Pseudotyping retrovirus and lentivirus vectors with different viral fusion proteins is a useful strategy to alter the host range of the vectors. Although lentivirus vectors are efficiently pseudotyped by Env proteins from several different subtypes of murine leukemia virus (MuLV), the related protein from gibbon ape leukemia virus (GaLV) does not form functional pseudotypes. We have determined that this arises because of an inability of GaLV Env to be incorporated into lentivirus vector particles. By exploiting the homology between the GaLV and MuLV Env proteins, we have mapped the determinants of incompatibility in the GaLV Env. Three modifications that allowed GaLV Env to pseudotype human immunodeficiency virus type 1 particles were identified: removal of the R peptide (C-terminal half of the cytoplasmic domain), replacement of the whole cytoplasmic tail with the corresponding MuLV region, and mutation of two residues upstream of the R peptide cleavage site. In addition, we have previously proposed that removal of the R peptide from MuLV Env proteins enhances their fusogenicity by transmitting a conformational change to the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Our analysis of chimeric MuLV/GaLV Env proteins provides further evidence in support of this model and suggests that proper Env function involves both interactions within the cytoplasmic tail and more long-range interactions between the cytoplasmic tail, the membrane-spanning region, and the ectodomain of the protein.Retrovirus vectors derived from murine leukemia virus (MuLV) are the most commonly used gene transfer vectors in current human gene therapy applications (reviewed in reference 5). Like those of their parental retroviruses, the host ranges of such vectors are influenced in large part by the properties of the fusion protein contained in the outer lipid envelope of the vector particle. An attractive property of these vectors is the relative ease with which different fusion proteins can be incorporated into particles in place of the native envelope (Env) protein, a process referred to as pseudotyping. This allows the vectors to transduce ranges of cells and tissues different from those that would be possible with just the native Env.There are many examples of pseudotyping in the literature. Both MuLV and retrovirus vectors derived from it can be pseudotyped by the Env proteins from other type C mammalian retroviruses. These include proteins from the different subtypes of MuLV, such as amphotropic (7,34,46), polytropic (15, 29), and xenotropic (3, 46) subtypes and 10A1 (35), as well as the Env proteins from gibbon ape leukemia virus (GaLV) (36, 46, 63) and feline leukemia virus type B (59). In addition, Env proteins from more-distantly related retroviruses can also pseudotype MuLV particles, such as feline endogenous virus RD114 (46, 59), Jaagsiekte sheep retrovirus (48), human Tcell-lymphotropic virus type 1 (HTLV-1) (63), and simian immunodeficiency virus (21). Finally, MuLV-based vectors can also be pseudot...

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.

No abstract