Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation

Abstract

We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-mediated Gag assembly at the plasma membrane. Consequently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demonstrate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endosomes/lysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autophagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus (MLV) assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by nonspecific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possibility that similar regulations can occur in other biological contexts.

Keywords

microRNA / Gag protein / autophagy

References

[1] Aldovini A, Young RA (1990) Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol 64:1920–1926

[2] Berkowitz RD, Luban J, Goff SP (1993) Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. J Virol 67:7190–7200

[3] Briggs JA, Simon MN, Gross I, Krausslich HG, Fuller SD, Vogt VM, Johnson MC (2004) The stoichiometry of Gag protein in HIV-1. Nat Struct Mol Biol 11:672–675

[4] Campbell S, Vogt VM (1995) Self-assembly in vitro of purified CANC proteins from Rous sarcoma virus and human immunodeficiency virus type 1. J Virol 69:6487–6497

[5] Chen AK, Sengupta P, Waki K, Van Engelenburg SB, Ochiya T, Ablan SD, Freed EO, Lippincott-Schwartz J (2014) MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production. Proc Natl Acad Sci USA 111:E2676–E2683

[6] Eiring AM, Harb JG, Neviani P, Garton C, Oaks JJ, Spizzo R, Liu S, Schwind S, Santhanam R, Hickey CJ (2010) miR-328 functions as an RNA decoy to modulate hnRNP E2 regulation of mRNA translation in leukemic blasts. Cell 140:652–665

[7] Fabbri M, Paone A, Calore F, Galli R, Gaudio E, Santhanam R, Lovat F, Fadda P, Mao C, Nuovo GJ (2012) MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response. Proc Natl Acad Sci USA 109:E2110–E2116

[8] Feng Y, He D, Yao Z, Klionsky DJ (2014) The machinery of macroautophagy. Cell Res 24:24–41

[9] Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102–114

[10] Frankel LB, Lubas M, Lund AH (2017) Emerging connections between RNA and autophagy. Autophagy 13:3–23

[11] Gorelick RJ, Nigida SM Jr, Bess JW Jr, Arthur LO, Henderson LE, Rein A (1990) Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA. J Virol 64:3207–3211

[12] Jin J, Sherer NM, Heidecker G, Derse D, Mothes W (2009) Assembly of the murine leukemia virus is directed towards sites of cell-cell contact. Plos Biol 7:e1000163

[13] Jouvenet N, Neil SJD, Bess C, Johnson MC, Virgen CA, Simon SM, Bieniasz PD (2006) Plasma membrane is the site of productive HIV-1 particle assembly. Plos Biol 4:2296–2310

[14] Jouvenet N, Simon SM, Bieniasz PD (2009) Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles. Proc Natl Acad Sci USA 106:19114–19119

[15] Klionsky DJ (2007) Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 8:931–937

[16] Kutluay SB, Bieniasz PD (2010) Analysis of the initiating events in HIV-1 particle assembly and genome packaging. PLoS Pathog 6: e1001200

[17] Kutluay SB, Zang T, Blanco-Melo D, Powell C, Jannain D, Errando M, Bieniasz PD (2014) Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis. Cell 159:1096–1109

[18] Lehmann SM, Kruger C, Park B, Derkow K, Rosenberger K, Baumgart J, Trimbuch T, Eom G, Hinz M, Kaul D (2012) An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nat Neurosci 15:827–835

[19] Linial ML (1999) Foamy viruses are unconventional retroviruses. J Virol 73:1747–1755

[20] Liu K, Czaja MJ (2013) Regulation of lipid stores and metabolism by lipophagy. Cell Death Differ 20:3–11

[21] Mizushima N (2007) Autophagy: process and function. Gene Dev 21:2861–2873

[22] Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451:1069–1075

[23] Mullers E (2013) The foamy virus Gag proteins: what makes them different? Viruses-Basel 5:1023–1041

[24] Muriaux D, Mirro J, Harvin D, Rein A (2001) RNA is a structural element in retrovirus particles. Proc Natl Acad Sci USA 98:5246–5251

[25] Muriaux D, Costes S, Nagashima K, Mirro J, Cho E, Lockett S, Rein A (2004) Role of murine leukemia virus nucleocapsid protein in virus assembly. J Virol 78:12378–12385

[26] Ono A, Waheed AA, Joshi A, Freed EO (2005) Association of human immunodeficiency virus type 1 gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel gag multimerization assay. J Virol 79:14131–14140

[27] Prud’homme GJ, Glinka Y, Lichner Z, Yousef GM (2016) Neuropilin-1 is a receptor for extracellular miRNA and AGO2/miRNA complexes and mediates the internalization of miRNAs that modulate cell function. Oncotarget 7:68057–68071

[28] Rambold AS, Lippincott-Schwartz J (2011) Mechanisms of mitochondriaand autophagy crosstalk. Cell Cycle 10:4032–4038

[29] Ranganathan P, Ngankeu A, Zitzer NC, Leoncini P, Yu XY, Casadei L, Challagundla K, Reichenbach DK, Garman S, Ruppert AS (2017) Serum miR-29a is upregulated in acute graft-versus-host disease and activates dendritic cells through TLR binding. J Immunol 198:2500–2512

[30] Rein A, Harvin DP, Mirro J, Ernst SM, Gorelick RJ (1994) Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia-virus. J Virol 68:6124–6129

[31] Rulli SJ Jr, Hibbert CS, Mirro J, Pederson T, Biswal S, Rein A (2007) Selective and nonselective packaging of cellular RNAs in retrovirus particles. J Virol 81:6623–6631

[32] Sanchez-Wandelmer J, Reggiori F (2013) Amphisomes: out of the autophagosome shadow? EMBO J 32:3116–3118

[33] Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

[34] Sharp PA (2009) The centrality of RNA. Cell 136:577–580

[35] Shaw KL, Lindemann D, Mulligan MJ, Goepfert PA (2003) Foamy virus envelope glycoprotein is sufficient for particle budding and release. J Virol 77:2338–2348

[36] Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13:423–433

[37] Wong E, Bejarano E, Rakshit M, Lee K, Hanson HH, Zaarur N, Phillips GR, Sherman MY, Cuervo AM (2012) Molecular determinants of selective clearance of protein inclusions by autophagy. Nat Commun 3:1240

[38] Yelamanchili SV, Lamberty BG, Rennard DA, Morsey BM, Hochfelder CG, Meays BM, Levy E, Fox HS (2015) MiR-21 in extracellular vesicles leads to neurotoxicity via TLR7 signaling in SIV neurological disease. PLoS Pathog 11:e1005032

[39] Zhao D, Yang Y, Qu N, Chen M, Ma Z, Krueger CJ, Behlke MA, Chen AK (2016) Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2’-Omethyl RNA molecular beacons. Biomaterials 100:172–183

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2017 The Author(s) 2017. This article is an open access publication