Vpu-dependent block to incorporation of GaLV Env into lentiviral vectors

Abstract

Background: 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. 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.

Results: We 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 beta 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.

Conclusions: Similar to the mechanism whereby Vpu targets BST-2/tetherin for degradation, these findings implicate beta-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.

Figure 1

Schematic representation of retroviral (pCgp) and lentiviral (R8.2, R8.2ΔVpu, R8.91 and R8.91Vpu) packaging constructs. All lentiviral vectors express Gag, Pol, Tat and Rev; inclusion of Vif, Nef, Vpu and Vpr is as indicated (grey boxes).

Figure 2

GaLV Env levels and titers of pseudotyped vectors. A. Western blot of a representative experiment showing levels of GaLV TM, HIV-1 p24, MLV p30 and actin in cell lysates and pelleted supernatants from 293T cells co-transfected with plasmids expressing GaLV Env, together with pCgp, R8.2, R8.91 or a control (Ctrl.) plasmid, as indicated. Also shown is a quantitative analysis of the steady-state levels of GaLV TM in cell lysates, made relative to the levels in the presence of control plasmid. Results are mean of three independent experiments. B. Titers of GaLV Env and VSV G pseudotyped retroviral (pCgp) and lentiviral (R8.2 and R8.91) vectors, as indicated, expressed as transducing units per ml (TU/ml). * indicates no detectable titer.

Figure 3

Representative Western blot and quantitative analysis of signal density on blots from three independent experiments analyzing GaLV TM levels in lysates of 293T cells co-transfected with GaLV Env, pHR' and indicated derivatives of R8.2 and R8.91. Immunological detection of cellular HIV-1 p24 and actin levels were included as controls.

Figure 4

Representative Western blot and quantitative analysis of signal density on blots from three independent experiments analyzing GaLV TM levels in lysates of 293T cells co-transfected with GaLV Env, pHR' and indicated derivatives of R8.2 or R8.91, with (+) expression of Vpu in trans, from plasmid CΔEvpu. Immunological detection of HIV-1 p24 and actin levels were included as controls.

Figure 5

Representative Western blot and quantitative analysis of signal density on blots from three independent experiments analyzing GaLV TM levels in lysates of 293T cells co-transfected with GaLV Env, pHR' and the indicated packaging plasmids. (SN) indicates Vpu containing substitutions S52N and S56N. Immunological detection of HIV-1 p24 and cellular actin levels were included as controls.

Figure 6

Western blot and quantitative analysis of GaLV TM levels for wild-type and mutant versions of GaLV Env in the presence of pHR' and either control plasmid pGEM, or plasmids R8.2 or R8.91. Graphs indicate relative levels of GaLV TM compared to the pGEM control, shown as the mean of 3-5 independent experiments. p-values were calculated, and statistical significance (p < 0.05) is indicated by an asterisk. HIV-1 p24 immunological detection was included as control.

Figure 7

Analyses of levels of GaLV Env Pr85 precursor and SU subunit. A. Schematic representation of FLAG-tagged GaLV Env, showing precursor (Pr85), mature SU and TM subunits, signal peptide (SP) and R peptide (R). B. Representative Western blots and quantitative analysis of ratio of levels of FLAG-tagged GaLV Env Pr85 precursor and SU subunit, in presence of pHR' and either control plasmid pGEM, or packaging plasmids R8.2, R8.91 or R8.2ΔVpu. Samples were deglycosylated with either PGNase F or endoH prior to SDS-PAGE, and analyzed by immunoblotting with anti-FLAG antibody.

Figure 8

Representative Western blots of levels of FLAG-tagged GaLV Env Pr85 precursor and SU subunit in presence of pHR' and either control plasmid pGEM (Ctrl.), R8.2 or R8.2ΔVpu. Cells were treated with either the proteasomal inhibitor MG132, the lysosomal inhibitor bafilomycin A1, or DMSO. Samples were deglycosylated using endoH prior to SDS-PAGE, and analyzed by immunoblotting with anti-FLAG antibody.