Crystal Structures of Beta- and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry

Abstract

Membrane fusion is a key step in the life cycle of all envelope viruses, but this process is energetically unfavorable; the transmembrane fusion subunit (TM) of the virion-attached glycoprotein actively catalyzes the membrane merger process. Retroviral glycoproteins are the prototypical system to study pH-independent viral entry. In this study, we determined crystal structures of extramembrane regions of the TMs from Mason-Pfizer monkey virus (MPMV) and xenotropic murine leukemia virus-related virus (XMRV) at 1.7-Å and 2.2-Å resolution, respectively. The structures are comprised of a trimer of hairpins that is characteristic of class I viral fusion proteins and now completes a structural library of retroviral fusion proteins. Our results allowed us to identify a series of intra-and interchain electrostatic interactions in the heptad repeat and chain reversal regions. Mutagenesis reveals that charge-neutralizing salt bridge mutations significantly destabilize the postfusion six-helix bundle and abrogate retroviral infection, demonstrating that electrostatic stapling of the fusion subunit is essential for viral entry. Our data indicate that salt bridges are a major stabilizing force on the MPMV and XMRV retroviral TMs and likely provide the key energetics for viral and host membrane fusion. The fusion of the viral lipid bilayer with the host membrane is indispensable for the obligate intracellular life cycle of all enveloped viruses. Viral-host cell fusion is typically catalyzed by one or more virion-associated surface envelope glycoproteins, which are structurally organized into three classes: classes I, II, and III (reviewed in references 1 and 2). Regardless of the class, the glycoprotein is displayed on the surface of the virus in a metastable, prefusion conformation, and depending on the virus, the glycoprotein is activated for fusion through one of three principal mechanisms: (i) receptor binding in a pH-independent fashion, (ii) low pH, or (iii) a combination of both receptor binding and low pH (reviewed in references 2 and 3).Retroviruses are a distinct family of enveloped RNA viruses that are classified into seven genera: alpha-, beta-, delta-, epsilon-, and gammaretroviruses; lentivirus; and spumavirus. The retroviral envelope glycoproteins (termed Env) belong to the class I viral fusion protein family. Env is synthesized as a single-chain polypeptide precursor and posttranslationally cleaved to yield a receptor-binding surface (SU) domain and a fusion-active transmembrane (TM) domain. Mature Env is incorporated as a metastable trimer of SU-TM heterodimers on the surface of the virus. Initial attachment to host cells is mediated through the interaction of the SU domain with a host cell receptor. In many retroviruses, this is thought to trigger a conformational change that allows the TM subunit to form an extended prehairpin conformation that facilitates the insertion of its hydrophobic fusion peptide into the host plasma membrane. Subsequently, two ␣-helical heptad repeat regions (HR1 and HR2) in the TM ...

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

“…As also observed in the previously reported gamma-type TM E structures [22,24,25], the 6HB of the syncytins features two "belts" or networks of hydrogen bonds: belt 1 ties the N-helix at turn 7 to its adjacent protomers in the trimer and belt 2 at turns 9 and 10, close to the bottom of the bundle (Fig. 3A).…”

“…In the 6HB of MPMV and XMRV TM E , numerous electrostatic interactions were described between charged residues mapping to the same region ( Fig. 3D) [22]. In addition, the syn1 6HB contains a string of 8 inter-chain and 1 intra-chain salt bridges that tie the first 5 turns of the N-helix to the C-helix of the adjacent protomer in a vertical fashion, i.e.…”

“…1A). We used structural information available for a number of postfusion retroviral TM E conformations [22,24,25] together with aa sequence alignments and secondary structure/hydrophobicity plots [30,31] to define the boundaries of the constructs we used for the structural studies. These analyses ( Fig.…”

“…In this scheme, the conserved salt bridge connects R þ2 with E þ20 . Both residues were shown to be essential for the fusion activity of XMRV, MPMV, and HTLV Envs [22,34]. The TM E of the deltaretroviruses contains a second, upstream hydrophilic layer, which is in the case of HTLV-1 gp21 organized around Asn364 that traps a second chloride ion, and in BLV gp21 around Thr353 that chelates a water molecule on the 3-fold axis.…”

Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell

“…The full-length structure in the viral membrane was determined by electron cryo tomography (cET) followed by sub-volume averaging (Förster et al, 2005) and after detergent extraction using cEM single particle analysis (Wu et al, 2008) from MoMLV produced in the mouse fibroblast cell lines CL-1 and MOV-3, respectively. Additionally, parts of the TM domain of MoMLV Env (Fass et al, 1996) and a xenotropic MLV (Aydin et al, 2014) have been crystallized in their post-fusion state, which contains a six helix bundle. Still, information about the changes that MLV Env undergoes upon interaction with the host cell is lacking.…”