The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Lentiviral Bipartite Nuclear Localization Signal Harboring an Atypical Spacer Sequence

The bovine immunodeficiency virus (BIV) Rev protein (186 amino acids [aa] in length) is involved in the nuclear exportation of partially spliced and unspliced viral RNAs. Previous studies have shown that BIV Rev localizes in the nucleus and nucleolus of infected cells. Here we report the characterization of the nuclear/nucleolar localization signals (NLS/NoLS) of this protein. Through transfection of a series of deletion mutants of BIV Rev fused to enhanced green fluorescent protein and fluorescence microscopy analyses, we were able to map the NLS region between aa 71 and 110 of the protein. Remarkably, by conducting alanine substitution of basic residues within the aa 71 to 110 sequence, we demonstrated that the BIV Rev NLS is bipartite, maps to aa 71 to 74 and 95 to 101, and is predominantly composed of arginine residues. This is the first report of a bipartite Rev (or Rev-like) NLS in a lentivirus/retrovirus. Moreover, this NLS is atypical, as the length of the sequence between the motifs composing the bipartite NLS, e.g., the spacer sequence, is 20 aa. Further mutagenesis experiments also identified the NoLS region of BIV Rev. It localizes mainly within the NLS spacer sequence. In addition, the BIV Rev NoLS sequence differs from the consensus sequence reported for other viral and cellular nucleolar proteins. In summary, we conclude that the nucleolar and nuclear localizations of BIV Rev are mediated via novel NLS and NoLS motifs.