Modular organization of the Friend murine leukemia virus envelope protein underlies the mechanism of infection

Abstract

Retrovirus infection is initiated by receptor-dependent fusion of the envelope to the cell membrane. The modular organization of the envelope protein of C type retroviruses has been exploited to investigate how binding of the surface subunit (SU) to receptor triggers fusion mediated by the transmembrane (TM) subunit. We show that deletion of the receptor-binding domain (RBD) from SU of Friend murine leukemia virus (Fr-MLV) abolishes infection that is restored by supplying RBD as a soluble protein. Infection by this mechanism remains dependent on receptor expression. When membrane attachment of the virus lacking RBD is reestablished by inserting the hormone erythropoietin, infection remains dependent on the RBD͞receptor complex. However, infection increases 50-fold to 5 ؋ 10 5 units͞ml on cells that also express the erythropoietin receptor. Soluble RBD from Fr-MLV also restores infection by amphotropic and xenotropic MLVs in which RBD is deleted. These experiments demonstrate that RBD has two functions: mediating virus attachment and activating the fusion mechanism. In addition, they indicate that receptor engagement triggers fusion by promoting a subgroup-independent functional interaction between RBD and the remainder of SU and͞or TM. R etrovirus infection is initiated by fusion of the virus envelope to the host cell membrane, resulting in delivery of the virion core into the cytoplasm. Fusion is triggered when the viral envelope proteins bind to receptors expressed on the host cell membrane. The retroviral envelope protein is a trimer of heterodimers formed between the surface (SU) and the transmembrane (TM) subunits (1, 2). At present, it is unclear how virus binding to receptor, which is mediated by SU, is coupled to fusion, which is mediated by TM. There is currently no evidence that the SU͞receptor complex participates in membrane fusion, nor that the receptor makes direct contact with TM. Therefore, critical intra-and possibly interenvelope interactions must occur that link receptor binding to the fusion mechanism.To investigate how this functional linking occurs, we have exploited the modular organization of the mammalian C-type retroviral envelope proteins (Fig. 1A). Sequence-based alignment of these proteins reveals a proline-rich region in the middle of SU that separates the N-terminal segment, which varies among murine leukemia virus (MLV) subgroups, from the C-terminal segment, which is relatively conserved (3-5). Chimeric envelope proteins, created by combining segments from viruses that use different receptors, are often functional and use the receptor specified by the N-terminal segment (4, 6, 7). In the SU of Friend MLV (Fr-MLV), the N-terminal 236 residues before the proline-rich region form a discrete domain (receptorbinding domain, RBD) that binds to receptor with high affinity and 1:1 stoichiometry (4,8,9). From functional studies informed by the atomic structure (Fig. 1B) (10), a pocket has been identified at the top of Fr-MLV RBD that is required for receptor binding and likely forms a cri...

Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering

“…According to the prevailing model, the receptor triggers changes in the SU subunit, including isomerization of the intersubunit disulfide (14)(15)(16). As a result, the TM subunits can refold, probably first into the putative extended form that can reach the target cell membrane with its N-terminal fusion peptide, and then into the backfolded form, the formation of which is thought to drag the viral and cell membranes together for fusion.…”

“…Receptor binding has been suggested to trigger a cascade of conformational signals from RBD to TM via the C-terminal SU domain (14,15). The SU and TM are coupled by a disulfide bond, and activation requires its isomerization (16).…”

Identification of a Novel Subgroup of Koala Retrovirus from Koalas in Japanese Zoos

“…The CETTG motif, which is involved in fusion activity (18), was disrupted by mutations in both KoRV-A and KoRV-J. KoRV-J had a replacement of Gln with Arg in the PHQ motif, which is important for the fusion activity (19)(20)(21)(22). Other important motifs that influence the functions of Env and/or the viral infectivity were highly conserved between the two subgroups.…”

Surface-engineering of lentiviral vectors

“…Examples of insertion sites in type C mammalian retrovirus glycoproteins -mainly the ecotropic and amphotropic MLV glycoproteins -that have been characterised and found to be functional, at least for retargeted binding, are shown in Figure 1. They include modifications of the glycoprotein such as (1) domain replacement [74][75][76], (2) peptide insertion in pre-folded domains [68,[77][78][79][80], and (3) display of polypeptides as additional folded domains [81][82][83][84][85][86][87][88][89][90][91][92]. Many of these chimeric glycoproteins fold correctly, are stably incorporated on virions and allow efficient retargeted virion binding to the expected cell-surface molecules.…”

“…Closer examination of the molecular mechanism that couples binding to the natural retroviral receptors to fusion activation has in fact revealed the very sophisticated solution adopted by retroviruses to coordinate these two functions. This coupling involves complex interrelations between the different subdomains of the glycoprotein complex [66,74,[101][102][103][104][105]. Attempts to reconstitute this coupling via the interaction of a non-viral target receptor to a non-viral ligand have so far been unsuccessful, even when inserted into a fusion-competent retroviral glycoprotein [106].…”