Cell Host & Microbe, Volume 14 Supplemental Information HIV-1 Induces the Formation of Stable Microtubules to Enhance Early Infection Yosef Sabo, Derek Walsh, Denis S. Barry, Sedef Tinaztepe, Kenia de los Santos, Stephen P. Goff, Gregg G. Gundersen, and Mojgan H. Naghavi
A. Supplemental Data Supplemental Figure Legends Figure S1, related to Figure 1. HIV-1 infection induces stable MT formation in human cells. (A) 293A cells were infected with either mock virus preparation (mock) or HIV-1-VSV at m.o.i. 3. Mock infected or HIV-1-VSV infected cells were fixed at the indicated h.p.i. and stained using anti-tyr-tubulin and anti-ac-tubulin antibodies. Scale bar, 10µm. (B-E) Quantification of the fluorescence signal intensity from IF images in Figure 1 and S1A; (B) HIV-1 infected U87.CD4.CCR5 (from Figure 1A), (C) NHDFs (from Figure 1B), (D) 293A (from S1A), or (E) CHME3 cells (from Figure 1C) normalized to that of uninfected cells. n 250 cells. (F) Quantification of the number of HIV-1 infected primary human macrophages (from Figure 1D) containing increased levels of AC-MTs. n 100 cells. (G-K) Heat-inactivated and nonenveloped viruses do not increase stable MT levels. 293A cells were infected with either mock virus preparation (mock), heat inactivated HIV-1-VSV (H.I.), HIV-1 carrying no envelope glycoproteins (No Env) or HIV-1-VSV. (G) Cells infected with heat-inactivated virus (H.I.) or HIV-1-VSV were fixed at the indicated h.p.i. and stained using anti-tyr-tubulin and anti-ac-tubulin antibodies. (H) Quantification of the fluorescence signal intensity in samples shown in G. normalized to that of the uninfected cells. n>250 cells. (I) Cells were infected with mock virus preparation (mock), No Env virus or HIV-1-VSV and processed as described in G. (J) Quantification of the fluorescence signal intensity in samples shown in I. normalized to that of the uninfected cells. n>250 cells. Scale bar, 10 µm. (K) WB analysis of HIV-1 Gag polyprotein p55 and capsid p24 levels using anti- HIV-1 p24 antibody in stock preparations of mock virus, No Env virus or HIV-1-VSV. This illustrates that viral particles lacking envelope were present in virus preparations used to infect
samples in C. and D. (L-N) HIV-1 infection increases stable Glu-MT levels in human cells. (L) 293A cells infected with mock virus preparation (mock) or HIV-1-VSV were fixed at the indicated h.p.i. then stained using anti-tyrosinated tubulin (Tyr-MTs) and anti-detyrosinated tubulin (Glu-MTs) antibodies. Scale bar, 10 µm. (M-N) WB analysis of the levels of acetylated (top panels) or detyrosinated (middle panels) MTs in mock infected and infected 293A (M) and CHME3 (N) cells using anti-acetylated (AC-MTs) and anti-detyrosinated (Glu-MTs) tubulin antibodies, respectively. eif4e (lower panels) was used as loading control. (O-T) HIV-1 infection increases stable MT levels in human cells regardless of the route of viral entry. 293A cells (O and R), CHME3 cells (P and S) or primary NHDFs (Q and T) were infected with mock virus preparation (mock) or HIV-1 carrying a MuLV amphotropic (HIV-1-Ampho) envelope. (O-Q) Cells were fixed at the indicated h.p.i. then stained using antityrosinated tubulin (Tyr-MTs) and anti-acelytated tubulin (AC-MTs) antibodies. Scale bar, 10 µm. (R-T) Quantification of the fluorescence signal intensity in samples in A- C normalized to that of uninfected cells. n>250 cells. Figure S2, related to Figure 4. EB1 is required for HIV-1 mediated MT stabilization. Quantification of the fluorescence (Alexafluor 488) signal intensity in IF images of mock infected and HIV-1-VSV infected cells treated with control GFP or EB1 sirnas (shown in Figures 4A and 4B, respectively) normalized to that of uninfected cells. n 250 cells. Data are represented as mean +/- SEM. Figure S3, related to Figure 6. EB1 depletion suppresses AC-MT induction by HIV-1. NHDFs were treated with control GFP or EB1 sirnas then infected with HIV-1-VSV-GFP-Vpr at m.o.i. 1 for 1h, 2h or 4h. Fixed samples were stained for (A)
Try-MTs (red) and EB1 (green) or (B) AC-MTs (green) and Tyr-MTs (red). Nuclei were stained with DAPI. Representative images are shown. Scale bar, 10µm. Figure S4, related to Figure 7. Expression of retroviral MA induces MT acetylation in human cells. (A) NHDF cells were transfected with control pcdna plasmid or pcdna plasmids encoding HA-tagged forms of HIV-1 MA or SIV MA. Whole cell extracts were analyzed by WB using anti-ha (to detect MA) and antiacelytated tubulin (AC-MTs) antibodies. eif4e served as loading control. (B) Kif4 is required for HIV-1-induced MT stabilization. CHME3 cells were transfected with control or Kif4 sirnas then infected with HIV-1-VSV for 6h. Fixed samples were stained for AC-MTs (green) and Tyr-MTs (red). Nuclei were stained with Hoechst. B. Supplemental Experimental Procedures Generation of viral vectors and expression constructs pnl4-3.zsgreen.r -.E - was created by replacing the luciferase gene in the pnl4-3.luc.r -.E - (AIDS Reagent Repository number 3418) (Connor et al., 1995) with the ZsGreen gene. Briefly, the ZsGreen ORF was amplified from plvx-zsgreen-n1 (Clontech) using primers 1 and 11 in Table S1 and ligated into pjet1.2. pjet1.2- zsgreen and pnl4-3.luc.r -.E - were digested with NotI and XhoI and the ZsGreen fragment was ligated into pnl4-3 to create pnl4-3.zsgreen.r -.E -. To generate HIV- 1 carrying a luciferase reporter or ZsGreen marker, pnl4-3.luc.r -.E - or pnl4-3.zsgreen.r -.E - were transfected into 293T cells together with plasmids expressing VSV-G (PMDG), MuLV amphotropic (phit456) or HIV-1 CCR5 tropic (pci-env) envelope glycoprotein (Naldini et al., 1996; Pugach et al., 2007; Soneoka et al.,
1995). Pseudotyped HIV-1-zsGreen virus was quantified as described (Sabo et al. 2011). Expression constructs encoding C-terminally HA-tagged HIV-1 Gag and MA were generated by PCR amplification from Rev-independent pgag-egfp (Hermida- Matsumoto and Resh, 2000), using primers 2 and 12 or 2 and 13 in Table S1, respectively, cloned into pcdna3.1-. The pcdna3.1 Gag-HA construct was then used as PCR template to generate constructs lacking 20 amino acids at the C terminus (-20) or the N terminus (-100) in the MA domain using the primers indicated in Table S1 (primers 8, 18 and 9,19, respectively). PCR products were sequenced and used to replace the wt sequence using the AgeI and XbaI restriction sites. Gag-HA with a single point mutation (N-Myr) MA G1A was created by annealing primers 7 and 17 (Table S1) and replacing the wt MA sequence using ClaI and XbaI in the pcdna3.1gag-ha construct. C-terminally HA-tagged SIV mac239 MA was generated by PCR amplification from VI-SIVmc(FL) (Zhang et al., 2009) plasmid, using primers 10 and 20 (Table S1), cloned into pcdna3.1- using XbaI and EcoRI. C-terminal and Full-length C-terminally Flag-tagged EB1 was amplified using cdna from 293T cells as template using the primers 4 and 10 in Table S1. This construct was then used as template to produce N-terminally truncated EB1 containing a C terminal Flag-tag using the primers 5 and 10 in Table S1. The restriction enzyme sites are in italics, and the Flag peptide sequence is underlined. EB1-Flag (full-length EB1) or EB1-C-Flag (truncated EB1) PCR products were then cloned into a MuLV based retroviral vector pqcxin (Clontech), and the inserts were confirmed by sequencing. VSV-G pseudotyped viral vectors (MuLV-VSV-neo) encoding EB1-Flag and EB1-C- Flag were produced by co-transfection of HEK239T cells with these pqcxin
plasmids together with MoMuLV Gag-Pol expressing vector (pcmvinteron) and VSV-G (PMDG) constructs (Naldini et al., 1996). HIV-1 fusion measurement Cells were infected for 2h with ZsGreen-HIV-1-VSV containing the BlaM-Vpr fusion protein (Addgene plasmid 21950; (Cavrois et al., 2002)) then washed with CO 2 independent medium and loaded with the CCF2/AM substrate together with 2.5mM probenecid for 2h according to the manufacturers instructions (Invitrogen). Samples were washed and incubated with CO 2 independent medium supplemented with 10% FBS for 16h at RT then washed with PBS and fixed with 1.2% paraformaldehyde overnight. CCF2 cleavage was monitored using a five-laser BD LSRII (Becton Dickinson, San Jose, CA). C. Supplemental References Cavrois, M., De Noronha, C., and Greene, W. C. (2002). A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20, 1151-1154. Connor, R. I., Chen, B. K., Choe, S., and Landau, N. R. (1995). Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206, 935-944. Hermida-Matsumoto, L., and Resh, M. D. (2000). Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging. J Virol 74, 8670-8679.
Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-267. Pugach, P., Marozsan, A. J., Ketas, T. J., Landes, E. L., Moore, J. P., and Kuhmann, S. E. (2007). HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry. Virology 361, 212-228. Sabo, Y., Ehrlich, M., and Bacharach, E. (2011). The conserved YAGL motif in human metapneumovirus is required for higher-order cellular assemblies of the matrix protein and for virion production. Journal of virology 85, 6594-6609. Soneoka, Y., Cannon, P. M., Ramsdale, E. E., Griffiths, J. C., Romano, G., Kingsman, S. M., and Kingsman, A. J. (1995). A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res 23, 628-633. Zhang, F., Wilson, S. J., Landford, W. C., Virgen, B., Gregory, D., Johnson, M. C., Munch, J., Kirchhoff, F., Bieniasz, P. D., and Hatziioannou, T. (2009). Nef proteins from simian immunodeficiency viruses are tetherin antagonists. Cell Host Microbe 6, 54-67.
Fold change intensity normalized to N.I. 10 8 6 4 2 GFP Mock GFP HIV-1 EB1 Mock EB1 HIV-1 H.P.I. 2 6
Table S1. Primer sequences Forward Primers Number and Name Sequence (5' 3') * 1. ZsGreen ORF-S ACGAATAGCGCGGCCGCATGGCCCAGTCCAAGCACGGCCTGACC 2. XbaI-Kozak-Gag/MA GAGCGTCTAGAACCATGGGTGCGAGAGCGTCAGTA 3. heb1-s CTGTATGCCACAGATGAAGG 4. heb1-s-noti GCAACTGCGGCCGCCATGGCAGTGAACGTATACTCAAC 5. heb1-c-s-noti GCAACTGCGGCCGCCATGTCAACACAGAGAACCGCTGC 6. MA fw TCTAGAACCATGGGTGCGAGAGCGTCAGTATTAAGCGGG 7. MA no myr fw CTAGAACCATGGCTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGAT 8. MA del 20c fw GATAGAGGAAGAGCAAAACAAGTCCAAGAAGCCTATAGTGCAGAACATCCAGGGGC 9. MA del100c fw GGGAAAGAAGAAGTACAAGCTAAAGCACATCGAAGGCTGTAGACAAATACTGGGACAG 10. SIV MA fw CTCTAGAACCATGGGCGTGAGAAACTCCGTCTTGTCAGGG Reverse Primers 11. ZsGreen ORF-A AAACTCGAGTTAGGGCAAGGCGGAGCCGGAGG 12. Gag-HA-TAA-EcoRI TTGCCGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTATTGTGACGAGGGGTCGTTG 13. MA-HA-TAA-EcoRI TTCTGGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTAGTAATTTTGGCTGACCTG 14. heb1-a CCAGACACAATGTCAAACGC 15. heb1-a-flag-bamhi GCAACGGGATCCTTACTTGTCGTCATCGTCTTTGTAGTCATACTCTTCTTGCTCCTCCTG 16. MA rev ACCGGTCTACATAGTCTCTAAAGGGTTCTTTTGGTCC 17. MA no myr rev CGATCTAATTCTCCCCCGCTTAATACTGACGCTCTCGCAGCCATGGTT 18. MA del 20c rev GCCCCTGGATGTTCTGCACTATAGGCTTCTTGGACTTGTTTTGCTCTTCCTCTATC 19. MA del100 rev CTGTCCCAGTATTTGTCTACAGCCTTCGATGTGCTTTAGCTTGTACTTCTTCTTTCCC 20. SIV MA-HA rev GGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTAGTAATTTCCTCCTCTGCCGC *: Restriction sites are shown in bold; tag sequences (HA or Flag) are underlined.