title: Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells.
abstract: BACKGROUND
  The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs.
  
  
  RESULTS
  Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication.
  
  
  CONCLUSIONS
  HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known.
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland.
authors.forename: Claudia F
authors.initials: CF
authors.lastname: Althaus
authors.forename: Valentina
authors.initials: V
authors.lastname: Vongrad
authors.forename: Barbara
authors.initials: B
authors.lastname: Niederöst
authors.forename: Beda
authors.initials: B
authors.lastname: Joos
authors.forename: Francesca
authors.initials: F
authors.lastname: Di Giallonardo
authors.forename: Philip
authors.initials: P
authors.lastname: Rieder
authors.forename: Jovan
authors.initials: J
authors.lastname: Pavlovic
authors.forename: Alexandra
authors.initials: A
authors.lastname: Trkola
authors.forename: Huldrych F
authors.initials: HF
authors.lastname: Günthard
authors.forename: Karin J
authors.initials: KJ
authors.lastname: Metzner
authors.forename: Marek
authors.initials: M
authors.lastname: Fischer
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D015497:HIV-1 / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D008264:Macrophages / Q000821:virology*; D058727:RNA, Small Untranslated / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism
chemical_list: D058727:RNA, Small Untranslated; D012367:RNA, Viral