The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Nuclear Import Pathway and Nuclear Export Signal among Retroviral Rev/Rev-Like Proteins

Abstract

The Rev protein is essential for the replication of lentiviruses. Rev is a shuttling protein that transports unspliced and partially spliced lentiviral RNAs from the nucleus to the cytoplasm via the nucleopore. To transport these RNAs, the human immunodeficiency virus type 1 (HIV-1) Rev uses the karyopherin β family importin β and CRM1 proteins that interact with the Rev nuclear localization signal (NLS) and nuclear exportation signal (NES), respectively. Recently, we reported the presence of new types of bipartite NLS and nucleolar localization signal (NoLS) in the bovine immunodeficiency virus (BIV) Rev protein. Here we report the characterization of the nuclear import and export pathways of BIV Rev. By using an in vitro nuclear import assay, we showed that BIV Rev is transported into the nucleus by a cytosolic and energy-dependent importin α/β classical pathway. Results from glutathione S -transferase (GST) pulldown assays that showed the binding of BIV Rev with importins α3 and α5 were in agreement with those from the nuclear import assay. We also identified a leptomycin B-sensitive NES in BIV Rev, which indicates that the protein is exported via CRM1 like HIV-1 Rev. Mutagenesis experiments showed that the BIV Rev NES maps between amino acids 109 to 121 of the protein. Remarkably, the BIV Rev NES was found to be of the cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) type instead of the HIV-1 Rev type. In summary, our data showed that the nuclear import mechanism of BIV Rev is novel among Rev proteins characterized so far in lentiviruses.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

“…Consistent with this observation, we recently showed using long-term (Ͼ24 h) live cell imaging that Rev/RRE-dependent transcripts typically build up in the nucleoplasm prior to a punctuated, CRM1-dependent en masse nuclear export event (76). Heterologous NES peptides have long been known to support Rev activity either in the context of Rev trafficking or RRE-dependent gene expression (52,71,72,81,82). Accordingly, and based on the hypothesis that the timing of viral RNA nuclear export is crucial to rates of Gag/Gag-Pol synthesis and genome encapsidation, we anticipated that altering the strength or number of Rev-CRM1 interactions via NES modulation would negatively impact rates of infectious virion production.…”

“…Despite conserved activity in mediating CRM1 binding, the Rev and PKI NES peptides differ in terms of CRM1 binding strength (71)(72)(73), are structurally distinct in the context of how they interface with CRM1's NES binding pocket (34), and may be involved in recruiting alternative cellular factors in addition to CRM1 (e.g., the HIV cofactor eIF5A) (74). Despite these differences, replacement of the Rev NES with the PKI NES in the context of Rev-mChe, Rev-mChe-NES or RevM10-mChe-NES yielded wild-type levels of infectious virion production (Fig.…”

MLV based viral-like-particles for delivery of toxic proteins and nuclear transcription factors

“…To address the relocalization of the precursor Gag-TF proteins to the nucleus (Fig. 2a), we tested whether a MLV Gag-TF chimera could be restored to localize to the cytoplasm and generate VLPs through the incorporation of optimized cyclic-AMP-dependent-kinase-inhibitor (PKI)-type NESs [24,25]. NES sequences were inserted within the p12 and NC genes at positions tolerant of insertions [4] (Fig.…”

“…Data in this study demonstrates that MLV Gag is directed to the nucleus by the insertion of foreign NLS-containing proteins and leads to the nuclear accumulation of the Gag chimera and thus decreasing the level of VLPs released from the plasma membrane of the infected cell. With the addition of the NES sequence within the p12 and NC genes and the co-expression of wild type Gag or Gag-Pol protein, the Gag chimera can cycle out of the nucleus using the CRM-1 dependent pathway defined for PKI-type NESs [24,25] and are capable of generating VLPs. Taken together, the modification of MLV Gag precursor protein with an NLS is counterbalanced by the NES sequence yielding little overall change to the MLV fitness.…”

Identification of the membrane-spanning domain of glycoprotein 45 in bovine immunodeficiency virus

“…The BIV genome, similar to human immunodeficiency virus (HIV) and other lentiviruses, contains the structural genes gag, pol, env and several accessory genes tat, rev, vif, vpw, vpy, and tmx. However, although some studies on BIV are focused on tat (Guo et al, 2013), vif (Zhang et al, 2014) rev (Gomez Corredor and Archambault, 2009;Gomez Corredor and Archambault, 2012) and others (Albernaz et al, 2015;Liu et al, 2015), very scarce attention has been paid to env. The Env of BIV is composed of two subunits: gp100, a surface subunit (SU) that binds host cell receptors, and gp45, a transmembrane subunit (TM) that ultimately inserts into the host cell membrane and promotes the fusion.…”