Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L.; Bulling, Manuela; Klingström, Jonas; Mancini, R. C.; Lozach, Pierre‐Yves; Tischler, Nicole D.

doi:10.1128/jvi.03118-13

Cited by 42 publications

(40 citation statements)

References 39 publications

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“…These mutants were all expressed correctly and reached the cell surface (S5 Fig), but syncytium formation and cell entry by the corresponding pseudotyped SIV particles was abrogated (Fig 4D and 4E). In order to further explore whether trimerisation of the mutants is impaired, we made virion-like particles (VLPs) of Andes virus [60] harboring the mutations D11A and H14Y in Gc. We harvested VLPs from cells transfected with wild type Gn/Gc or with wild type Gn and the mutant Gc, which secreted VLPs at similar levels (S5 Fig).…”

Section: Resultsmentioning

confidence: 99%

“…VLPs were harvested from supernatants of 293FT cells transfected with the pI.18/GPC wt or the different mutant constructs at 48 h post-transfection as previously established [60]. Subsequently, VLPs were concentrated by ultracentrifugation for one hour at 135,000 g and detected by reducing SDS PAGE and western blot using anti-Gc MAb as described above.…”

Section: Methodsmentioning

confidence: 99%

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Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

et al. 2016

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Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

et al. 2016

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“…R1074 (R417 in Gc numbering) is the only residue at the base of PUUV sG C stem that maintains side chain intramolecular contacts with domain II and it is highly conserved among hantaviruses (except HNTV and SEOV where it is substituted with lysine of similar properties, S2 Fig). To investigate the role of R1074 in membrane fusion, we introduced an alanine substitution of R1074 in both, PUUV and ANDV GPC in order to test their activity in the available in vitro systems established mostly for ANDV [17, 58]. The R1074A mutants of ANDV and PUUV G C were expressed as the wild type proteins, localized on the cell surface and assembled into virus like particles (VLPs) (S4A Fig).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation

et al. 2016

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Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens.

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“…However, not all 3 proteins are necessary for VLP formation. Rodrigo Acuna et al showed that Gn and Gc co-expressed Gn and Gc in mammalian cells assemble into VLPs, suggesting that the NP protein is not necessary for VLP assembly (Acuna et al, 2014). VLPs with other members of the Bunyaviridae family can also form after Gn and Gc co-expression (Overby et al, 2006;Mandell et al, 2010).…”

Section: Bunyaviridae Familymentioning

confidence: 99%

Virus like particle-based vaccines against emerging infectious disease viruses

et al. 2016

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Emerging infectious diseases are major threats to human health. Most severe viral disease outbreaks occur in developing regions where health conditions are poor. With increased international travel and business, the possibility of eventually transmitting infectious viruses between different countries is increasing. The most effective approach in preventing viral diseases is vaccination. However, vaccines are not currently available for numerous viral diseases. Virus-like particles (VLPs) are engineered vaccine candidates that have been studied for decades. VLPs are constructed by viral protein expression in various expression systems that promote the selfassembly of proteins into structures resembling virus particles. VLPs have antigenicity similar to that of the native virus, but are non-infectious as they lack key viral genetic material. VLP vaccines have attracted considerable research interest because they offer several advantages over traditional vaccines. Studies have shown that VLP vaccines can stimulate both humoral and cellular immune responses, which may offer effective antiviral protection. Here we review recent developments with VLP-based vaccines for several highly virulent emerging or re-emerging infectious diseases. The infectious agents discussed include RNA viruses from different virus families, such as the Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Togaviridae families.

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Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

Cited by 42 publications

References 39 publications

Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation

Virus like particle-based vaccines against emerging infectious disease viruses

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