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Differential Effects of Strategies to Improve the Transduction Efficiency of Lentiviral Vector that Conveys an Anti-HIV Protein, Nullbasic, in Human T Cells

Virologica Sinica volume 33, pages 142–152 (2018)Cite this article

Abstract

Nullbasic is a mutant form of HIV-1 Tat that has strong ability to protect cells from HIV-1 replication by inhibiting three different steps of viral replication: reverse transcription, Rev export of viral mRNA from the nucleus to the cytoplasm and transcription of viral mRNA by RNA polymerase II. We previously showed that Nullbasic inhibits transduction of human cells including T cells by HIV-1-based lentiviral vectors. Here we investigated whether the Nullbasic antagonists huTat2 (a Tat targeting intrabody), HIV-1 Tat or Rev proteins or cellular DDX1 protein could improve transduction by a HIV-1 lentiviral vector conveying Nullbasic-ZsGreen1 to human T cells. We show that overexpression of huTat2, Tat-FLAG and DDX1-HA in virus-like particle (VLP) producer cells significantly improved transduction efficiency of VLPs that convey Nullbasic in Jurkat cells. Specifically, co-expression of Tat-FLAG and DDX1-HA in the VLP producer cell improved transduction efficiency better than if used individually. Transduction efficiencies could be further improved by including a spinoculation step. However, the same optimised protocol and using the same VLPs failed to transduce primary human CD4+ T cells. The results imply that the effects of Nullbasic on VLPs on early HIV-1 replication are robust in human CD4+ T cells. Given this significant block to lentiviral vector transduction by Nullbasic in primary CD4+ T cells, our data indicate that gammaretroviral, but not lentiviral, vectors are suitable for delivering Nullbasic to primary human T cells.

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References

  • Abbud RA, Finegan CK, Guay LA, Rich EA (1995) Enhanced production of human immunodeficiency virus type 1 by in vitro- infected alveolar macrophages from otherwise healthy cigarette smokers. J Infect Dis 172:859–863

    Article  CAS  PubMed  Google Scholar 

  • Ahn J (2016) Functional organization of human SAMHD1 and mechanisms of HIV-1 restriction. Biol Chem 397:373–379

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Apolloni A, Meredith LW, Suhrbier A, Kiernan R, Harrich D (2007) The HIV-1 Tat protein stimulates reverse transcription in vitro. Curr HIV Res 5:473–483

    Article  PubMed  Google Scholar 

  • Apolloni A, Lin MH, Sivakumaran H, Li D, Kershaw MH, Harrich D (2013) A mutant Tat protein provides strong protection from HIV-1 infection in human CD4+ T cells. Hum Gene Ther 24:270–282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bauer G, Anderson JS (2014) Gene therapy vectors. In: Gene therapy for HIV. Springer, pp 27–33

  • Braun SE, Taube R, Zhu Q, Wong FE, Murakami A, Kamau E, Dwyer M, Qiu G, Daigle J, Carville A, Johnson RP, Marasco WA (2012) In vivo selection of CD4(+) T cells transduced with a gamma-retroviral vector expressing a single-chain intrabody targeting HIV-1 tat. Hum Gene Ther 23:917–931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Connor RI, Chen BK, Choe S, Landau NR (1995) Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology 206:935–944

    Article  CAS  PubMed  Google Scholar 

  • Fang J, Kubota S, Yang B, Zhou N, Zhang H, Godbout R, Pomerantz RJ (2004) A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev. Virology 330:471–480

    Article  CAS  PubMed  Google Scholar 

  • Forestell SP, Dando JS, Bohnlein E, Rigg RJ (1996) Improved detection of replication-competent retrovirus. J Virol Methods 60:171–178

    Article  CAS  PubMed  Google Scholar 

  • Guo J, Wang W, Yu D, Wu Y (2011) Spinoculation triggers dynamic actin and cofilin activity that facilitates HIV-1 infection of transformed and resting CD4 T cells. J Virol 85:9824–9833

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harrich D, Ulich C, Garcia-Martinez LF, Gaynor RB (1997) Tat is required for efficient HIV-1 reverse transcription. EMBO J 16:1224–1235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • He J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR (1995) Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 69:6705–6711

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hulme AE, Perez O, Hope TJ (2011) Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription. Proc Natl Acad Sci USA 108:9975–9980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jin H, Li D, Sivakumaran H, Lor M, Rustanti L, Cloonan N, Wani S, Harrich D (2016) Shutdown of HIV-1 transcription in T cells by nullbasic, a mutant Tat protein. MBio 7:e00518-16

    Article  PubMed  PubMed Central  Google Scholar 

  • Kamori D, Ueno T (2017) HIV-1 tat and viral latency: what we can learn from naturally occurring sequence variations. Front Microbiol 8:80

    Article  PubMed  PubMed Central  Google Scholar 

  • Kang W, Marasco WA, Tong HI, Byron MM, Wu C, Shi Y, Sun S, Sun Y, Lu Y (2014) Anti-tat Hutat2: Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages. J Neuroinflammation 11:195

    Article  PubMed  PubMed Central  Google Scholar 

  • Knight S, Zhang F, Mueller-Kuller U, Bokhoven M, Gupta A, Broughton T, Sha S, Antoniou MN, Brendel C, Grez M, Thrasher AJ, Collins M, Takeuchi Y (2012) Safer, silencing-resistant lentiviral vectors: optimization of the ubiquitous chromatin-opening element through elimination of aberrant splicing. J Virol 86:9088–9095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lin MH, Sivakumaran H, Apolloni A, Wei T, Jans DA, Harrich D (2012) Nullbasic, a potent anti-HIV tat mutant, induces CRM1-dependent disruption of HIV Rev trafficking. PLoS ONE 7:e51466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lin MH, Sivakumaran H, Jones A, Li D, Harper C, Wei T, Jin H, Rustanti L, Meunier FA, Spann K, Harrich D (2014) A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1. Retrovirology 11:121

    Article  PubMed  PubMed Central  Google Scholar 

  • Lin MH, Apolloni A, Cutillas V, Sivakumaran H, Martin S, Li D, Wei T, Wang R, Jin H, Spann K, Harrich D (2015) A mutant tat protein inhibits HIV-1 reverse transcription by targeting the reverse transcription complex. J Virol 89:4827–4836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marasco WA, LaVecchio J, Winkler A (1999) Human anti-HIV-1 Tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS. J Immunol Methods 231:223–238

    Article  CAS  PubMed  Google Scholar 

  • Meredith LW, Sivakumaran H, Major L, Suhrbier A, Harrich D (2009) Potent inhibition of HIV-1 replication by a Tat mutant. PLoS ONE 4:e7769

    Article  PubMed  PubMed Central  Google Scholar 

  • Mhashilkar AM, Bagley J, Chen S, Szilvay A, Helland D, Marasco W (1995a) Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies. EMBO J 14:1542

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mhashilkar AM, Bagley J, Chen SY, Szilvay AM, Helland DG, Marasco WA (1995b) Inhibition of HIV-1 Tat-mediated LTR transactivation and HIV-1 infection by anti-Tat single chain intrabodies. EMBO J 14:1542–1551

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mhashilkar AM, LaVecchio J, Eberhardt B, Porter-Brooks J, Boisot S, Dove JH, Pumphrey C, Li X, Weissmahr RN, Ring DB, Ramstedt U, Marasco WA (1999) Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4+ mononuclear cells expressing murine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies [see comments]. Hum Gene Ther 10:1453–1467

    Article  CAS  PubMed  Google Scholar 

  • Naldini L, Verma IM (2000) Lentiviral vectors. Adv Virus Res 55:599–609

    Article  CAS  PubMed  Google Scholar 

  • Okitsu T, Kobayashi N, Totsugawa T, Maruyama M, Noguchi H, Watanabe T, Matsumura T, Fujiwara T, Tanaka N (2003) Lentiviral vector mediated gene delivery into non-dividing isolated islet cells. Transplant Proc 35:483

    Article  CAS  PubMed  Google Scholar 

  • Pearson L, Garcia J, Wu F, Modesti N, Nelson J, Gaynor R (1990) A transdominant tat mutant that inhibits tat-induced gene expression from the human immunodeficiency virus long terminal repeat. Proc Natl Acad Sci USA 87:5079–5083

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Robertson-Anderson RM, Wang J, Edgcomb SP, Carmel AB, Williamson JR, Millar DP (2011) Single-molecule studies reveal that DEAD box protein DDX1 promotes oligomerization of HIV-1 Rev on the Rev response element. J Mol Biol 410:959–971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ruffin N, Brezar V, Ayinde D, Lefebvre C, Schulze Zur Wiesch J, van Lunzen J, Bockhorn M, Schwartz O, Hocini H, Lelievre JD, Banchereau J, Levy Y, Seddiki N (2015) Low SAMHD1 expression following T-cell activation and proliferation renders CD4+ T cells susceptible to HIV-1. AIDS 29:519–530

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ryan MD, King AM, Thomas GP (1991) Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen Virol 72:2727–2732

    Article  CAS  PubMed  Google Scholar 

  • Schambach A, Zychlinski D, Ehrnstroem B, Baum C (2013) Biosafety features of lentiviral vectors. Hum Gene Ther 24:132–142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taylor MP, Koyuncu OO, Enquist LW (2011) Subversion of the actin cytoskeleton during viral infection. Nat Rev Microbiol 9:427–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ulich C, Harrich D, Estes P, Gaynor RB (1996) Inhibition of human immunodeficiency virus type 1 replication is enhanced by a combination of transdominant Tat and Rev proteins. J Virol 70:4871–4876

    CAS  PubMed  PubMed Central  Google Scholar 

  • Varmus HE (1982) Form and function of retroviral proviruses. Science 216:812–820

    Article  CAS  PubMed  Google Scholar 

  • Varmus H (1988) Retroviruses. Science 240:1427–1435

    Article  CAS  PubMed  Google Scholar 

  • Zhou M, Deng L, Kashanchi F, Brady JN, Shatkin AJ, Kumar A (2003) The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA. Proc Natl Acad Sci USA 100:12666–12671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zufferey R, Donello JE, Trono D, Hope TJ (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73:2886–2892

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by the National Health and Medical Research Council Project Grant (1085359). LR was supported by Prime Minister’s Australia Asia Endeavour Postgraduate (Ph.D.) Award funded by the Australian Government, Department of Education and Training, UQ international scholarship (UQI) and UQ Centenial scholarship (UQCent). We thank the QIMR Berghofer Flow Cytometry and Imaging Facility for technical expertise with cell sorting and flow cytometry. We thank Wayne Marasco for providing the sFvhutat2 expression plasmid for this study.

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Authors and Affiliations

  1. Faculty of Medicine, The University of Queensland, Herston, QLD, 4029, Australia

    Lina Rustanti

  2. Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, QLD, 4029, Australia

    Lina Rustanti, Hongping Jin, Dongsheng Li, Mary Lor, Haran Sivakumaran & David Harrich

  3. National Institute of Health Research and Development, the Ministry of Health of Republic of Indonesia, Central Jakarta, DKI Jakarta, 10560, Indonesia

    Lina Rustanti

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  1. Lina Rustanti
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  2. Hongping Jin
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  3. Dongsheng Li
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  4. Mary Lor
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  5. Haran Sivakumaran
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  6. David Harrich
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Contributions

DH and LR originated and guided the study. DL, DH, LR and HJ designed the experiments. LR, ML and HJ performed experiments. LR, DH, HJ and DL wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to David Harrich.

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The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

“Buffy coat” human blood cells were obtained from the Australian Red Cross. All samples were provided by donors with informed consent.

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Rustanti, L., Jin, H., Li, D. et al. Differential Effects of Strategies to Improve the Transduction Efficiency of Lentiviral Vector that Conveys an Anti-HIV Protein, Nullbasic, in Human T Cells. Virol. Sin. 33, 142–152 (2018). https://doi.org/10.1007/s12250-018-0004-7

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Keywords

  • HIV-1
  • Transduction
  • Lentiviral vector
  • Nullbasic
  • Tat
  • Rev
  • DDX1
  • Intrabody