13, 14, 16 D5S818 9 10, 11 12 11 D13S317 8, 12 12 10, 11 10, 11, 12 D7S820 8, 12 8, 10 9, 10 11 D16S539 11 9, 12 12, 13 9, 12 CSF1PO 10, 11, 12, 13 11, 12 10 10, 11, 12 Penta D 11, 13 10, 13 11, 15 12 vWA 18, 19 17, 18 15, 16 16,17,18,19 D8S1179 12, 13, 14, 15 10, 15 11, 14 13,14 TPOX 8, 10 11 11 9 FGA 20, 21 23 21, 22 19, 20, 21 AMEL X,Y X,Y X,Y X,Y

% Match to Cellosaurus 87.88 100 98.31 82.35 reference

source NIH-ARP NIH-ARP NIH-ARP unknown catalog no. 177 120 404 & 11395 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 2. and is also made available for use under a CC0 license.

A) B) C8166 M8166 1,500 1,500 Mock 1,250 SIVmac239 WT 1,250 L L µ 1,000 µ 1,000 750 750 RT-CPM/ RT-CPM/ 500 500 250 250 0 2 5 7 9 12 14 16 19 21 23 25 28 2 5 7 9 12 14 16 19 21 23 25 28 days post-transfection days post-transfection bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 3. and is also made available for use under a CC0 license.

A) 708 856 - -

gp41 CT: LLP2 LLP3 LLP1 710 WT NL4-3 QGYSPL CTdel144 QGY**

B) E) MT-4 Jurkat E6.1 8,000 mock 2,000 WT CTdel144 6,000 L 1,500 µ

4,000 1,000 RT-CPM/µL RT-CPM/ 2,000 500

0 0 2 3 4 5 7 9 11 14 16 18 3 5 7 9 11 14 16 18 days post-transfection C) days post-transfection F) C8166 SupT1 40,000 12,500

10,000 30,000 L L µ µ 7,500 20,000 5,000 RT-CPM/ RT-CPM/ 10,000 2,500

0 0 2 5 7 9 11 13 15 17 3 5 7 9 11 14 16 18 days post-transfection days post-transfection D) G) M8166 hPBMC 20,000 12,500 mock Env- WT 15,000 10,000 CTdel144 L L µ µ 7,500 10,000 5,000 RT-CPM/ 5,000 RT-CPM/ 2,500

0 0 2 5 7 9 11 13 15 17 3 5 7 10 12 14 17 19 days post-transfection days post-infection bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 4. and is also made available for use under a CC0 license.

A) 293T

ATL) (Tax- Tax GFP - - 55T - Om1 -4 -

kDa kDa SupT1 C8166 M8166 pHR'CMV pHR’CMV MT C8166 ED ATL TL SupT1 43- -xTa 43- -Tax 34- 34- -GFP 43- 26- -β- actin 43- -β-actin

B) MT -4 versus SupT1 NF-훋B-target genes IRF3 -target genes 15 10

5 10 NF-훋B genes 0 5

Log2(FC) -5

Illumina RNA-seq 0 -10 IL4 IRF8 TNF BAX RELFOS IRF4 CALU FAN1 XBP1 CCL5CCL3 CD44EGFR BCL3 JUNB RELARELB ACTR2 MADD STAT6 TRAF1TRAF2 DTYMKGIMAP6 MAPK7 ERBB2CFLARPTPN13 CEBPDNFKBIENFKBIANFKBIZSTAT5APIK3CAALCAMNFKB2NFKB1 TP53BP1 UBE2E1 TNFSF15TNFSF10 WNT10B LGALS1 TNFRSF1B C) C8166 versus SupT1 NF-훋B-target genes IRF3 -target genes 15 10

NF-훋B genes 5 10 0 5

Log2(FC) -5

Illumina RNA-seq 0 -10

IL4 IRF8 TNF BAX RELFOS IRF4 CALU FAN1 XBP1 CCL5CCL3 CD44EGFR BCL3 JUNB RELARELB ACTR2 MADD STAT6 TRAF1TRAF2 DTYMKGIMAP6 MAPK7 ERBB2CFLARPTPN13 CEBPDNFKBIENFKBIANFKBIZSTAT5APIK3CAALCAMNFKB2NFKB1 TP53BP1 UBE2E1 TNFSF15TNFSF10 WNT10B LGALS1 TNFRSF1B D) M8166 versus SupT1 NF-훋B-target genes IRF3 -target genes 15 10

5 10 NF-훋B genes 0 5

Log2(FC) -5

Illumina RNA-seq 0 -10

IL4 IRF8 TNF BAX RELFOS IRF4 CALU FAN1 XBP1 CCL5CCL3 CD44EGFR BCL3 JUNB RELARELB ACTR2 MADD STAT6 TRAF1TRAF2 DTYMKGIMAP6 MAPK7 ERBB2CFLARPTPN13 CEBPDNFKBIENFKBIANFKBIZSTAT5APIK3CAALCAMNFKB2NFKB1 TP53BP1 UBE2E1 TNFSF15TNFSF10 WNT10B LGALS1 TNFRSF1B bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 5. and is also made available for use under a CC0 license.

A) B) 400 500 ** D) 400 VSV-G 300 2.0 300 n.s. 1.5 200 *

CD4 MFI 200 1.0

CXCR4 MFI * 100 100 **** 0.5 ** ** ** 0 0 0.0 Viral Entry (% Cleaved CCF2)

MT-4 MT-4 MT-4 SupT1 C8166M8166 SupT1 C8166M8166 SupT1 C8166M8166 Jurkat E6.1 Jurkat E6.1 Jurkat E6.1

C) mock Env– Env– + VSV-G WT CTdel144 E) WT 8 **

SupT1 6 ** * 4 *

2

Jurkat E6.1 0 Viral Entry (% Cleaved CCF2)

MT-4 SupT1 C8166M8166

Jurkat E6.1 -4 MT

F) CTdel144 15

** Uncleaved CCF2

C8166 ** 10 ** **

5 M8166 -

AM 0 Viral Entry (% Cleaved CCF2)

MT-4 SupT1 C8166M8166 Cleaved CCF2-AM Jurkat E6.1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 6. and is also made available for use under a CC0 license.

A) 80 SupT1 Jurkat E6.1 MT-4 **** C8166 60 M8166 +

40 % eGFP *** 20 n.s.

*** 0 12 24 48 hours post-transduction B) SupT1 Jurkat E6.1 MT4 - C8166 M8166

eGFP+ eGFP-

n.s. C) * D) n.s. 100 3 SupT1 ** Jurkat E6.1 MT-4 75 C8166 * M8166 2 *** **** n.s. 50 n.s. **** eGFP MFI eGFP ******** 1 n.s.

25 Relative to SupT1 *

** Fold-Change HIV-1 RT Activity RT Fold-Change HIV-1 0 0 0 12 24 48 MT-4 hours post-transduction SupT1 C8166 M8166

Jurkat E6.1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 7. and is also made available for use under a CC0 license.

A) SupT1 D) * * SupT1 1.0 * MT-4

0.8

WT CTdel144 WT CTdel144 WT CTdel144 n.s. kDa 46- 0.6 }gp160 -1gp4 -gp120- 0.4

32- 0.2 -gp41.t p66(RT)- Cellular Env Processing 0.0 -Pr55Gag 25- -p24(CA) WT WT 22- CTdel144 CTdel144 virion-associated

100 gp120 p32(IN)- 80 gp41 ** 60 E) * SupT1 0.8 n.s. MT-4 -p24(CA)- 40 20 0.6 % relative to WT cell-associated virion-associated WT CTdel144 0.4

B) MT4 - 0.2 Cellular gp120:Total Gag Cellular gp120:Total 0.0

WT WT WT CTdel144 WT CTdel144 WT CTdel144 kDa CTdel144 CTdel144

}gp160 46- -gp120- -1gp4 F) * 32- 0.8 p66(RT)- -gp41.t 0.6 -Pr55Gag 25- 22- -p24(CA) 0.4 virion-associated 0.2 p32(IN)- 100 gp120 80 gp41 Cellular Gag Processing 0.0 -p24(CA)- 60 MT-4 40 SupT1 20 cell-associated virion-associated % relative to WT WT CTdel144

C) 100 SupT1 MT-4 gp120/p24 kDa SupT1 MT-4 80 gp41/p24 46- -1gp4 - gp120 60 *** 32- **** - p24(CA) 25- 40 -p24(CA) 22- [ 35S]Cys Radio-IP 20 % relative to SupT1 chemiluminescence SupT1 MT-4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 8. and is also made available for use under a CC0 license.

A) SupT1 Jurkat E6.1 MT-4 C8166 M8166 100

75 **** 50 *** **** 25 **** **** Cell-Free Infectivity 0

WT WT WT WT WT

CTdel144 CTdel144 CTdel144 CTdel144 CTdel144 B) C) **** **** **** **** 200 **** 800 ****

150 600

100 400 n.s. **** n.s. 50 **** Relative to SupT1 200 Relative to SupT1 n.s. WT Cell-Free Infectivity **** 1.6

0 CTdel144 Cell-Free Infectivity 0

MT-4 MT-4 SupT1 C8166 M8166 SupT1 C8166 M8166

Jurkat E6.1 Jurkat E6.1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 9. and is also made available for use under a CC0 license.

A) SupT1 Jurkat E6.1 MT4 - C8166 M8166 WT Env–

CTdel144 Env–

Env (b12+2G12)

B) * * ** ** * 50 * WT 40 CTdel144

30 ** n.s. 20 * **

Surface Env MFI 10

0 SupT1 Jurkat E6.1 MT-4 C8166 M8166 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 10. and is also made available for use under a CC0 license.

A) B) Tfn (blue) Env (magenta) GFP (yellow) Merge 1.5×107 Surface Env Internal Env

1.0×107

Total Env 5.0×106 (Fluorescence Intensity) 0.0

WT WT

CTdel144 CTdel144 MT-4 SupT1

C) **** **** **** *** 80

60

40

20 % Env internalized

0

WT WT

CTdel144 CTdel144 MT-4 SupT1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.06.190801; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 Figure 11. and is also made available for use under a CC0 license.

A) Co-culture (cell-free + cell-to-cell) SupT1 MT-4 C8166 M8166 70 n.s. * 60 * 50 ** + 40 * 30 % eGFP 20 10 0 – – – – WT WT WT WT Env Env Env Env

B) Transwell (cell-free)

SupT1 MT-4 C8166 M8166 40 n.s. n.s. n.s. * 30 * +

20 n.s. % eGFP 10

0 – – – – WT WT WT WT Env Env Env Env

C) Cell-to-cell 50 n.s. * 40 *

+ * 30

20 % eGFP

10

0

MT-4 SupT1 C8166 M8166