Abstract
HIV-1 evades host immunity via a number of virally encoded mediators. Here we show that the HIV-1 antisense protein (ASP) evades host immunity by interfering with the type I interferon (IFN-I) response. Mechanistically, host prolyl hydroxylase 3 (PHD3) hydroxylates ASP at Pro47, enabling the recruitment of the RING finger protein 114 (RNF114) to TANK-binding kinase 1 (TBK1). Subsequently, RNF114 mediates K6-linked ubiquitination of TBK1 at Lys236, suppressing TBK1 activation and the downstream IFN-I response. Conversely, mutation of ASP at Pro47 abolishes this inhibitory effect. In humanized mice, either ASP deletion or treatment with the RNF114 inhibitor EN219 or the PHD3 inhibitor Molidustat enhances antiviral immunity and reduces viral replication. Clinically, RNF114 and PHD3 transcript levels exhibit a positive correlation with viral load in treatment-naive patients. Here we show a distinct HIV-1 immune evasion mechanism involving proline hydroxylation and K6-linked ubiquitination, highlighting therapeutic potential.
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Data availability
The RNA-seq data generated in this study have been deposited in the Sequence Read Archive (SRA) database under accession codes PRJNA1417155 and PRJNA1417156. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD073921. All newly generated materials in this study are available from the corresponding author upon request. All data necessary for confirming the conclusions of the article are present within the article or the Supplementary information. Source data are provided in this paper.
References
Gallo, R. C. HIV/AIDS Research for the future. Cell Host Microbe 27, 499–501 (2020).
Siddiqui, M. A. & Yamashita, M. Toll-like receptor (TLR) signaling enables cyclic GMP-AMP synthase (cGAS) sensing of HIV-1 infection in macrophages. mBio 12, e0281721 (2021).
Cai, B., Wu, J., Yu, X., Su, X. Z. & Wang, R. F. FOSL1 inhibits type I interferon responses to malaria and viral infections by blocking TBK1 and TRAF3/TRIF interactions. mBio 8, https://doi.org/10.1128/mbio.02161-16 (2017).
Hopfner, K. P. & Hornung, V. Molecular mechanisms and cellular functions of cGAS-STING signalling. Nat. Rev. Mol. Cell Biol. 21, 501–521 (2020).
Zhang, X., Bai, X. -c & Chen, Z. J. Structures and mechanisms in the cGAS-STING innate immunity pathway. Immunity 53, 43–53 (2020).
Gondim, M. V. P. et al. Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption. Sci. Transl. Med. 13, https://doi.org/10.1126/scitranslmed.abd8179 (2021).
Liu, H. et al. HIV infection suppresses TLR3 activation-mediated antiviral immunity in microglia and macrophages. Immunology 160, 269–279 (2020).
Evans, D. T. et al. N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages. PLOS Pathog. 17, e1009421 (2021).
Nevels, M. et al. Disruption of type I interferon induction by HIV infection of T cells. Plos ONE 10, e0137951 (2015).
Dhamanage, A., Thakar, M. & Paranjape, R. Human immunodeficiency virus-1 Impairs IFN-Alpha Production Induced by TLR-7 Agonist in Plasmacytoid Dendritic Cells. Viral Immunol. 30, 28–34 (2017).
Harman, A. N. et al. HIV blocks interferon induction in human dendritic cells and macrophages by dysregulation of TBK1. J. Virol. 89, 6575–6584 (2015).
Nan, Y., Wu, C. & Zhang, Y.-J. Interplay between janus kinase/signal transducer and activator of transcription signaling activated by type I interferons and viral antagonism. Front. Immunol. 8, https://doi.org/10.3389/fimmu.2017.01758 (2017).
Savoret, J., Mesnard, J. M., Gross, A. & Chazal, N. Antisense transcripts and antisense protein: a new perspective on human immunodeficiency virus type 1. Front. Microbiol. 11, 625941 (2020).
Affram, Y. et al. The HIV-1 antisense protein ASP is a transmembrane protein of the cell surface and an integral protein of the viral envelope. J. Virol. 93, https://doi.org/10.1128/jvi.00574-19 (2019).
Bet, A. et al. The HIV-1 antisense protein (ASP) induces CD8 T cell responses during chronic infection. Retrovirology 12, https://doi.org/10.1186/s12977-015-0135-y (2015).
Pavesi, A. & Romerio, F. Creation of the HIV-1 antisense gene asp coincided with the emergence of the pandemic group M and is associated with faster disease progression. Microbiol. Spectr. 12, e0380223 (2024).
Gholizadeh, Z., Iqbal, M. S., Li, R. & Romerio, F. The HIV-1 antisense gene ASP: the new kid on the block. Vaccines 9, https://doi.org/10.3390/vaccines9050513 (2021).
Zapata, J. C. et al. The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly. Virology 506, 34–44 (2017).
Abe, T. & Barber, G. N. Cytosolic-DNA-mediated, STING-dependent proinflammatory gene induction necessitates canonical NF-kappaB activation through TBK1. J. Virol. 88, 5328–5341 (2014).
Gao, D. et al. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science 341, 903–906 (2013).
Izumida, M., Togawa, K., Hayashi, H., Matsuyama, T. & Kubo, Y. Production of vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vector is enhanced by ezrin silencing. Front. Bioeng. Biotechnol. 8, https://doi.org/10.3389/fbioe.2020.00368 (2020).
Clerc, I. et al. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells. Retrovirology 8, https://doi.org/10.1186/1742-4690-8-74 (2011).
Hesselman, M. C. et al. Rare twin cysteine residues in the HIV-1 envelope variable region 1 link to neutralization escape and breadth development. Cell Host Microbe 33, 279–293 (2025).
Caetano, D. G. et al. Patterns of diversity and humoral immunogenicity for HIV-1 antisense protein (ASP). Vaccines 12, 771 (2024).
Larabi, A. et al. Crystal structure and mechanism of activation of TANK-binding kinase 1. Cell Rep. 3, 734–746 (2013).
Song, G. et al. E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1. Nat. Immunol. 17, 1342–1351 (2016).
Michel, M. A., Swatek, K. N., Hospenthal, M. K. & Komander, D. Ubiquitin linkage-specific affimers reveal insights into K6-linked ubiquitin signaling. Mol. Cell 68, 233–246 (2017).
Lee, S. B. et al. Proline hydroxylation primes protein kinases for autophosphorylation and activation. Mol. Cell 79, 376–389 (2020).
Semenza, G. L. HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell 107, 1–3 (2001).
Rajashekar, J. K. et al. Modulating HIV-1 envelope glycoprotein conformation to decrease the HIV-1 reservoir. Cell Host Microbe 29, 904–916 (2021).
Vabret, N. et al. Y RNAs are conserved endogenous RIG-I ligands across RNA virus infection and are targeted by HIV-1. iScience 25, 104599 (2022).
Yin, X. et al. Sensor sensibility—HIV-1 and the innate immune response. Cells 9, https://doi.org/10.3390/cells9010254 (2020).
Fonseca, D., Pisanelli, G., Seoane, R., Miorin, L. & García-Sastre, A. TRIM65 regulates innate immune signaling by enhancing K6-linked ubiquitination of IRF3 and its chromatin recruitment. Cell Rep. 43, 114960 (2024).
Yang, B. et al. RNF144A promotes antiviral responses by modulating STING ubiquitination. EMBO Rep.24, https://doi.org/10.15252/embr.202357528 (2023).
Guo, J. et al. A genome-wide base-editing screen uncovers a pivotal role of paxillin δ ubiquitination in influenza virus infection. Cell Rep. 44, 115748 (2025).
Strowitzki, M., Cummins, E. & Taylor, C. Protein hydroxylation by hypoxia-inducible factor (HIF) hydroxylases: unique or ubiquitous? Cells 8, 384 (2019).
Duette, G. et al. Induction of HIF-1α by HIV-1 infection in CD4 + T cells promotes viral replication and drives extracellular vesicle-mediated inflammation. mBio 9, https://doi.org/10.1128/mbio.00757-18 (2018).
Porter, K. M. et al. Human immunodeficiency virus-1 transgene expression increases pulmonary vascular resistance and exacerbates hypoxia-induced pulmonary hypertension development. Pulm. Circul. 3, 58–67 (2013).
Saksela, K. Interactions of the HIV/SIV pathogenicity factor Nef with SH3 domain-containing host cell proteins. Curr. HIV Res. 9, 531–542 (2011).
Cassan, E., Arigon-Chifolleau, A.-M., Mesnard, J.-M., Gross, A. & Gascuel, O. Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic. Proc. Natl. Acad. Sci. USA 113, 11537–11542 (2016).
Kirchhoff, F. Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses. Cell Host Microbe 8, 55–67 (2010).
Sauter, D. et al. Tetherin-driven adaptation of Vpu and Nef function and the evolution of pandemic and nonpandemic HIV-1 strains. Cell Host Microbe 6, 409–421 (2009).
Doyle, T., Goujon, C. & Malim, M. H. HIV-1 and interferons: who’s interfering with whom?. Nat. Rev. Microbiol. 13, 403–413 (2015).
Huang, X., Liu, Y., Ling, G. & Cao, X. Mitochondrial Lon protease promotes CD4+ T cell activation by activating the cGAS-STING-TBK1 axis in systemic lupus erythematosus. Int. Immunopharmacol. 123, https://doi.org/10.1016/j.intimp.2023.110519 (2023).
Yu, J. et al. Regulation of T-cell activation and migration by the kinase TBK1 during neuroinflammation. Nat. Commun. 6, https://doi.org/10.1038/ncomms7074 (2015).
Fukasaku, Y. et al. Novel immunological approach to asses donor reactivity of transplant recipients using a humanized mouse model. Hum. Immunol. 81, 342–353 (2020).
Lubow, J. et al. Mannose receptor is an HIV restriction factor counteracted by Vpr in macrophages. Elife 9, https://doi.org/10.7554/elife.51035 (2020).
Iwamura, T. et al. Induction of IRF-3/-7 kinase and NF-κB in response to double-stranded RNA and virus infection: common and unique pathways. Genes Cells 6, 375–388 (2001).
Wang, Y. et al. Decreased expression of the host long-noncoding RNA-GM facilitates viral escape by inhibiting the kinase activity TBK1 via S-glutathionylation. Immunity 53, 1168–1181 (2020).
Mijit, A. et al. Mapping synthetic binding proteins epitopes on diverse protein targets by protein structure prediction and protein-protein docking. Comput. Biol. Med. 163, 107183 (2023).
Yan, Y., Tao, H., He, J. & Huang, S.-Y. The HDOCK server for integrated protein–protein docking. Nat. Protoc. 15, 1829–1852 (2020).
Yan, Y., Zhang, D., Zhou, P., Li, B. & Huang, S.-Y. HDOCK: a web server for protein–protein and protein–DNA/RNA docking based on a hybrid strategy. Nucleic Acids Res. 45, W365–W373 (2017).
Acknowledgements
We thank Dr. B. Ge (Tongji University, Shanghai, China) for generously providing the expression constructs Flag-N-RIG-I, Flag-TBK1, Flag-IRF3-5D, Flag-cGAS, and Flag-STING. We are also grateful to Dr. D. Sauter (Ulm University, Meierhof-strasse, Germany) for providing the plasmids pYU2, pNL4-3 and VSV-G-pseudotyped NL4.3-Δenv used in this study. The study was supported by a grant from the Shanghai Pilot Program for Basic Research - Fudan University (21TQ1400100), the National Natural Science Foundation of China (32270973, 32572032, 32571090), the National Natural Science Foundation Youth Program (32301986), the Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project (2025ZD01904400), the Shuguang Program of Shanghai Education Development Foundation, Shanghai Municipal Education Commission, In-hospital Research Project of Shanghai Public Health Clinical Center (KY-GW-2024-14).
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X.L. and D.Y. designed this study; X.L. performed the experiments, assisted by W.Z., M.L., C.Z., J.X., and W.H.; J.C., Y.Z., and X.L. contributed to discussions and agreement with the conclusions; X.L. and D.Y. analyzed the data and wrote the manuscript; and all authors discussed the results and commented on the manuscript.
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Lin, X., Zhang, W., Li, M. et al. Hydroxylation of the HIV-1 antisense protein promotes immune evasion of HIV-1 via modulation of TBK1. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71807-z
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DOI: https://doi.org/10.1038/s41467-026-71807-z
