title: Investigation of the HIV-1 matrix interactome during virus replication.
abstract: OBJECTIVE
  Like all viruses, human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors.
  
  
  METHODS
  A Strep-tag was inserted into the C-terminus of the matrix (MA) region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. MA complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS, data were log2 -transformed, and Student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays.
  
  
  RESULTS
  A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. X-ray repair cross-complementing protein 6 (Ku70), X-ray repair cross-complementing protein 5 (Ku80), and Y-box binding protein 1 (YB-1) were confirmed to interact with MA by immunoblot. Knockdown of two candidates, EZRIN and Y-box binding protein 1, enhanced HIV infection in vitro.
  
  
  CONCLUSIONS
  The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV host -cell interactions.
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Frederick
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Haverland
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA.|The Nebraska Center for Virology, University of Nebraska, Lincoln, NE, USA.
authors.forename: Pawel
authors.initials: P
authors.lastname: Ciborowski
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE, USA.|The Nebraska Center for Virology, University of Nebraska, Lincoln, NE, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Belshan
mesh_terms: D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006678:HIV / Q000254:growth & development* / Q000378:metabolism*; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006801:Humans; D019169:Jurkat Cells; D014779:Virus Replication
chemical_list: D016326:Extracellular Matrix Proteins
keywords: EZRIN; HIV; Ku70; Ku80; Matrix; SWATH; YB-1