Multimerization Potential of the Cytoplasmic Domain of the Human Immunodeficiency Virus Type 1 Transmembrane Glycoprotein gp41

Abstract

We previously demonstrated that an envelope mutant of human immunodeficiency virus type 1 lacking the entire cytoplasmic domain interferes in trans with the production of infectious virus by inclusion of the mutant envelope into the wild-type envelope complex. We also showed that the envelope incorporation into virions is not affected when the wild-type envelope is coexpressed with the mutant envelope. These results suggest that an oligomeric structure of the cytoplasmic domain is functionally required for viral infectivity. To understand whether the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 has the potential to self-assemble as an oligomer, in the present study we fused the coding sequence of the entire cytoplasmic domain at 3 to the Escherichia coli malE gene, which encodes a monomeric maltose-binding protein. The expressed fusion protein was examined by chemical cross-linking, sucrose gradient centrifugation, and gel filtration. The results showed that the cytoplasmic domain of gp41 assembles into a high-ordered structural complex. The intersubunit interaction of the cytoplasmic domain was also confirmed by a mammalian two-hybrid system that detects protein-protein interactions in eucaryotic cells. A cytoplasmic domain fragment expressed in eucaryotic cells was pulled down by glutathione-Sepharose 4B beads via its association with another cytoplasmic domain fragment fused to the C terminus of the glutathione S-transferase moiety. We also found that sequences encompassing the lentiviral lytic peptide-1 and lentiviral lytic peptide-2, which are located within residues 828 -856 and 770 -795, respectively, play a critical role in cytoplasmic domain selfassembly. Taken together, the results from the present study indicate that the cytoplasmic domain of gp41 by itself is sufficient to assemble into a multimeric structure. This finding supports the hypothesis that a multimeric form of the gp41 cytoplasmic domain plays a crucial role in virus infectivity.

Wild-Type-Like Viral Replication Potential of Human Immunodeficiency Virus Type 1 Envelope Mutants Lacking Palmitoylation Signals

“…To construct the 764S 837S and 764A 837A double mutants, the 764S and 764A PCR products were used as templates, and the paired sense and antisense oligonucleotides for generating the 837S and 837A mutants were used as internal primers, respectively. All PCR amplifications were performed using Vent DNA polymerase (New England BioLabs, Beverly, MA) according to a previously described amplification program (26). The amplified mutated BamHI-XhoI fragments were cloned into the pHXB2RU3 provirus to generate various mutant proviruses.…”

“…We previously showed that the C-terminal twothirds of the cytoplasmic domain confers multimerization and plays a role in membrane association (13,26). It is likely that minor amino acid differences between the env clones used in the studies by Rousso et al (50), Bhattacharya et al (5), and us may be responsible for the differential effects of the deacylated Env proteins on lipid raft association, Env incorporation, and viral infectivity observed by the three groups.…”

Effect of Extension of the Cytoplasmic Domain of Human Immunodeficiency Type 1 Virus Transmembrane Protein gp41 on Virus Replication

“…For generation of pEBG clones encoding glutathione S-transferase (GST)-cytoplasmic subdomain fusion proteins, a series of cytoplasmic tail truncation mutant pSVE7puro plasmids (5) were used as templates in PCR as previously described (28). For construction of pEBG constructs that encoded GST fusion proteins containing elongated cytoplasmic domains, 706fSpeI (28) and oligonucleotide 5Ј-CCCGGTACCGCTCC CACCCCATCTGCTGC-3Ј were used as the sense and antisense primers, respectively, and various mutant HXB2RU3 proviruses were used as templates to generate the DNA fragments that were subsequently cloned into the pEBG vector. pHIVgptGag/30LE was constructed by substituting the NarI-SpeI fragment isolated from pNL4-3/30LE for the corresponding fragment in pHIVgptGag.…”

“…The three highly conserved amphipathic ␣-helical segments, designated lentiviral lytic peptide 1 (LLP)-1, LLP-2, and LLP-3, located in the C terminus of the cytoplasmic tail are thought to be associated with the inner surfaces of viral and cellular membranes (33,45). We previously showed that the multimerization potential and membrane binding ability of the cytoplasmic tail may play a crucial role in virus replication (3,5,7,28) and that the N-terminal segment of LLP-1 contains a structural determinant critical for modulating Env stability (27).…”

“…pCMVgag directs the synthesis of the Gag precursor driven by a CMV promoter. pEBG/706-856 encodes a mammalian elongation factor 1␣ promoter-driven cytoplasmic tail fragment fused to the C terminus of GST (28). GST/706-856 but not GST itself was incorporated into the Gag virus-like particles (Fig.…”