Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41

We previously demonstrated that an envelope mutant of human immunodeficienc y virus type 1 lacking the entire cytoplasmic domain interferes in trans wi th the production of infectious virus by inclusion of the mutant envelope i nto the wild-type envelope complex. We also showed that the envelope incorp oration into virions is not affected when the wild-type envelope is coexpre ssed with the mutant envelope. These results suggest that an oligomeric str ucture of the cytoplasmic domain is functionally required for viral infecti vity, To understand whether the cytoplasmic domain of human immunodeficienc y virus type 1 transmembrane protein gp41 has the potential to self-assembl e as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3' to the Escherichia coil malE gene, which en codes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and g el filtration. The results showed that the cytoplasmic domain of gp41 assem bles into a high-ordered structural complex. The intersubunit interaction o f the cytoplasmic domain was also confirmed by a mammalian two-hybrid syste m that detects protein-protein interactions in eucaryotic cells. A cytoplas mic domain fragment expressed in eucaryotic cells was pulled down by glutat hione-Sepharose 4B beads via its association with another cytoplasmic domai n fragment fused to the C terminus of the glutathione S-transferase moiety, We also found that sequences encompassing the lentiviral lytic peptide-1 a nd lentiviral lytic peptide-2, which are located within residues 828-856 an d 770-795, respectively, play a critical role in cytoplasmic domain self-as sembly. Taken together, the results from the present study indicate that th e cytoplasmic domain of gp41 by itself is sufficient to assemble into a mul timeric structure. This finding supports the hypothesis that a multimeric f orm of the gp41 cytoplasmic domain plays a crucial role in virus infectivit y.