Role of I182, R187 and K188 Amino Acids of the Catalytic Domain of HIV-1 Integrase in the Processes of Reverse Transcription and Integration

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Abstract

The structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of a subunit of the second dimer. It is the tetrameric structure that allows both ends of viral DNA to be correctly positioned relative to cellular DNA and to implement the catalytic functions of integrase, namely 3′-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of newly formed virions. HIV-1 integrase is proposed to be a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying the interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain I182, R187 and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for the formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in the formation of the integrase structure, which is important for effective reverse transcription.

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About the authors

T. F. Kikhai

Lomonosov Moscow State University

Author for correspondence.
Email: kih.t1996@yandex.ru
Russian Federation, Moscow

Yu. Yu. Agapkina

Lomonosov Moscow State University

Email: kih.t1996@yandex.ru
Russian Federation, Moscow

T. A. Prikazchikova

Lomonosov Moscow State University

Email: kih.t1996@yandex.ru
Russian Federation, Moscow

M. V. Vdovina

Lomonosov Moscow State University

Email: kih.t1996@yandex.ru
Russian Federation, Moscow

S. P. Shekhtman

Lomonosov Moscow State University

Email: kih.t1996@yandex.ru
Russian Federation, Moscow

S. V. Fomicheva

Lomonosov Moscow State University

Email: kih.t1996@yandex.ru
Russian Federation, Moscow

S. P. Korolev

Lomonosov Moscow State University

Email: kih.t1996@yandex.ru
Russian Federation, Moscow

M. B. Gottikh

Lomonosov Moscow State University

Email: gottikh@belozersky.msu.ru
Russian Federation, Moscow

References

  1. Wiskerchen, M., and Muesing, M. A. (1995) Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells, J. Virol., 69, 376-386, https://doi.org/10.1128/JVI.69.1.376-386.1995.
  2. Engelman, A. N., and Kvaratskhelia, M. (2022) Multimodal functionalities of HIV-1 integrase, Viruses, 14, 926, https://doi.org/10.3390/v14050926.
  3. Wu, X., Liu, H., Xiao, H., Conway, J. A., Hehl, E., Kalpana, G. V., Prasad, V., and Kappes, J. C. (1999) Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex, J. Virol., 73, 2126-2135, https://doi.org/10.1128/JVI.73.3. 2126-2135.1999.
  4. Engelman, A., Englund, G., Orenstein, J. M., Martin, M. A., and Craigie, R. (1995) Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication, J. Virol., 69, 2729-2736, https://doi.org/10.1128/JVI.69.5.2729-2736.1995.
  5. Leavitt, A. D., Robles, G., Alesandro, N., and Varmus, H. E. (1996) Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection, J. Virol., 70, 721-728, https://doi.org/10.1128/JVI.70.2.721-728.1996.
  6. Elliott, J., Eschbach, J. E., Koneru, P. C., Li, W., Puray-Chavez, M., Townsend, D., Lawson, D. Q., Engelman, A. N., Kvaratskhelia, M., and Kutluay, S. B. (2020) Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis, Elife, 9, e54311, https://doi.org/10.7554/eLife.54311.
  7. Kessl, J. J., Kutluay, S. B., Townsend, D., Rebensburg, S., Slaughter, A., Larue, R. C., Shkriabai, N., Bakouche, N., Fuchs, J. R., Bieniasz, P. D., and Kvaratskhelia, M. (2016) HIV-1 integrase binds the viral RNA genome and is essential during virion morphogenesis, Cell, 166, 1257-1268.e12, https://doi.org/10.1016/j.cell.2016.07.044.
  8. Briones, M. S., Dobard, C. W., and Chow, S. A. (2010) Role of human immunodeficiency virus type 1 integrase in uncoating of the viral core, J. Virol., 84, 5181-5190, https://doi.org/10.1128/jvi.02382-09.
  9. Rozina, A., Anisenko, A., Kikhai, T., Silkina, M., and Gottikh, M. (2022) Complex relationships between HIV-1 integrase and its cellular partners, Int. J. Mol. Sci., 23, 12341, https://doi.org/10.3390/ijms232012341.
  10. Knyazhanskaya, E., Anisenko, A., Shadrina, O., Kalinina, A., Zatsepin, T., Zalevsky, A., Mazurov, D., and Gottikh, M. (2019) NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase, Retrovirology, 16, 30, https://doi.org/10.1186/s12977-019-0492-z.
  11. Агапкина Ю., Приказчикова Т., Смолов М., Готтих М. (2005) Структура и функции интегразы ВИЧ-1, Успехи биологической химии, 45, 87-112.
  12. Hare, S., Di Nunzio, F., Labeja, A., Wang, J., Engelman, A., and Cherepanov, P. (2009) Structural basis for functional tetramerization of lentiviral integrase, PLoS Pathog., 5, e1000515, https://doi.org/10.1371/journal.ppat.1000515.
  13. Passos, D. O., Li, M., Yang, R., Rebensburg, S. V., Ghirlando, R., Jeon, Y., Shkriabai, N., Kvaratskhelia, M., Craigie, R., and Lyumkis, D. (2017) Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome, Science, 355, 89-92, https://doi.org/10.1126/science.aah5163.
  14. Ballandras-Colas, A., Maskell, D. P., Serrao, E., Locke, J., Swuec, P., Jónsson, S. R., Kotecha, A., Cook, N. J., Pye, V. E., Taylor, I. A., Andrésdóttir, V., Engelman, A. N., Costa, A., and Cherepanov, P. (2017) A supramolecular assembly mediates lentiviral DNA integration, Science, 355, 93-95, https://doi.org/10.1126/science.aah7002.
  15. Berthoux, L., Sebastian, S., Muesing, M. A., and Luban, J. (2007) The role of lysine 186 in HIV-1 integrase multimerization, Virology, 364, 227-236, https://doi.org/10.1016/j.virol.2007.02.029.
  16. Knyazhanskaya, E. S., Smolov, M. A., Kondrashina, O. V., and Gottikh, M. B. (2009) Relative comparison of catalytic characteristics of human foamy virus and HIV-1 integrases, Acta Naturae, 1, 78-80, https://doi.org/ 10.32607/20758251-2009-1-2-78-80.
  17. Mazurov, D., Ilinskaya, A., Heidecker, G., Lloyd, P., and Derse, D. (2010) Quantitative comparison of HTLV-1 and HIV-1 cell-to-cell infection with new replication dependent vectors, PLoS Pathog., 6, e1000788, https://doi.org/ 10.1371/journal.ppat.1000788.
  18. Vandergeeten, C., Fromentin, R., Merlini, E., Lawani, M. B., DaFonseca, S., Bakeman, W., McNulty, A., Ramgopal, M., Michael, N., Kim, J. H., Ananworanich, J., and Chomont, N. (2014) Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies, J. Virol., 88, 12385-12396, https://doi.org/10.1128/jvi.00609-14.
  19. McKee, C. J., Kessl, J. J., Shkriabai, N., Dar, M. J., Engelman, A., and Kvaratskhelia, M. (2008) Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein, J. Biol. Chem., 283, 31802-31812, https://doi.org/10.1074/jbc.M805843200.
  20. Shadrina, O. A., Zatsepin, T. S., Agapkina, Yu. Yu., Isaguliants, M. G., and Gottikh, M. B. (2015) Influence of drug resistance mutations on the activity of HIV-1 subtypes A and B integrases: a comparative study, Acta Naturae, 7, 78-86, https://doi.org/10.32607/20758251-2015-7-1-78-86.
  21. Takahata, T., Takeda, E., Tobiume, M., Tokunaga, K., Yokoyama, M., Huang, Y.-L., Hasegawa, A., Shioda, T., Sato, H., Kannagi, M., and Masuda, T. (2017) Critical contribution of Tyr15 in the HIV-1 integrase (IN) in facilitating IN assembly and nonenzymatic function through the IN precursor form with reverse transcriptase, J. Virol., https:// doi.org/10.1128/jvi.02003-16.
  22. Lu, R., Limón, A., Devroe, E., Silver, P. A., Cherepanov, P., and Engelman, A. (2004) Class II integrase mutants with changes in putative nuclear localization signals are primarily blocked at a postnuclear entry step of human immunodeficiency virus type 1 replication, J. Virol., 78, 12735-12746, https://doi.org/10.1128/jvi.78.23.12735-12746.2004.
  23. Elliott, J. L., and Kutluay, S. B. (2020) Going beyond integration: the emerging role of HIV-1 integrase in virion morphogenesis, Viruses, 12, 1005, https://doi.org/10.3390/v12091005.
  24. Gallay, P., Hope, T., Chin, D., and Trono, D. (1997) HIV-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway (nuclear import human immunodeficiency virus), Proc. Natl. Acad. Sci. USA, 94, 9825-9830, https://doi.org/10.1073/pnas.94.18.9825.
  25. Tsurutani, N., Kubo, M., Maeda, Y., Ohashi, T., Yamamoto, N., Kannagi, M., and Masuda, T. (2000) Identification of critical amino acid residues in human immunodeficiency virus type 1 IN required for efficient proviral DNA Formation at steps prior to integration in dividing and nondividing cells the multiple effects of in mutations suggest that, J. Virol., 74, 4795-4806, https://doi.org/10.1128/jvi.74.10.4795-4806.2000.
  26. De Houwer, S., Demeulemeester, J., Thys, W., Taltynov, O., Zmajkovicova, K., Christ, F., and Debyser, Z. (2012) Identification of residues in the C-terminal domain of HIV-1 integrase that mediate binding to the transportin-SR2 protein, J. Biol. Chem., 287, 34059-34068, https://doi.org/10.1074/jbc.M112.387944.
  27. Liat Shimoni, H., and Glusker, J. P. (1995) Hydrogen bonding motifs of protein side chains: descriptions of binding of arginine and amide groups, Protein Sci., 4, 65-74, https://doi.org/10.1002/pro.5560040109.

Supplementary files

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2. Fig. 1. Relative luminescence level of firefly luciferase in cells transduced with pseudovirus with wild-type IN (IN_wt) and its mutant variants: IN_I182A, IN_R187A and IN_K188A. The luciferase signal was measured 24 h after transduction, and the results were normalised to the luciferase expression of pseudovirus with IN_wt. The mean values of three independent experiments are presented. Significance was determined by two-way analysis of variance with Shidak's correction for multiple comparisons; ** p < 0.01; *** p < 0.001

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3. Fig. 2. Relative amounts of total viral cDNA (a) and integrated viral cDNA (b) after transduction of HEK 293T cells with IN_wt pseudoviruses and mutant proteins. The mean values of three independent experiments are presented. Significance was determined by two-way analysis of variance with Shidak's correction for multiple comparisons; *** p < 0.001; ns - not significant

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4. Fig. 3. Electrophoretic analysis of the catalytic activity of IN_wt and mutants IN_I182A, IN_R187A, and IN_K188A in 3′-processing (a) and chain transfer (b) reactions. a, U5B/U5A duplex without IN (lane 1); in the presence of IN_wt (lanes 2, 3); in the presence of IN_K188A (lanes 4, 5); in the presence of IN_I182A (lanes 6-9); in the presence of IN_R187A (lanes 10-13). The reaction was performed with 5 nM DNA duplex and increasing concentrations of IN: 100 nM (lanes 2, 4, 6, 10); 200 nM (lanes 3, 5, 7, 11); 400 nM (lanes 8, 12) and 800 nM (lanes 9, 13). b - U5B-2/U5A duplex without IN (lane 1); in the presence of IN_wt (lanes 2, 3); in the presence of IN_K182A (lanes 4, 5); in the presence of IN_I187A (lanes 6, 7); and in the presence of IN_R188A (lanes 8, 9). The reaction was performed with 10 nM DNA duplex and IN concentrations of 100 nM (lanes 2, 4, 6, 8) and 200 nM (lanes 3, 5, 7, 9)

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5. Fig. 4. Effect of amino acid substitutions I182A, R187A and K188A in IN on the efficiency of its binding to DNA-substrate and to TAR-RNA. a - Binding curves of IN to the U5B/U5A DNA duplex obtained by in-gel inhibition. 5 nM DNA-substrate was incubated with IN at different concentrations: 0, 25, 50, 100, 200 and 500 nM. b - Binding curves of IN with TAR-RNA obtained by in-gel inhibition. 5 nM TAR-RNA was incubated with IN at different concentrations: 0, 25, 50, 100, 200, 500 nM

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6. Fig. 5. Analysis of the interaction of OT with wild-type IN (IN_wt) and with the I182A, R187A, and K188A substitutions containing the N-terminal GST-tag by co-precipitation on glutathione-Sepharose. OT in the absence of IN (lane 1); OT in the presence of IN_wt (lane 2); OT in the presence of IN_I182A (lane 3); OT in the presence of IN_R187A (lane 4); OT in the presence of IN_K188A (lane 5). Incubation of IN and OT at a concentration of 250 nM was performed for 30 min at 4 °C

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7. Fig. 6. Subunit exchange efficiency analysis for IN_wt and with I182A, R187A and K188A substitutions. 100 nM GST-IN was incubated for 2 h at room temperature with proteins containing His6-tag: IN_wt (lanes 1, 2), IN_K182A (lanes 3, 4), IN_I187A (lanes 5, 6), IN_R188A (lanes 7, 8). His6-tagged protein concentrations: 100 nM (lanes 1, 3, 5, 7) and 200 nM (lanes 2, 4, 6, 8). Lane 9 - control without GST-IN

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8. Fig. 7. Location of amino acids I182, R187 and K188 in the structure of IN (according to Passos et al. [13] (PDB 5U1C)). a - Structure of HIV-1 IN tetramer. Individual subunits of IN are marked in different colours. b - The location of amino acids I182, R187 and K188 (highlighted in bright yellow) of the catalytic domain of one subunit (highlighted in green) and the N-terminal domain of the other subunit (highlighted in turquoise) are marked with a rectangle and enlarged. c-d - The environment of amino acids I182, R187 and K188, respectively

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