Article Figures & Data
Figures
- FIG. 1.
Mutations introduced into CAEV-63 gp135. (A) Mature gp135 sequence with mutations shown for each amino acid position below the sequence. The sequence between parentheses indicates multiple substitutions in mutant HV2-GRQG. The CAEV gp135 regions homologous to β-strands 4 to 12 and 25 and loops LA to LD of HIV-1 gp120 (8, 15) are shown above the sequence. The gp135 sequences conserved in most or all lentiviruses (9) are highlighted. The mutations reducing MAb 29A binding are boxed. (B) Ribbon diagram of the CD4-bound gp120 core. The gp120 inner and outer domains are indicated. The region most conserved between HIV-1 gp120 and CAEV gp135 (β-strands 4 to 8 and 25 in the inner-proximal region) is indicated in black. The virion envelope is located toward the top. (C) Detail of the inner-proximal region of HIV-1 gp120 indicating the position of β-strands 4 to 8 and 25. The conserved β4-β5 and β25 regions highlighted in panel A are highlighted in black. The locations of gp120 residues corresponding to CAEV gp135 residues P117, Y118, P119, and V516 are shown. The ribbon diagrams in panels B and C were produced with the Pymol software.
- FIG. 2.
Formation of syncytia in human cells by CAEV envelope glycoprotein mutant P117D. Transfected 293T cells expressing the wild-type (top) and P117D mutant (bottom) CAEV-63 envelopes, respectively, were fixed in PBS containing 0.5% glutaraldehyde 24 h posttransfection and stained with hematoxylin and eosin.
- FIG. 3.
Immunoprecipitation of gp135 mutants with MAbs against CAEV-63 gp135. Supernatants of [35S]methionine-labeled 293T cells expressing the wild-type and mutant CAEV-63 envelopes were immunoprecipitated with 10 μl of immune serum from goat 8517 or 4 μg of MAb F7-299, 74A, or 29A. The amounts of gp135 in the supernatants were determined by ELISA with MAb F7-299 and HRP-labeled MAb 74A as capture and detection reagents, respectively, and 60 ng of gp135 was used in each reaction mixture. Representative results of at least two experiments are shown.
- FIG. 4.
Immunoprecipitation of envelope glycoproteins from cell lysates and supernatants of transfected 293T cells. (A) Sera from CAEV-63-infected goat 8517 (lanes 1 to 4) and uninfected goat 8505 (lanes 5 to 8) were used to immunoprecipitate envelope glycoproteins from 293T cells transfected with plasmid pCMVCO2 (CAEV-CO), pCMV63 (CAEV-63), or pCMV63S (F715/Stop) or control plasmid VR1012. Positions of 14C-labeled markers are shown on the right. The positions of gp135 and the envelope glycoprotein precursor (gPr150) are shown on the left. (B) Immunoprecipitation of envelope glycoproteins in supernatants (top) and cell lysates (bottom) of 293T cells expressing the mutant or wild-type CAEV-63 envelope glycoprotein with an excess amount of serum from goat 8517. Representative results of at least two experiments are shown.
- FIG. 5.
Immunoprecipitation of gp135 and gp150 envelope precursor by MAbs. Supernatants of 293T cells expressing full-length (lanes 2, 4, 6, and 8) and truncated (lanes 1, 3, 5, and 7) CAEV envelope glycoproteins were immunoprecipitated with serum from goat 9308 (lanes 1 and 2) or MAb F7-299 (lanes 3 and 4), 74A (lanes 5 and 6), or 29A (lanes 7 and 8). The positions of soluble gp135 and the soluble envelope glycoprotein precursor (gPr140) are shown on the left.
Tables
- TABLE 1.
Effects of envelope glycoprotein mutations on intersubunit association
Env variant MAbb Concn of gp135 (ng/ml) ina: Association indexc Cell lysate Supernatant CAEV-63 29A 8,332 2,083 1.00 F715/Stopd 29A 640 4,964 0.03 N96T 29A 4,959 1,223 1.01 HV2-GRQG 29A 6,290 1,405 1.12 L6R 29A 983 1,048 0.23 P117D 29A 909 1,857 0.12 Y118R 29A 781 1,012 0.19 Y118F 29A 1,563 1,132 0.35 V516F 29A 638 419 0.38 CAEV-63 74A 6,926 1,609 1.00 F715/Stopd 74A 1,170 3,030 0.09 N96T 74A 4,149 1,001 0.96 HV2-GRQG 74A 5,485 1,105 1.15 P119E 74A 605 650 0.22 Y521D 74A 1,361 753 0.42 ↵ a The culture medium of transfected cell cultures was replaced, and supernatants (4 ml) and cell lysates (1 ml) were harvested at 20 and 44 h posttransfection, respectively.
↵ b HRP-conjugated MAb used for detection of F7-299-captured SU.
↵ c The index of gp135 association to gp38 was calculated by the following formula: ([mutant gp135cell lysate] × [wild-type gp135supernatant])/([wild-type gp135cell lysate] × [mutant gp135supernatant]).
↵ d The F715/Stop mutant expresses an envelope glycoprotein truncated just before the transmembrane anchor of TM.
- TABLE 2.
Functional activity of mutant envelope glycoproteins
Env variant Fusion in GSM cellsa Infectivity (FFU/ml)b % Reduction (avg ± SD) of CAEV-AP(CO) infectivity by gp135 at: 50 ng/ml 100 ng/ml 200 ng/ml CAEV-63 +++ 1.5 × 105 42.1 ± 3.0 71.4 ± 1.4 95.7 ± 0.9 L6R − <10 41.9 ± 9.9 70.0 ± 2.5 81.0 ± 3.8 N96T +++ 9.5 × 104 P117D +++ 9.2 × 102 46.7 ± 3.0 74.3 ± 2.5 91.1 ± 2.2 Y118R − <10 44.3 ± 4.0 70.5 ± 6.3 89.9 ± 2.2 Y118F ++ 5.0 × 104 54.8 ± 1.6 82.3 ± 5.8 99.7 ± 2.2 P119E + 1.1 × 104 42.4 ± 4.4 62.9 ± 5.2 88.6 ± 7.6 HV2-GRQG ++ 1.3 × 105 V516F − <10 48.4 ± 5.8 68.3 ± 2.5 91.1 ± 5.8 Y521D − <10 48.9 ± 1.9 71.5 ± 6.5 91.1 ± 4.4 ↵ a Fusion of transfected 293T cells with GSM cells relative to CAEV-63 Env in at least two experiments: +++, >66% relative fusion; ++, 33 to 66% relative fusion; +, <33% relative fusion; −, no fusion.
↵ b Infectivity of CAEV-AP pseudotyped with indicated envelopes in GSM cells. Values are averages of two experiments.
