title: The HIV-1 Rev protein enhances encapsidation of unspliced and spliced, RRE-containing lentiviral vector RNA.
abstract: BACKGROUND
  During the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein.
  
  
  RESULTS
  Starting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly.
  
  
  CONCLUSIONS
  Rev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.
authors.affiliation: Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany. Bastian.Grewe@rub.de
authors.forename: Bastian
authors.initials: B
authors.lastname: Grewe
authors.forename: Katrin
authors.initials: K
authors.lastname: Ehrhardt
authors.forename: Bianca
authors.initials: B
authors.lastname: Hoffmann
authors.forename: Maik
authors.initials: M
authors.lastname: Blissenbach
authors.forename: Sabine
authors.initials: S
authors.lastname: Brandt
authors.forename: Klaus
authors.initials: K
authors.lastname: Uberla
mesh_terms: D021581:Active Transport, Cell Nucleus; D017398:Alternative Splicing; D005822:Genetic Vectors; D016679:Genome, Viral; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing; D012367:RNA, Viral / Q000235:genetics; D020218:Response Elements; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000502:physiology*
chemical_list: D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1; D012313:RNA