Background: Lentiviruses have transmembrane envelope glycoproteins (Env) with long cytoplasmic tails that have been implicated in the incorporation of the Env protein into virions. In SIV, gp41 CD is not absolutely required for viral replication. In vitro passage of SIVmac in certain human cell lines has been shown to select for variants with truncated cytoplasmic tails. The SIV Env CD contains multiple endocytosis signals that modulate its surface expression on infected cells. We created a number of mutations within the gp41 CD. We changed residue 767 to a stop codon; we changed residues 738 and 739 to stop codons; we mutated a prominent YXXf endocytosis motif (Y721G); and finally we created Q738stop, Q739stop, Y721G. The mutations were introduced into multiple genetic backgrounds: SIVmac239 (difficult-to-neutralize, CD4^-dependent) and M5 (easier-to-neutralize with 5 sites for N-linked glycosylation in gp120 eliminated). Selected mutations were also placed in SIV316 (highly macrophage-competent neutralization sensitive).

Methods: Env content in virions: 293T cells were transiently transfected by the calcium-phosphate method. Virions were purified from the clarified cell supernatant by ultracentrifugation and quantified with the p27 antigen capture. Identical quantities of p27 were analyzed by Western blotting. Infectivity: CEMx174 SIV-SEAP cells were exposed to increasing amounts of p27-containing virus. Secreted engineered alkaline phosphatase (SEAP) activity was measured using a Phosphalight kit. Neutralization: CEMx174-SEAP cells were used to measure the ability of SIV⁺ monkey sera to block infection.

Results: Truncations of the cytoplasmic domain of gp41 can have dramatic effects on Env incorporation. Env content in virions of SIVmac239 increased 25–50 fold by a stop codon at Env residue 767. 1) Increased Env content correlated with increased infectivity but increases in infectivity were not as great as the increases in Env content. 2) Both Env content and infectivity correlated inversely with sensitivity to antibody-mediated neutralization. 3) Truncation of gp41 had the most dramatic effect on the neutralization sensitivity of strain SIV316 where 50% neutralization went from 1:3,000 to 1:10.

Conclusions: Our results indicate that the sequences present in the cytoplasmic domain of gp41 can dramatically influence env content in virions and that env content in virions is an important determinant of relative sensitivity to antibody-mediated neutralization.