Expression of ABO transferases in cells producing HIV-1 particles leads to incorporation of ABO antigens into viral envelopes. (A) HIV-1-based vectors pseudotyped with the HXB2 envelope were produced in 293T cells cotransfected with the H (giving the O blood group) and A or B transferases, or the porcine α(1-3)galactosyltransferase. The envelope protein, gp120, from vectors was captured by immobilized anti-gp120 antibody. Captured ABO sugar was then detected with biotinylated ABO selective lectins H pomatia (A), BSI-B4 (B), or BSI (B and α(1-3)galactoside), or anti-HIV-1 human sera as a loading control. Background lectin binding was controlled by capturing media from ABO-expressing virus-negative 293T cells. Binding was detected by streptavidin-conjugated alkaline phosphates and expressed as relative light units. (B) Viral lysate was separated on a 10% SDS-PAGE gel and Western blotted using the same reagents as in panel A, this time detecting antigen with streptavidin-linked horseradish peroxidase. ABO-expressing 293T cell supernatant was used as a negative control. Arrows represent the 113-kDa molecular weight marker.