Characterizing the Murine Leukemia Virus Envelope Glycoprotein Membrane-Spanning Domain for Its Roles in Interface Alignment and Fusogenicity

Abstract

The membrane-proximal region of murine leukemia virus envelope (Env) is a critical modulator of its functionality. We have previously shown that the insertion of one amino acid (؉1 leucine) within the membrane-spanning domain (MSD) abolished protein functionality in infectivity assays. However, functionality could be restored to this ؉1 leucine mutant by either inserting two additional amino acids (؉3 leucine) or by deleting the cytoplasmic tail domain (CTD) in the ؉1 leucine background. We inferred that the ectodomain and CTD have protein interfaces that have to be in alignment for Env to be functional. Here, we made single residue deletions to the Env mutant with the ؉1 leucine insertion to restore the interface alignment (gain of functionality) and therefore define the boundaries of the two interfaces. We identified the glycine-proline pairs near the N terminus (positions 147 and 148) and the C terminus (positions 159 and 160) of the MSD as being the boundaries of the two interfaces. Deletions between these pairs restored function, but deletions outside of them did not. In addition, the vast majority of the single residue deletions regained function if the CTD was deleted. The exceptions were four hydroxyl-containing amino acid residues (T139, T140, S143, and T144) that reside in the ectodomain interface and the proline at position 148, which were all indispensable for functionality. We hypothesize that the hydroxyl-containing residues at positions T139 and S143 could be a driving force for stabilizing the ectodomain interface through formation of a hydrogen-bonding network. IMPORTANCEThe membrane-proximal external region (MPER) and membrane-spanning domains (MSDs) of viral glycoproteins have been shown to be critical for regulating glycoprotein fusogenicity. However, the roles of these two domains are poorly understood. We report here that point deletions and insertions within the MPER or MSD result in functionally inactive proteins. However, when the C-terminal tail domain (CTD) is deleted, the majority of the proteins remain functional. The only residues that were found to be critical for function regardless of the CTD were four hydroxyl-containing amino acids located at the C terminus of the MPER (T139 and T140) and at the N terminus of the MSD (S143 and T144) and a proline near the beginning of the MSD (P148). We demonstrate that hydrogen-bonding at positions T139 and S143 is critical for protein function. Our findings provide novel insights into the role of the MPER in regulating fusogenic activity of viral glycoproteins. E nveloped viruses require membrane-spanning cell surface glycoproteins to coordinate fusion between viral and host cell membranes. Retroviral envelope (Env) glycoproteins are produced as precursors that undergo trimerization in the endoplasmic reticulum (1-3). Subsequently, the precursor trimer is cleaved into two subdomains: the receptor-binding surface domain (SU) and the fusion-promoting transmembrane domain (TM). This cleavage process is mediated by a host-cell furin, or f...

In Vivo Analysis of Infectivity, Fusogenicity, and Incorporation of a Mutagenic Viral Glycoprotein Library Reveals Determinants for Virus Incorporation

“…Nonetheless, some positions were strikingly sensitive to mutation, such as S124, R134, and S143, whereas others displayed more modest patterns of sensitivity, such as T139, T140, P148, and several residues in the MSD. These results are in agreement with our recent examination of residues in the membrane-proximal ectodomain that are critical for MLV Env fusogenic activity (41).…”

“…The G147-P148 pair at the N terminus of the MSD may facilitate the molecular reordering of TM during this process. In a recent study where we examined the coordination between the MPER and CTD interfaces that contribute to MLV Env functionality, we independently identified the hydroxyl residues T139, T140, S143, and T144 as being critical modulators of Env fusogenicity without affecting incorporation into viral particles (41). We proposed that these residues contribute a hydrogen-bonding network that stabilizes the TM trimer (47)(48)(49)(50)(51)(52).…”

Epstein–Barr virus BORF2 inhibits cellular APOBEC3B to preserve viral genome integrity

“…BORF2 with a C-terminal eGFP tag was generated by high-fidelity PCR of pcDNA4-BORF2-3xFlag using primers RSH13422 5’-NNN NAT GCA TCA TGG CAA CGA CCA GTC ATG TC-3’ and RSH13424 5’-NNN NAC GCG TCC TTG GCA AGA TTC ACA GGC TCG-3’. PCR products were digested with Nsi I-HF (NEB R3127) and Mlu I-HF (NEB R3198) and cloned into the previously described pQCXIP (Clontech) with a C-terminal eGFP tag 41 . BORF2 with C-terminal 3x-Flag tag was cloned into an MLV-based pQCXIP lentivirus vector (Clontech) for complementation experiments by PCR of pcDNA4-BORF2-3xFlag using primers RSH13422 5’-NNN NAT GCA TCA TGG CAA CGA CCA GTC ATG TC-3’ and RSH13423 5’-NNN NTT AAT TAA TTA AAC GGG CCC CTT GTC GTC-3’.…”

Across the Hall from Pioneers

“…The roles of the different TM domains in membrane fusion have recently been further investigated by Marc Johnson and his colleagues, using detailed genetic mapping techniques [ 57 , 58 ]. Based on their results, they suggest that TM trimers contain coiled coils in their ectodomains and in their cytoplasmic tails and that these trimeric interfaces, which are on opposite sides of the membrane, must be aligned for successful TM function.…”