Binding to PI(4,5)P2 is indispensable for secretion of B-cell clonogenic HIV-1 matrix protein p17 variants
2021; Elsevier BV; Volume: 297; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2021.100934
ISSN1083-351X
AutoresAntonella Bugatti, Francesca Caccuri, Federica Filippini, Cosetta Ravelli, Arnaldo Caruso,
Tópico(s)Viral-associated cancers and disorders
ResumoHIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin's lymphoma (NHL) tissues of HIV-1–seropositive (HIV+) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala–Ala) or 125 to 126 (Gly–Asn), respectively, are secreted from HIV-1–infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55Gag) by cellular aspartyl proteases. Binding of Pr55Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing. HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin's lymphoma (NHL) tissues of HIV-1–seropositive (HIV+) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala–Ala) or 125 to 126 (Gly–Asn), respectively, are secreted from HIV-1–infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55Gag) by cellular aspartyl proteases. Binding of Pr55Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing. The HIV-1 matrix protein p17 (p17) is a 132 amino acids–long Gag-encoded protein that plays a key role in virus assembly and release (1Fiorentini S. Marini E. Caracciolo S. Caruso A. Functions of the HIV-1 matrix proteins p17.New Microbiol. 2006; 29: 1-10PubMed Google Scholar). Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human-specific adaptation (2Caccuri F. D'Ursi P. Uggeri M. Bugatti A. Mazzuca P. Zani A. Filippini F. Salmona M. Ribatti D. Slevin M. Orro A. Lu W. Liò P. Gallo R.C. Caruso A. Evolution toward beta common chain receptor usage links the matrix proteins of HIV-1 and its ancestors to human erythropoietin.Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2021366118Crossref PubMed Scopus (2) Google Scholar). Virion-free p17 is released from HIV-1–infected cells, and it can be detected in blood at nanomolar concentrations (1Fiorentini S. Marini E. Caracciolo S. Caruso A. Functions of the HIV-1 matrix proteins p17.New Microbiol. 2006; 29: 1-10PubMed Google Scholar, 3Fiorentini S. Riboldi E. Facchetti F. Avolio M. Fabbri M. Tosti G. Becker P.D. Guzman C.A. Sozzani S. Caruso A. Fiorentini S. HIV-1 matrix protein p17 induces human plasmacytoid dendritic cells to acquire a migratory immature cell phenotype.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3867-3872Crossref PubMed Scopus (44) Google Scholar). Moreover, p17 accumulates and persists in different organs and tissues of patients even under successful combined antiretroviral therapy (cART) (4Popovic M. Tenner-Racz K. Pelser C. Stellbrink H.-J. Van Lunzen J. Lewis G. Kalyanaraman V.S. Gallo R.C. Racz P. Popovic M. Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therap.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14807-14812Crossref PubMed Scopus (128) Google Scholar, 5Caccuri F. Giagulli C. Bugatti A. Benetti A. Alessandri G. Ribatti D. Marsico S. Apostoli P. Slevin M.A. Rusnati M. Guzman C.A. Fiorentini S. Caruso A. HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 14580-14585Crossref PubMed Scopus (64) Google Scholar, 6Dolcetti R. Giagulli C. He W. Selleri M. Caccuri F. Eyzaguirre L.M. Mazzuca P. Corbellini S. Campilongo F. Marsico S. Giombini E. Muraro E. Rozera G. De Paoli P. Carbone A. et al.Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 14331-14336Crossref PubMed Scopus (45) Google Scholar). In particular, a long-term p17 accumulation in lymph nodes of cART-treated patients has been shown, even after prolonged HIV-1 suppression (4Popovic M. Tenner-Racz K. Pelser C. Stellbrink H.-J. Van Lunzen J. Lewis G. Kalyanaraman V.S. Gallo R.C. Racz P. Popovic M. Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therap.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14807-14812Crossref PubMed Scopus (128) Google Scholar). Taken together, these findings strongly suggest that p17 may be chronically present in the tissue microenvironment of HIV-1–seropositive (HIV+) patients, even during the pharmacological control of viral replication. These findings are consistent with several reports showing that the latently infected resting CD4+ T cells transcribe and translate Gag proteins, even without cell stimulation (7Vandergeeten C. Quivy V. Moutschen M. Van Lint C. Piette J. Legrand-Poels S. HIV-1 protease inhibitors do not interfere with provirus transcription and host cell apoptosis induced by combined treatment TNF-alpha + TSA.Biochem. Pharmacol. 2007; 73: 1738-1748Crossref PubMed Scopus (19) Google Scholar, 8Pace M.J. Graf E.H. Agosto L.M. Mexas A.M. Male F. Brady T. Bushman F.D. O'Doherty U. Directly infected resting CD4+T cells can produce HIV Gag without spreading infection in a model of HIV latency.PLoS Pathog. 2012; 8e1002818Crossref PubMed Scopus (116) Google Scholar), and that defective HIV-1 proviruses in the CD4+ T cells of HIV+ individuals on cART produce intact Gag proteins in vitro and in vivo (9Imamichi H. Smith M. Adelsberger J.W. Izumi T. Scrimieri F. Sherman B.T. Rehm C.A. Imamichi T. Pau A. Catalfamo M. Fauci A.S. Lane H.C. Defective HIV-1 proviruses produce viral proteins.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 3704-3710Crossref PubMed Scopus (64) Google Scholar). Extracellularly, p17 has been found to deregulate the biological activity of different immune cells, which may be relevant in the context of viral pathogenesis. In particular, p17 is capable of triggering the activation, differentiation, and proliferation of different target immune cells as T cells (10De Francesco M.A. Caruso A. Fallacara F. Canaris A.D. Dima F. Poiesi C. Licenziati S. Corulli M. Martinelli F. Fiorentini S. Turano A. HIV p17 enhances lymphocyte proliferation and HIV-1 replication after binding to a human serum factor.AIDS. 1998; 12: 245-252Crossref PubMed Scopus (25) Google Scholar, 11De Francesco M.A. Baronio M. Fiorentini S. Signorini C. Bonfanti C. Poiesi C. Popovic M. Grassi M. Garrafa E. Bozzo L. Lewis G.K. Licenziati S. Gallo R.C. Caruso A. HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9972-9977Crossref PubMed Scopus (58) Google Scholar), natural killer cells (12Vitale M. Caruso A. De Francesco M.A. Rodella L. Bozzo L. Garrafa E. Grassi M. Gobbi G. Cacchioli A. Fiorentini S. HIV-1 matrix protein p17 enhances the proliferative activity of natural killer cells and increases their ability to secrete proinflammatory cytokines.Br. J. Haematol. 2003; 120: 337-343Crossref PubMed Scopus (34) Google Scholar), monocytes (13Marini E. Tiberio L. Caracciolo S. Tosti G. Guzman C.A. Schiaffonati L. Fiorentini S. Caruso A. HIV-1 matrix protein p17 binds to monocytes and selectively stimulates MCP-1 secretion: Role of transcriptional factor AP-1.Cell. Microbiol. 2008; 10: 655-666Crossref PubMed Scopus (35) Google Scholar), and plasmacytoid dendritic cells (3Fiorentini S. Riboldi E. Facchetti F. Avolio M. Fabbri M. Tosti G. Becker P.D. Guzman C.A. Sozzani S. Caruso A. Fiorentini S. HIV-1 matrix protein p17 induces human plasmacytoid dendritic cells to acquire a migratory immature cell phenotype.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3867-3872Crossref PubMed Scopus (44) Google Scholar). We also highlighted the capability of p17 to exert chemotactic activity on human primary B cells after binding to the C-X-C motif chemokine receptors 1 and 2 (14Giagulli C. Magieram A.K. Bugatti A. Caccuri F. Marsico S. Rusnati M. Vermi W. Fiorentini S. Caruso A. HIV-1 matrix protein p17 binds to the IL-8 receptor CXCR1 and shows IL-8–like chemokine activity on monocytes through Rho/ROCK activation.Blood. 2012; 119: 2274-2283Crossref PubMed Scopus (41) Google Scholar, 15Caccuri F. Giagulli C. Reichelt J. Martorelli D. Marsico S. Bugatti A. Barone I. Rusnati M. Guzman C.A. Dolcetti R. Caruso A. Simian immunodeficiency virus and human immunodeficiency virus type 1 matrix proteins specify different capabilities to modulate B cell growth.J. Virol. 2014; 88: 5706-5717Crossref PubMed Scopus (22) Google Scholar). Interestingly, we showed that a p17 variant (vp17) carrying various amino acid substitutions, named S75X, derived from an Ugandan HIV-1 A1 strain, differently from a p17 derived from clade B virus (strain BH10; refp17), activated the Akt signaling pathway in B cells, thereby promoting cell growth and clonogenicity (16Giagulli C. Marsico S. Magiera A.K. Bruno R. Caccuri F. Barone I. Fiorentini S. Andò S. Caruso A. Opposite effects of HIV-1 p17 variants on PTEN activation and cell growth in B cells.PLoS One. 2011; 6e17831Crossref PubMed Scopus (52) Google Scholar). This finding provided the first evidence on the existence of a vp17 with B-cell clonogenic activity on human B cells. More recently, in studies of p17 from human non-Hodgkin's lymphoma (NHL) cases, we characterized two categories of vp17s that promote B-cell growth and activate the Akt pathway: the first characterized by amino acid insertions at position 117 to 118 and the second characterized by amino acid insertions at position 125 to 126 (6Dolcetti R. Giagulli C. He W. Selleri M. Caccuri F. Eyzaguirre L.M. Mazzuca P. Corbellini S. Campilongo F. Marsico S. Giombini E. Muraro E. Rozera G. De Paoli P. Carbone A. et al.Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 14331-14336Crossref PubMed Scopus (45) Google Scholar). Ultradeep pyrosequencing showed that these two categories of vp17s are more frequently detected in plasma of HIV+ patients with NHL than without NHL (12Vitale M. Caruso A. De Francesco M.A. Rodella L. Bozzo L. Garrafa E. Grassi M. Gobbi G. Cacchioli A. Fiorentini S. HIV-1 matrix protein p17 enhances the proliferative activity of natural killer cells and increases their ability to secrete proinflammatory cytokines.Br. J. Haematol. 2003; 120: 337-343Crossref PubMed Scopus (34) Google Scholar). Moreover, we showed that vp17s characterized by an Ala–Ala insertion at position 117 to 118 (NHL-a101) and a Gly–Asn insertion at position 125 to 126 (NHL-a102), differently from refp17, were drastically destabilized. Because of misfolding, vp17s were found to expose a common functional epitope with B-cell clonogenic activity located at their N-terminal region (17He W. Mazzuca P. Yuan W. Varney K. Bugatti A. Cagnotto A. Giagulli C. Rusnati M. Marsico S. Diomede L. Salmona M. Caruso A. Lu W. Caccuri F. Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants.Biochim. Biophys. Acta Gen. Subj. 2018; 1863: 13-24Crossref PubMed Scopus (14) Google Scholar). Recently, we demonstrated that the B-cell clonogenic activity of both NHL-a101 and NHL-a102 is mediated by activation of the protease-activated receptor 1, which triggers transactivation of the epidermal growth factor receptor (18Giagulli C. Caccuri F. Zorzan S. Bugatti A. Zani A. Filippini F. Manocha E. D'Ursi P. Orro A. Dolcetti R. Caruso A. B-cell clonogenic activity of HIV-1 p17 variants is driven by PAR1-mediated EGF transactivation.Cancer Gene Ther. 2020; 28: 649-666Crossref PubMed Scopus (4) Google Scholar). Collectively, our data support the hypothesis that certain vp17s may play a role in lymphoma pathogenesis. It is worth noting that in the cART era, B-cell lymphomas, and NHLs in particular, comprise more than 50% of all AIDS-defining cancers (19Shiels M.S. Pfeiffer R.M. Gail M.H. Hall H.I. Li J. Chaturvedi A.K. Bhatia K. Uldrick T.S. Yarchoan R. Goedert J.J. Engels E.A. Cancer burden in the HIV-infected population in the United States.J. Natl. Cancer Inst. 2011; 103: 753-762Crossref PubMed Scopus (536) Google Scholar) and are still the most frequent cause of death in HIV+ patients (20Vandenhende M.A. Roussillon C. Henard S. Morlat P. Oksenhendler E. Aumaitre H. Georget A. May T. Rosenthal E. Salmon D. Cacoub P. Costagliola D. Chêne G. Bonnet F. ANRS EN20 Mortalité 2010 Study GroupCancer-related causes of death among HIV-infected patients in France in 2010: Evolution since 2000.PLoS One. 2015; 10e0129550Crossref PubMed Scopus (64) Google Scholar), being often characterized by clinical aggressiveness (21Coté T.R. Biggar R.J. Rosenberg P.S. Devesa S.S. Percy C. Yellin F.J. Lemp G. Hardy C. Geodert J.J. Blattner W.A. Non-Hodgkin's lymphoma among people with AIDS: Incidence, presentation and public health burden. AIDS/Cancer Study Group.Int. J. Cancer. 1997; 73: 645-650Crossref PubMed Scopus (250) Google Scholar, 22Knowles D.M. Etiology and pathogenesis of AIDS-related non-Hodgkin's lymphoma.Hematol. Oncol. Clin. North. Am. 2003; 17: 785-820Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Despite the lack of signal sequence, similarly to other viral proteins (17He W. Mazzuca P. Yuan W. Varney K. Bugatti A. Cagnotto A. Giagulli C. Rusnati M. Marsico S. Diomede L. Salmona M. Caruso A. Lu W. Caccuri F. Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants.Biochim. Biophys. Acta Gen. Subj. 2018; 1863: 13-24Crossref PubMed Scopus (14) Google Scholar, 23Johnson K.A. Taghon G.J. Scott J.L. Stahelin R.V. The Ebola virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress.Sci. Rep. 2016; 6: 19125Crossref PubMed Scopus (48) Google Scholar, 24Mori T. Mineta Y. Aoyama Y. Sera T. Efficient secretion of the herpes simplex virus tegument protein VP22 from living mammalian cells.Arch. Virol. 2008; 153: 1191-1195Crossref PubMed Scopus (7) Google Scholar, 25Alefantis T. Mostoller K. Jain P. Harhaj E. Grant C. Wigdahl B. Secretion of the human T cell leukemia virus type I transactivator protein tax.J. Biol. Chem. 2005; 280: 17353-17362Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), p17 is secreted in a biologically active form by an unconventional pathway (26Caccuri F. Iaria M.L. Campilongo F. Varney K. Rossi A. Mitola S. Schiarea S. Bugatti A. Mazzuca P. Giagulli C. Fiorentini S. Lu W. Salmona M. Caruso A. Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17.Sci. Rep. 2016; 6: 38027Crossref PubMed Scopus (11) Google Scholar). Its secretion takes place at the plasma membrane of Gag-expressing cells, in the absence of active viral proteases. Secretion of p17 was found to be dependent on the interaction of Gag precursor polyprotein (Pr55Gag) with phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) and its subsequent cleavage from Pr55Gag operated by cellular aspartyl proteases. We also highlighted that targeting Pr55Gag to the plasma membrane through PI(4,5)P2 interactions is the only feasible mechanism for p17 secretion (26Caccuri F. Iaria M.L. Campilongo F. Varney K. Rossi A. Mitola S. Schiarea S. Bugatti A. Mazzuca P. Giagulli C. Fiorentini S. Lu W. Salmona M. Caruso A. Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17.Sci. Rep. 2016; 6: 38027Crossref PubMed Scopus (11) Google Scholar). Several groups have shown the role played by the highly basic region (HBR) located at the p17 N-terminal region in the p17 interaction with PI(4,5)P2 (27Saad J.S. Miller J. Tai J. Kim A. Ghanam R.H. Summers M.F. Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11364-11369Crossref PubMed Scopus (428) Google Scholar, 28Chukkapalli V. Hogue I.B. Boyko V. Hu W.S. Ono A. Interaction between the human immunodeficiency virus type 1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient Gag membrane binding.J. Virol. 2008; 82: 2405-2417Crossref PubMed Scopus (193) Google Scholar, 29Chukkapalli V. Oh S.J. Ono A. Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 1600-1605Crossref PubMed Scopus (168) Google Scholar, 30Shkriabai N. Datta S.A. Zhao Z. Hess S. Rein A. Kvaratskhelia M. Interactions of HIV-1 Gag with assembly cofactors.Biochemistry. 2006; 45: 4077-4083Crossref PubMed Scopus (120) Google Scholar). Moreover, some Pr55Gag mutants in the HBR showed impaired Pr55Gag–PI(4,5)P2 interaction and relocalization of Pr55Gag from the plasma membrane to intracellular compartments, thus impairing or abolishing p17 secretion (26Caccuri F. Iaria M.L. Campilongo F. Varney K. Rossi A. Mitola S. Schiarea S. Bugatti A. Mazzuca P. Giagulli C. Fiorentini S. Lu W. Salmona M. Caruso A. Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17.Sci. Rep. 2016; 6: 38027Crossref PubMed Scopus (11) Google Scholar). Because of their partial unfolding state and remodeling of their N-terminal region to expose the B-cell clonogenic epitope (17He W. Mazzuca P. Yuan W. Varney K. Bugatti A. Cagnotto A. Giagulli C. Rusnati M. Marsico S. Diomede L. Salmona M. Caruso A. Lu W. Caccuri F. Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants.Biochim. Biophys. Acta Gen. Subj. 2018; 1863: 13-24Crossref PubMed Scopus (14) Google Scholar), we asked whether interaction of Pr55Gag carrying vp17s with PI(4,5)P2 occurs, leading to Pr55Gag cleavage by cellular or viral proteases and secretion as biologically active proteins. This is critical to hypothesize a role of vp17s in producing a microenvironment that fosters lymphoma development, progression, and metastasis. Herein, we report that biologically active NHL-a101 and NHL-a102 vp17s are released by cells nucleofected with plasmid-expressing Gag or viral mutants. Finally, we show for the first time that secreted refp17 and vp17s are, at least in part, tethered to the cell surface through heparan sulfate proteoglycans (HSPGs). Our first aim was to assess if vp17s secretion occurs during active virus replication. Jurkat cells, which are similar to most HIV-1–infected CD4+ T cells (31Stebbing J. Gazzard B. Douek D.C. Where does HIV live?.N. Engl. J. Med. 2004; 350: 1872-1880Crossref PubMed Scopus (129) Google Scholar), were transiently nucleofected with pNL4-3 plasmids expressing refp17, NHL-a101, or NHL-a102 (Jurkat-refp17, Jurkat-NHL-a101, and Jurkat-NHL-a102, respectively). Mock nucleofected cells were used as negative control (Jurkat). Twenty-four hours after nucleofection, 7.5 × 105 cells/well were cultured for additional 24 h in complete medium in anti-p17 mAb MBS-15–coated wells of 96-well plates. Plate-bound p17, secreted by seeded Jurkat-nucleofected cells, was then quantified by ELISA (26Caccuri F. Iaria M.L. Campilongo F. Varney K. Rossi A. Mitola S. Schiarea S. Bugatti A. Mazzuca P. Giagulli C. Fiorentini S. Lu W. Salmona M. Caruso A. Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17.Sci. Rep. 2016; 6: 38027Crossref PubMed Scopus (11) Google Scholar). As previously reported, p17 was easily detected in the supernatant of Jurkat-refp17, and the amount of secreted p17 ranged from 3.30 to 3.55 nM (mean ± SD: 3.41 ± 0.10 nM). At the same time, vp17s were also secreted by Jurkat-NHL-a101 (from 1.65 to 2.86 nM, mean ± SD: 2.29 ± 0.57 nM) and Jurkat-NHL-a102 (2.48–3.59 nM, mean ± SD: 3.1 ± 0.39 nM) (Fig. 1). This result attests for the active secretion of refp17 and vp17s from infected cells. We previously demonstrated that differently from refp17, NHL-a101 and NHL-a102 are able to induce B-cell growth and clonogenicity (6Dolcetti R. Giagulli C. He W. Selleri M. Caccuri F. Eyzaguirre L.M. Mazzuca P. Corbellini S. Campilongo F. Marsico S. Giombini E. Muraro E. Rozera G. De Paoli P. Carbone A. et al.Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 14331-14336Crossref PubMed Scopus (45) Google Scholar). To determine if vp17s are secreted in their biologically active form, we performed a single cell cloning assay on Raji B cells resuspended with supernatants collected from HeLa cells nucleofected with Gag-expressing plasmids carrying refp17 (HeLa-refp17), NHL-a101 (HeLa-NHL-a101), or NHL-a102 (HeLa-NHL-a102). As representatively shown in Figure 2A, a visible single colony was spontaneously developing in >60% of seeded wells at day 8 of culture, attesting for active cell proliferation. As expected, in the presence of vp17s released from nucleofected cells, colonies showed a significantly larger size than colonies cultured in medium of mock- or refp17-nucleofected cells. To demonstrate that the observed B-cell growth and clone expansion was specifically mediated by vp17s, the B-cell cloning assay was performed in the presence or absence of the p17-neutralizing mAb MBS-3, which recognizes the clonogenic epitope of vp17s (17He W. Mazzuca P. Yuan W. Varney K. Bugatti A. Cagnotto A. Giagulli C. Rusnati M. Marsico S. Diomede L. Salmona M. Caruso A. Lu W. Caccuri F. Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants.Biochim. Biophys. Acta Gen. Subj. 2018; 1863: 13-24Crossref PubMed Scopus (14) Google Scholar). An unrelated mAb (Ctrl mAb) was used as negative control in the assay. As representatively shown in Figure 2B, the presence of mAb MBS-3 (1 μg/ml) completely inhibited the clonogenic activity of both vp17s, whereas the Ctrl mAb (1 μg/ml) turned out to be ineffective. We have recently shown that vp17s are biologically active on endothelial cells (ECs) (5Caccuri F. Giagulli C. Bugatti A. Benetti A. Alessandri G. Ribatti D. Marsico S. Apostoli P. Slevin M.A. Rusnati M. Guzman C.A. Fiorentini S. Caruso A. HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 14580-14585Crossref PubMed Scopus (64) Google Scholar, 32Mazzuca P. Marsico S. Schulze K. Mitola S. Pils M.C. Giagulli C. Guzman C.A. Caruso A. Caccuri F. Role of autophagy in HIV-1 matrix protein p17-driven lymphangiogenesis.J. Virol. 2017; 91: e00801-e00817Crossref PubMed Scopus (12) Google Scholar). To evaluate whether secreted vp17s were active on ECs, we performed a wound healing assay, by cocultivation of nucleofected HeLa cells and human umbilical vein endothelial cells (HUVECs). A Culture-Insert 2 Well was used to provide two reservoirs for culturing cells that were separated by a 500 μm thick wall. The HUVEC and nucleofected HeLa cells were seeded in the reservoirs and cultured until they attached and formed a monolayer. Removal of the silicone insert from the surface was resulting in two precisely defined cell patches, which were separated by a zone that is exactly the same width as the separation wall. Cell migration of HUVECs was calculated as a percentage of wound observed over a period of 12 h. The number of HUVECs in the wound area increased more quickly (Fig. 3A), and the wound area decreased more rapidly in refp17- and vp17s-treated cells as compared with control cells (mock). As shown in Figure 3B, HUVECs cocultivated with mock HeLa cells reached approximately 47% sealing (range from 45 to 49%) after 12 h of culture, whereas, at the same time, HUVECs cocultivated with nucleofected HeLa cells expressing refp17 or its variants reached 80 to 90% sealing. This finding attests for the capability of viral proteins to promote EC migratory activity. Taken together, these data demonstrate that vp17s are secreted from infected cells in their biologically active form. Recently, we demonstrated that refp17 can be detected inside the cell as a punctate staining at the plasma membrane (26Caccuri F. Iaria M.L. Campilongo F. Varney K. Rossi A. Mitola S. Schiarea S. Bugatti A. Mazzuca P. Giagulli C. Fiorentini S. Lu W. Salmona M. Caruso A. Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17.Sci. Rep. 2016; 6: 38027Crossref PubMed Scopus (11) Google Scholar). To evaluate the cellular localization of vp17s, we carried out an indirect immunofluorescence assay and confocal microscopy analysis. HeLa cells were nucleofected with Gag-expressing plasmids as described previously (HeLa-refp17, HeLa-NHL-a101, and HeLa-NHL-a102), then fixed, and permeabilized or not before staining with the anti-p17 mAb MBS-3. To avoid mAb MBS-3 internalization, live and not permeabilized cells were maintained on ice during the staining step. Representative confocal images of permeabilized cells show that vp17s are localized at the plasma membrane as a punctate staining (Fig. 4A), as for refp17. On the contrary, in not permeabilized HeLa cells, both vp17s are confined on cell surface only (Fig. 4B). Previously, we have shown that refp17 is able to bind the HSPGs because of the presence of two distinct heparin-binding domains (33Bugatti A. Giagulli C. Urbinati C. Caccuri F. Chiodelli P. Oreste P. Fiorentini S. Orro A. Milanesi L. D'Ursi P. Caruso A. Rusnati M. Molecular interaction studies of hiv-1 matrix protein p17 and heparin identification of the heparin-binding motif of p17 as a target for the development of multitarget antagonists.J. Biol. Chem. 2012; 288: 1150-1161Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To scrutinize the binding of vp17s to HSPGs, we performed a surface plasmon resonance (SPR) assay with heparin bound on the sensor chip. As shown in Figure 5A, both vp17s were found to bind heparin in a dose-dependent manner. The sensorgram overlay shown in Figure 5A allowed the calculation of association (Kon) and dissociation (Koff) rates and of Kd (as Koff/Kon ratio) (Table 1). Based on this evidence, we hypothesize that HSPGs could be involved in vp17s binding on the cellular surface. Then, live and not permeabilized nucleofected HeLa cells were maintained for 24 h in the presence or absence of soluble heparin (10 μg/ml) or washed with 2 M NaCl (salt wash) before staining for indirect immunofluorescence assay. As shown in Figure 5B, not permeabilized cells treated with soluble heparin or salt washed, completely lost the expression of refp17 and vp17s on the cell surface, as compared with not treated cells. This finding suggests for the key role played by HSPGs in tethering refp17 and vp17s on cellular surface following their secretion.Figure 5P17s associate with cellular HSPGs. A, binding of p17s to heparin analyzed by SPR. Refp17 and its variants were injected on heparin sensor chip surface. All the proteins were tested at increasing concentrations (from 62.5 to 1000 nM). Affinity values of interaction were determined by BIAevaluation software. B, HeLa cells were nucleofected with Gag-expressing plasmids carrying refp17 (HeLa-refp17), NHL-a101 (HeLa-NHL-a101), or NHL-a102 (HeLa-NHL-a102) and then either incubated for 24 h in the presence of soluble heparin (10 μg/ml) or washed with PBS supplemented with 2 M NaCl (salt wash). Not permeabilized cells were then stained with anti-p17 mAb MBS-3 followed by Alexa Fluor 488–conjugated antimouse IgG and 4′,6-diamidino-2-phenylindole. Analysis was performed by fluorescence microscopy. Images display mAb MBS-3 signals in green and cell nuclei in blue. The scale bar represents 5 μm. HSPG, heparan sulfate proteoglycan; SPR, surface plasmon resonance.View Large
Referência(s)