title: Replication of biotinylated human immunodeficiency viruses.
abstract: Previous work demonstrated recently the adaptation of the Escherichia coli biotin ligase BirA - biotin acceptor sequence (BAS) labeling system to produce human immunodeficiency virus type 1 viruses with biotinylated integrase (NLXIN(B)) and matrix (NLXMA(B)) proteins (Belshan et al., 2009). This report describes the construction of an HIV permissive cell line stably expressing BirA (SupT1.BirA). Consistent with the results in the previous report, NLXMA(B) replicated similar to wild-type levels and expressed biotinylated Gag and MA proteins in the SupT1.BirA cells, whereas the replication of NLXIN(B) was reduced severely. Three additional HIV type 2 (HIV-2) viruses were constructed with the BAS inserted into the vpx and vpr accessory genes. Two BAS insertions were made into the C-terminal half of the Vpx, including one internal insertion, and one at the N-terminus of Vpr. All three viruses were replication competent in the SupT1.BirA cells and their target proteins biotinylated efficiently and incorporated into virions. These results demonstrate the potential utility of the biotinylation system to label and capture HIV protein complexes in the context of replicating virus.
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA. michaelbelshan@creighton.edu
authors.forename: Michael
authors.initials: M
authors.lastname: Belshan
authors.forename: John M
authors.initials: JM
authors.lastname: Matthews
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Madson
mesh_terms: D001710:Biotin / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D002460:Cell Line; D029968:Escherichia coli Proteins / Q000378:metabolism*; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D015498:HIV-2 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C514800:p17 protein, Human Immunodeficiency Virus Type 2; D001710:Biotin; D019427:HIV Integrase; C514802:p31 integrase protein, Human immunodeficiency virus 2; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli