Abstract
BackgroundThe gibbon ape leukemia virus (GaLV) Env protein mediates entry into a wide range of human cells and is frequently used to pseudotype retroviral vectors. However, an incompatibility exists between GaLV Env and lentiviral vectors that results in decreased steady-state levels of the mature GaLV Env in cells and prevents its incorporation into lentiviral vector particles.ResultsWe identified the HIV-1 Vpu protein as the major cause of the depletion in GaLV Env levels that occurs when lentiviral vector components are present. This activity of Vpu targeted the mature (cleaved) form of the GaLV Env that exists within or beyond the trans-Golgi. The activity required two conserved phospho-serines in the cytoplasmic tail of Vpu that are known to recruit β TrCP, a substrate adaptor for an SCF E3 ubiquitin ligase complex, and could be blocked by mutation of lysine 618 in the GaLV Env tail. Moreover, the Vpu-mediated decrease of GaLV Env levels was inhibited by the lysosomal inhibitor, bafilomycin A1. Interestingly, this activity of Vpu was only observed in the presence of other lentiviral vector components.ConclusionsSimilar to the mechanism whereby Vpu targets BST-2/tetherin for degradation, these findings implicate β-TrCP-mediated ubiquitination and the endo-lysosomal pathway in the degradation of the GaLV Env by lentiviral vector components. Possibly, the cytoplasmic tail of the GaLV Env contains features that mimic bona fide targets of Vpu, important to HIV-1 replication. Furthermore, the lack of effect of Vpu on GaLV Env in the absence of other HIV-1 proteins, suggests that a more complex interaction may exist between Vpu and its target proteins, with the additional involvement of one or more component(s) of the HIV-1 replication machinery.
Highlights
The gibbon ape leukemia virus (GaLV) Env protein mediates entry into a wide range of human cells and is frequently used to pseudotype retroviral vectors
GaLV TM subunit levels are decreased by lentiviral packaging constructs expressing human immunodeficiency virus type 1 (HIV-1) accessory proteins We have previously shown that levels of the GaLV Env trans-membrane (TM) subunit in cell lysates are strongly reduced in the presence of the lentiviral packaging plasmid pCMVΔR8.2 (R8.2) and that GaLV Env is unable to pseudotype lentiviral vector particles [13]
The GaLV Env protein is processed from a polypeptide precursor protein, Pr85, into SU and TM subunits that remain non-covalently linked in the viral particle [24]
Summary
The gibbon ape leukemia virus (GaLV) Env protein mediates entry into a wide range of human cells and is frequently used to pseudotype retroviral vectors. An incompatibility exists between GaLV Env and lentiviral vectors that results in decreased steady-state levels of the mature GaLV Env in cells and prevents its incorporation into lentiviral vector particles Both human immunodeficiency virus type 1 (HIV-1)based lentiviral and murine leukemia virus (MuLV)based retroviral vectors are used clinically in human gene therapies. A key feature of both lentiviral and retroviral vectors is their ability to incorporate heterologous fusion proteins [reviewed in [5]], in particular the broadly-tropic vesicular stomatitis virus (VSV) G protein [6,7], in a process known as pseudotyping. This allows user-defined host targeting of these vectors, depending on their downstream purpose. We observed that coexpression of lentiviral vector components led to decreased levels of GaLV Env in cells when compared to the expression levels observed in the presence of retroviral elements [13], suggesting a basis for this incompatibility
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