title: Detection of the HIV-1 minus-strand-encoded antisense protein and its association with autophagy.
abstract: HIV-1 proteins are synthesized from a single transcript in an unspliced form or following splicing, but the existence of an antisense protein (ASP) expressed from an antisense polyadenylated transcript has been suggested. Difficulties linked to the detection of this protein in mammalian cells led us to codon optimize its cDNA. Codon-optimized ASP was indeed efficiently detected in various transfected cell lines following flow cytometry and confocal microscopy analyses. Western blot analyses also led to the detection of optimized ASP in transfected cells but also provided evidence of its instability and high multimerization potential. ASP was mainly distributed in the cytoplasm in a punctate manner, which was reminiscent of autophagosomes. In agreement with this observation, a significant increase in ASP-positive cells and loss of its punctate distribution was observed in transfected cells when autophagy was inhibited at early steps. Induction of autophagy was confirmed by Western blot analyses that showed an ASP-mediated increase in levels of LC3b-II and Beclin 1, as well as colocalization and interaction between ASP and LC3. Interestingly, Myc-tagged ASP was detected in the context of proviral DNA following autophagy inhibition with a concomitant increase in the level and punctate distribution of LC3b-II. Finally, 3-methyladenine treatment of transfected or infected U937 cells decreased extracellular p24 levels in wild-type proviral DNA and to a much lesser extent in ASP-mutated proviral DNA. This study provides the first detection of ASP in mammalian cells by Western blotting. ASP-induced autophagy might explain the inherent difficulty in detecting this viral protein and might justify its presumed low abundance in infected cells.
authors: Département des Sciences Biologiques and Centre de Recherche BioMed, Université du Québec à Montréal, Montréal, Québec, Canada.
authors: Cynthia
authors: C
authors: Torresilla
authors: Émilie
authors: É
authors: Larocque
authors: Sébastien
authors: S
authors: Landry
authors: Marilène
authors: M
authors: Halin
authors: Yan
authors: Y
authors: Coulombe
authors: Jean-Yves
authors: JY
authors: Masson
authors: Jean-Michel
authors: JM
authors: Mesnard
authors: Benoit
authors: B
authors: Barbeau
mesh_terms: D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy; D000071186:Beclin-1; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology* / Q000821:virology*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; C477012:MAP1LC3A protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D012367:RNA, Viral; D014764:Viral Proteins