Targeting retroviral vectors to CD34-expressing cells: Binding to CD34 does not catalyze virus-cell fusion

We have attempted to engineer murine leukemia virus (MuLV)-based retroviral vectors to specifically transduce cells expressing human CD34, an antigen present on the surface of undifferentiated hematopoietic stem cells. A numb er of chimeric ecotropic MuLV envelope (Env) proteins were constructed that contained anti-CD34 single-chain antibody variable fragments (scFvs). The scFv-Env proteins were generated either by replacing the receptor-binding d omain of Env with the scFv or by inserting the scFv into the N terminus of the Env protein. Only chimeric Env proteins with scFv insertions between am ino acids 6 and 7 were incorporated into viral particles, and coexpression of native MuLV Env did not rescue incorporation-defective proteins. In addi tion, the efficiency of incorporation varied with the specific anti-CD34 sc Fv that was used, Retroviral vectors containing the scFv-Env proteins bound to CD34(+) cells and transduced NIH 3T3 cells expressing human CD34 (3T3-C D34 cells) at approximately twice the efficiency of the parental NIH 3T3 ce lls. However, the introduction of the mutation D84K, which prevents binding to the ecotropic MuLV receptor meat-1, prevented transduction of both NIH 3T3 and 3T3-CD34 cells. Complementation cell-cell fusion assays [Zhao et al , (1997). J. Virol. 71, 6967-6972] in 3T3-CD34 cells revealed that although the scFv-Env proteins could contribute postbinding entry functions when bo und to meat-1, they were unable to do so when bound to CD34. Taken together , these data suggest that although the interaction with CD34 effectively in creased the concentration of virus on 3T3-CD34 cells, entry could occur onl y through an interaction with meat-1; CD34 alone was not capable of trigger ing the appropriate postbinding changes that lead to viral entry.