pressing T7 RNA polymerase (20), was kindly provided by T. Fuerst and B.Moss, and the HIV-1 (NL-43) gp160 cDNA was from W.Garten and H.-D.Klenk (Institute of Virology, University of Marburg, Germany). The G-deficient Street-Alabama-Dufferin RV mutant, SAD ΔG, was recovered and propagated after deletion of the entire G protein coding region from a full-length RV cDNA in cells expressing RV G from a transfected plasmid in the vaccinia/T7 polymerase system (7).
Construction of Expression Plasmids. An expression plasmid encoding wild-type HIV-1 Env (pEnv) was generated by insertion of the BbsI-XhoI-Klenow fragment spanning the entire gp160 coding region from the HIV NL-43 cDNA in the EcoRV site of pT7T (21). To replace the HIV cytoplasmic domain, an HpaI site (underlined) was introduced by PCR using a primer (5′-GCTCTAGACTAGTTAACTATAGAAAGTACAGC-3′) overlapping the transmembrane/ cytoplasmic domain-encoding region and a second primer (5′-AACAATTACACAAGCTTAAT-3′) spanning an upstream unique HindIII site (underlined) of the Env cDNA. An HpaI site was also introduced by PCR (5′-AAGTCGACCGTTAACAGAAGAGTCAATCGATCA-3′) upstream of the pT7T-G sequence encoding the RV SAD B19 G cytoplasmic tail. After ligation of the HIV-derived HindIII-HpaI fragment and the G-derived HpaI-PstI fragment, the construct was used to replace the sequence downstream of HindIII in pEnv to yield pEnv-RVG. The removal of an HpaI fragment of pEnv resulted in pEnv-Δ107; the encoded protein possessed a deletion of 107 amino acids adjacent to the transmembrane domain (see Fig. 1). The Env construct possessing a carboxyl-terminal truncation (pEnv-44) was generated by introduction of a translational stop codon (complementary sequence underlined) into the cytoplasmic tail-encoding sequence of pHIV-Env by PCR mutagenesis using a primer with the sequence 5′-ACGAATTCATTAGTTCACTAATCGAATG-3′.
Expression of Envelope Proteins. BSR cells were first infected with vTF7–3 at a multiplicity of infection of 5. After 1 hr of infection, cells were transfected with CsCl-purified plasmids by using the Stratagene mammalian transfection kit as described (21). To adjust the level of cell surface expression, various concentrations of plasmid DNA were used for transfection (see below). After incubation for 16 hr, cells were fixed with 4% paraformaldehyde and incubated at 4°C for 30 min with human anti-HIV-1 IgG (1:500 dilution) or a monoclonal antibody directed against RV G protein (diluted 1:100). Cells were stained with fluorescein isothiocyanate-conjugated goat anti-human IgG or goat anti-mouse IgG (Dianova, Hamburg, Germany). For double staining, rhodamine-conjugated goat anti-mouse IgG (Dianova) was used in combination with fluorescein isothiocyanate-conjugated goat anti-human IgG.
Complementation of SAD ΔG with Envelope Proteins. BSR cells were infected at a multiplicity of infection of 1 with SAD ΔG phenotypically complemented with RV G protein and were then superinfected with vTF7–3. Plasmids encoding the spike proteins, pT7T-G, pEnv, pEnv-Δ107, pEnv-44, and pEnv-RVG (1, 7.5, 5, 3, and 3 μg, respectively) were then transfected as described above. After incubation at 37°C for 24 hr, cell culture media were harvested and cleared of cell debris. To determine the infectious titers of pseudotype viruses, supernatants were serially diluted and used for inoculation of confluent monolayers of CD4+ or CD8+ HeLa cells. After 24 hr of infection cells were fixed with 80% acetone and expression of RV nucleoprotein was examined by direct immunofluorescence after staining with an fluorescein isothiocyanateconjugate directed against RV nucleoprotein (Centocor).
Sucrose Gradients and Western Blotting. Supernatants from complementation experiments (approximately 6×106 cells) were harvested 24 hr after transfection, and virions were pelleted through a 10% sucrose cushion at 19,000 rpm in a Beckman SW 41 rotor. Pellets were resuspended in TEN buffer (10 mM Tris, pH 7.4/50 mM NaCl/1 mM EDTA), layered on continuous 10–50% sucrose gradients, and centrifuged at 27,000 rpm in an SW 41 rotor for 1 hr. Virus proteins from 12 equal gradient fractions were resolved by SDS/PAGE and transferred to nitrocellulose membranes using a semidry transfer apparatus (Hoefer). After incubation with a blocking solution (2.5% dry milk/0.05% Tween 20 in PBS), the blot was incubated overnight with a mixture of human HIV-1 IgG (1:10,000) and rabbit sera raised against purified RV G protein (S72, 1:20,000), RV ribonucleoprotein complex (S50, 1:20,000) or a peptide deduced from the the RV matrix protein sequence (M1–B4, 1:10,000) in PBST. After three successive washes in PBST, the blot was incubated for 2 hr with a mixture of peroxidase-conjugated goat anti-human and goat anti-rabbit IgG (Dianova) diluted 1:10,000 in PBST. The blot was washed as above, stained with an Enhanced Chemiluminescence Western blot detection kit (Amersham) for 1 min, and exposed to x-ray films (Biomax MR, Kodak).
The HIV-1 env protein is synthesized as a precursor (gp160) that is cleaved during transport to the plasma membrane into two noncovalently associated subunits, gp120, which is involved in binding to the HIV receptor, and gp41, which contains the membrane anchor and a carboxyl-terminal cytoplasmic domain of 150 amino acids (22, 23). To determine the influence of both sequence and length of the cytoplasmic domain on formation of RV(HIV) pseudotypes, various cDNA constructs resulting in altered cytoplasmic domains were prepared in the expression plasmid pT7T (21), which is under the control of the T7 RNA polymerase promoter. Starting from a plasmid encoding the authentic HIV-1 NL-43 Env protein (pEnv), we prepared a construct that encodes a protein in which the entire Env cytoplasmic domain is replaced by the 44 amino acid cytoplasmic domain of RV G (EnvRVG). To investigate whether the considerable length of the HIV Env cytoplasmic domain may affect incorporation into RV particles, two other plasmids encoding proteins with short Env cytoplasmic domains, similar in length to that of RV G, were constructed. Env-44 represented a protein with a carboxyl-terminally truncated cytoplasmic domain, whereas in pEnv-Δ107 an internal deletion removed the membrane-proximal 107 amino acids and fused the carboxyl-terminal 43 residues to the membrane anchor domain (Fig. 1).
Transient Expression of Glycoproteins. To analyze expression of the recombinant glycoproteins on the cell surface, which represents a prerequisite for incorporation into RV particles, plasmids were transfected into BSR cells that had been infected with the recombinant vaccinia virus vTF7–3 providing T7 RNA polymerase (20). Surface expression was compared by indirect immunofluorescence with human anti-HIV IgG (18) after transfection of equal amounts of protein-encoding plasmids. Interestingly, compared with the authentic HIV-Env, the level of cell surface expression was higher for all Env constructs with modified cytoplasmic domains (data not shown). To obtain similar levels of expression, the amounts of the plasmids in transfection experiments were therefore adjusted. After transfection of 7.5 μg pT7T-Env and of 5, 3, and 3 μg of pEnv-Δ107, pEnv-44, and pEnv-RVG, respectively, surface fluorescence intensity and the number of expressing cells were similar. Representative micrographs are shown in Fig. 2. By double labeling cells expressing both RV G and each of the plasmids encoding an Env protein, no differences in the distribution of proteins on the cell surface were observed (data not shown).
We next determined whether the introduced mutations affected the function of the HIV envelope proteins in attachment to the HIV receptor and induction of membrane fusion. The adjusted amounts of plasmids were transfected into HeLa