title: Balanced splicing at the Tat-specific HIV-1 3'ss A3 is critical for HIV-1 replication.
abstract: BACKGROUND
  The viral regulatory protein Tat is essential for establishing a productive transcription from the 5'-LTR promoter during the early phase of viral gene expression. Formation of the Tat-encoding mRNAs requires splicing at the viral 3'ss A3, which has previously been shown to be both negatively and positively regulated by the downstream splicing regulatory elements (SREs) ESS2p and ESE2/ESS2. However, using the novel RESCUE-type computational HEXplorer algorithm, we were recently able to identify another splicing enhancer (ESE(5807-5838), henceforth referred to as ESE tat ) located between ESS2p and ESE2/ESS2. Here we show that ESE tat has a great impact on viral tat-mRNA splicing and that it is fundamental for regulated 3'ss A3 usage.
  
  
  RESULTS
  Mutational inactivation or locked nucleic acid (LNA)-directed masking of the ESE tat sequence in the context of a replication-competent virus was associated with a failure (i) to activate viral 3'ss A3 and (ii) to accumulate Tat-encoding mRNA species. Consequently, due to insufficient amounts of Tat protein efficient viral replication was drastically impaired. RNA in vitro binding assays revealed SRSF2 and SRSF6 as candidate splicing factors acting through ESE tat and ESE2 for 3'ss A3 activation. This notion was supported by coexpression experiments, in which wild-type, but not ESE tat -negative provirus responded to higher levels of SRSF2 and SRSF6 proteins with higher levels of tat-mRNA splicing. Remarkably, we could also find that SRSF6 overexpression established an antiviral state within provirus-transfected cells, efficiently blocking virus particle production. For the anti-HIV-1 activity the arginine-serine (RS)-rich domain of the splicing factor was dispensable.
  
  
  CONCLUSIONS
  Based on our results, we propose that splicing at 3'ss A3 is dependent on binding of the enhancing SR proteins SRSF2 and SRSF6 to the ESE tat and ESE2 sequence. Mutational inactivation or interference specifically with ESE tat activity by LNA-directed masking seem to account for an early stage defect in viral gene expression, probably by cutting off the supply line of Tat that HIV needs to efficiently transcribe its genome.
authors.affiliation: Institute of Virology, Heinrich-Heine-University Düsseldorf, D-40225, Düsseldorf, Germany. Steffen.Erkelenz@med.uni-duesseldorf.de.
authors.forename: Steffen
authors.initials: S
authors.lastname: Erkelenz
authors.affiliation: Institute of Virology, Heinrich-Heine-University Düsseldorf, D-40225, Düsseldorf, Germany. Frank.Hillebrand@med.uni-duesseldorf.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Hillebrand
authors.affiliation: Institute of Virology, Heinrich-Heine-University Düsseldorf, D-40225, Düsseldorf, Germany. Marek.Widera@uk-essen.de.|Current address: Institute of Virology, University Hospital Essen, Essen, Germany. Marek.Widera@uk-essen.de.
authors.forename: Marek
authors.initials: M
authors.lastname: Widera
authors.affiliation: Institute of Clinical Neuroscience and Medical Psychology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany. theiss@uni-duesseldorf.de.
authors.forename: Stephan
authors.initials: S
authors.lastname: Theiss
authors.affiliation: Plant Biology Laboratories, Michigan State University, Michigan, USA. fayyazan@msu.edu.
authors.forename: Anaam
authors.initials: A
authors.lastname: Fayyaz
authors.affiliation: Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, D-40225, Düsseldorf, Germany. Daniel.Degrandi@uni-duesseldorf.de.
authors.forename: Daniel
authors.initials: D
authors.lastname: Degrandi
authors.affiliation: Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, D-40225, Düsseldorf, Germany. klaus.pfeffer@uni-duesseldorf.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Pfeffer
authors.affiliation: Institute of Virology, Heinrich-Heine-University Düsseldorf, D-40225, Düsseldorf, Germany. schaal@uni-duesseldorf.de.
authors.forename: Heiner
authors.initials: H
authors.lastname: Schaal
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis; D015870:Gene Expression; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D012261:Ribonucleoproteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D012261:Ribonucleoproteins; C489470:SRSF6 protein, human; D054322:tat Gene Products, Human Immunodeficiency Virus; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors