title: Inhibition of interferon response by cystatin B: implication in HIV replication of macrophage reservoirs.
abstract: Cystatin B and signal transducer and activator of transcription-1 (STAT-1) phosphorylation have recently been shown to increase human immunodeficiency virus-1 (HIV-1) replication in monocyte-derived macrophages (MDM), but the molecular pathways by which they do are unknown. We hypothesized that cystatin B inhibits the interferon (IFN) response and regulates STAT-1 phosphorylation by interacting with additional proteins. To test if cystatin B inhibits the IFN-β response, we performed luciferase reporter gene assays in Vero cells, which are IFN deficient. Interferon-stimulated response element (ISRE)-driven expression of firefly luciferase was significantly inhibited in Vero cells transfected with a cystatin B expression vector compared to cells transfected with an empty vector. To determine whether cystatin B interacts with other key players regulating STAT-1 phosphorylation and HIV-1 replication, cystatin B was immunoprecipitated from HIV-1-infected MDM. The protein complex was analyzed by liquid chromatography tandem mass spectrometry. Protein interactions with cystatin B were verified by Western blots and immunofluorescence with confocal imaging. Our findings confirmed that cystatin B interacts with pyruvate kinase M2 isoform, a protein previously associated cocaine enhancement of HIV-1 replication, and major vault protein (MVP), an IFN-responsive protein that interferes with JAK/STAT signals. Western blot studies confirmed the interaction with pyruvate kinase M2 isoform and MVP. Immunofluorescence studies of HIV-1-infected MDM showed that upregulated MVP colocalized with STAT-1. To our knowledge, the current study is the first to demonstrate the coexpression of cystatin B, STAT-1, MVP, and pyruvate kinase M2 isoform with HIV-1 replication in MDM and thus suggests novel targets for HIV-1 restriction in macrophages, the principal reservoirs for HIV-1 in the central nervous system.
authors.affiliation: Department of Microbiology and NeuroAIDS Program, University of Puerto Rico-Medical Sciences Campus, San Juan, Puerto Rico 00935, USA.
authors.forename: L
authors.initials: L
authors.lastname: Rivera-Rivera
authors.forename: J
authors.initials: J
authors.lastname: Perez-Laspiur
authors.forename: Krystal
authors.initials: K
authors.lastname: Colón
authors.forename: L M
authors.initials: LM
authors.lastname: Meléndez
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D055313:Cystatin B / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D015658:HIV Infections / Q000276:immunology / Q000378:metabolism / Q000821:virology; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D015850:Interleukin-6 / Q000494:pharmacology*; D049409:Luciferases, Firefly; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology / Q000821:virology; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020394:Vault Ribonucleoprotein Particles / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects; D000099187:Major Vault Protein
chemical_list: D055313:Cystatin B; D015850:Interleukin-6; D049409:Luciferases, Firefly; D011770:Pyruvate Kinase; D050794:STAT1 Transcription Factor; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein; C524994:CSTB protein, human; C494079:STAT1 protein, human