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

bRetrovirus infection starts with the binding of envelope glycoproteins to host cell receptors. Subsequently, conformational changes in the glycoproteins trigger fusion of the viral and cellular membranes. Some retroviruses, such as avian sarcoma/leukosis virus (ASLV), employ a two-step mechanism in which receptor binding precedes low-pH activation and fusion. We used cryoelectron tomography to study virion/receptor/liposome complexes that simulate the interactions of ASLV virions with cells. Binding the soluble receptor at neutral pH resulted in virions capable of binding liposomes tightly enough to alter their curvature. At virion-liposome interfaces, the glycoproteins are ϳ3-fold more concentrated than elsewhere in the viral envelope, indicating specific recruitment to these sites. Subtomogram averaging showed that the oblate globular domain in the prehairpin intermediate (presumably the receptor-binding domain) is connected to both the target and the viral membrane by 2.5-nm-long stalks and is partially disordered, compared with its native conformation. Upon lowering the pH, fusion took place. Fusion is a stochastic process that, once initiated, must be rapid, as only final (postfusion) products were observed. These fusion products showed glycoprotein spikes on their surface, with their interiors occupied by patches of dense material but without capsids, implying their disassembly. In addition, some of the products presented a density layer underlying and resolved from the viral membrane, which may represent detachment of the matrix protein to facilitate the fusion process.

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confidence: 99%

Visualization of the Two-Step Fusion Process of the Retrovirus Avian Sarcoma/Leukosis Virus by Cryo-Electron Tomography

Cardone

Brecher

Fontana

et al. 2012

“…This suggests that the sequence just proximal to the MSD can modulate functionality. MLV Env is trimeric in the ectodomain and also is modeled to be trimeric in the CTD prior to R-peptide cleavage (11,29). A recent study by Löving et al (29) demonstrated that the membrane-proximal ectodomain of MLV Env is tightly packed prior to R-peptide cleavage.…”

Section: Resultsmentioning

confidence: 99%

“…MLV Env is trimeric in the ectodomain and also is modeled to be trimeric in the CTD prior to R-peptide cleavage (11,29). A recent study by Löving et al (29) demonstrated that the membrane-proximal ectodomain of MLV Env is tightly packed prior to R-peptide cleavage. These pieces of data led us to hypothesize that there are protein contacts in both the glycoprotein ectodomain and CTD that contribute to Env trimerization and that glycoprotein function is inhibited if these two trimer interfaces are not synchronous with each other.…”

Section: Resultsmentioning

confidence: 99%

“…Cleavage of the R-peptide allows for receptor-dependent fusion of the Env protein to the target cell (25,28). Recent cryo-electron microscopy (cryo-EM) imaging data suggest that the CTD of murine leukemia virus (MLV) Env holds the MLV Env ectodomain in a tight conformation, but upon cleavage of the R-peptide, the TM legs are splayed, and this allows fusogenic activation of Env (29).…”

mentioning

confidence: 99%

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Retrovirus Glycoprotein Functionality Requires Proper Alignment of the Ectodomain and the Membrane-Proximal Cytoplasmic Tail

Janaka

Gregory

Johnson

2013

Nonnative viral glycoproteins, including Friend murine leukemia virus envelope (F-MLV Env) are actively recruited to HIV-1 assembly sites by an unknown mechanism. Because interactions with the lipid microenvironment at budding sites could contribute to recruitment, we examined the contribution of the hydrophobicity of the F-MLV Env membrane-spanning domain (MSD) to its incorporation into HIV-1 particles. A series of F-MLV Env mutants that added or deleted one, two, or three leucines in the MSD were constructed. All six mutants retained the ability to be incorporated into HIV-1 particles, but the ؊1L, ؊2L, ؊3L, ؉1L, and ؉2L mutants were not capable of producing infectious particles. Surprisingly, the ؉3L Env glycoprotein was able to produce infectious particles and was constitutively fusogenic. However, when the cytoplasmic tail domains (CTDs) in the Env constructs were deleted, all six of the MSD mutants were able to produce infectious particles. Further mutational analyses revealed that the first 10 amino acids of the CTD is a critical regulator of infectivity. A similar phenotype was observed in HIV-1 Env upon addition of leucines in the MSD, with ؉1 and ؉2 leucine mutations greatly reducing Env activity, but ؉3 leucine mutations behaving similar to the wild type. Unlike F-MLV Env (؉1L and ؉2L), HIV-1 Env (؉1L and ؉2L) infectivity was not restored by deletion of the CTD. We hypothesize that the CTD forms a coiled-coil that disrupts the protein's functionality if it is not in phase with the trimer interface of the ectodomain. E nveloped viruses assemble by the confluence of structural proteins, genetic material, and surface glycoproteins. Glycoprotein-deficient viruses have the potential to be complemented by diverse, nonnative viral glycoproteins in a process termed pseudotyping (reviewed in references 1 to 4). The molecular mechanisms underlying the recruitment of foreign glycoproteins to budding viral particles are not understood. However, such pseudotyped viruses can be used as a gene delivery tool to direct viruses to a specific cell type (5-10).Retroviral Env glycoproteins are produced as precursors that trimerize in the endoplasmic reticulum (ER). Subsequently, the precursor protein is cleaved by furin or a furin-like protease into its constituent subdomains: the surface subunit (SU) containing the receptor binding domain and the transmembrane subunit (TM) containing the fusion-promoting domain. The mature Env is thus a trimer of heterodimers. SU and TM are held together by an intersubunit disulfide bond in the case of alpha-, gamma-, and deltaretroviruses (11-16) and noncovalently associated in the case of lentiviruses and betaretroviruses (17, 18). Upon receptor binding by SU, several conformational changes take place in Env, promoting coreceptor binding and/or accession of the host cell membrane by the fusion peptide in TM (19). For gammaretroviruses, fusogenicity is controlled by the cytoplasmic tail domain (CTD) of Env and isomerization of the disulfide bond between SU and TM (20-22). The C termini ...

“…In the case of P148, it is possible that this residue, in combination with glycine at position 147, forms a molecular hinge that allows for a conformational change within the transmembrane domain (p15E/TM) stalk during activation of the fusion mechanism. Recent cryoelectron microscopy imaging data indicate that the CTD of MLV Env holds the ectodomain in a tight conformation; however, cleavage of the R peptide results in a conformational rearrangement in TM wherein the trimer helices are splayed apart, allowing for fusogenic activation of Env (46). The G147-P148 pair at the N terminus of the MSD may facilitate the molecular reordering of TM during this process.…”

Section: Discussionmentioning

confidence: 99%

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

Salamango

Alam

Burke

et al. 2016

Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into nonnative viral particles in a process termed pseudotyping; however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. In this study, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the membrane-proximal external region (MPER), particularly W127 and W137, and the residues in the membranespanning domain (MSD) and also immediately flanking it (T140 to L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly. IMPORTANCEResearchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest that pseudotyping occurs through specific lipid-protein interactions at the viral assembly site. Formation of infectious retroviral particles requires a symphony of viral and host cell proteins to coordinate in a spatial and temporal manner. Typically, this is an exclusive process wherein components from a given virus assemble only with components from the same or closely related viruses, such as HIV-1 and HIV-2 (1-5). However, this exclusivity is not typical for the incorporation of viral glycoproteins. Incorporation of foreign glycoproteins into nonnative viral particles is termed pseudotyping (6-8), and researchers from numerous fields routinely exploit this ability to manipulate viral tropism. Although very little is known about the molecular mechanisms that govern this process, it is clearly not random. For example, we have shown using scanning electron microscopy (SEM) that both vesicular...