The structure of RVFV fusion protein reveals a lipid binding pocket conserved in class-II fusion proteins

ABSTRACT The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.

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Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus

Journal of Virology ◽

10.1128/jvi.78.7.3514-3523.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3514-3523 ◽

Cited By ~ 20

Author(s):

Don L. Gibbons ◽

Brigid Reilly ◽

Anna Ahn ◽

Marie-Christine Vaney ◽

Armelle Vigouroux ◽

...

Keyword(s):

Fusion Protein ◽

Fusion Proteins ◽

Semliki Forest Virus ◽

Three Dimensional ◽

Class Ii ◽

Class I ◽

Structural Basis ◽

Viral Fusion ◽

Purification Method ◽

Viral Fusion Proteins

ABSTRACT The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.

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Site-Directed Antibodies against the Stem Region Reveal Low pH-Induced Conformational Changes of the Semliki Forest Virus Fusion Protein

Journal of Virology ◽

10.1128/jvi.01054-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9599-9607 ◽

Cited By ~ 9

Author(s):

Maofu Liao ◽

Margaret Kielian

Keyword(s):

Fusion Protein ◽

Conformational Changes ◽

Fusion Proteins ◽

Semliki Forest Virus ◽

Transmembrane Domain ◽

Polyclonal Antibodies ◽

Class Ii ◽

Low Ph ◽

Protein Domain ◽

Domain Iii

ABSTRACT The E1 envelope protein of the alphavirus Semliki Forest virus (SFV) is a class II fusion protein that mediates low pH-triggered membrane fusion during virus infection. Like other class I and class II fusion proteins, during fusion E1 inserts into the target membrane and rearranges to form a trimeric hairpin structure. The postfusion structures of the alphavirus and flavivirus fusion proteins suggest that the “stem” region connecting the fusion protein domain III to the transmembrane domain interacts along the trimer core during the low pH-induced conformational change. However, the location of the E1 stem in the SFV particle and its rearrangement and functional importance during fusion are not known. We developed site-directed polyclonal antibodies to the N- or C-terminal regions of the SFV E1 stem and used them to study the stem during fusion. The E1 stem was hidden on neutral pH virus but became accessible after low pH-triggered dissociation of the E2/E1 heterodimer. The stem packed onto the trimer core in the postfusion conformation and became inaccessible to antibody binding. Generation of the E1 homotrimer on fusion-incompetent membranes identified an intermediate conformation in which domain III had folded back but stem packing was incomplete. Our data suggest that E1 hairpin formation occurs by the sequential packing of domain III and the stem onto the trimer core and indicate a tight correlation between stem packing and membrane merger.

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The C. Elegans fusion protein EFF-1 is homologous to viral class II proteins

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331408406x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1593-C1593

Author(s):

Félix Rey

Keyword(s):

Fusion Protein ◽

Genetic Information ◽

Fusion Proteins ◽

Class Ii ◽

Icosahedral Symmetry ◽

Viral Fusion ◽

Ancestral Gene ◽

Viral Membrane ◽

C Elegans ◽

Viral Fusion Protein

Class II proteins are viral membrane fusogenic molecules folded essentially as β-sheet and having an internal fusion peptide. In particular, they lack the characteristic central alpha-helical coiled coil present in the post-fusion conformation of all other viral fusion proteins. The regular, icosahedrally symmetric enveloped viruses that have been studied so far, such as flaviviruses, alphaviruses and phleboviruses have been shown to have class II fusion proteins, which in their pre-fusion conformation make an icosahedral shell surrounding the viral membrane. Yet despite having very similar envelope proteins, these viruses belong to three different viral families with totally different genome replication machineries. We have recently identified the rubella virus fusion a belonging to class II, although the virus particles appear pleomorphic and lack icosahedral symmetry. In spite of the lack of any detectable sequence conservation, the available structures indicate that class II proteins have undergone divergent evolution from a distal, ancestral gene. We have now discovered that the cellular fusion protein EFF-1, involved in syncytium formation during the genesis of the skin in nematodes (C. elegans) and in other multicellular organisms, is also folded as a class II viral fusion protein, thereby indicating common ancestry, highlighting an unprecedented amount of exchange of genetic information between viruses and cells. My talk will discuss the implications of this finding, which highlights the intricate exchange of genetic information that has taken place between viruses and cells during evolution. This analysis also suggests a mechanism for the homotypic cell-cell fusion process, which has not been studied so far.

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Involvement of Lipids in Different Steps of the Flavivirus Fusion Mechanism

Journal of Virology ◽

10.1128/jvi.77.14.7856-7862.2003 ◽

2003 ◽

Vol 77 (14) ◽

pp. 7856-7862 ◽

Cited By ~ 65

Author(s):

Karin Stiasny ◽

Christian Koessl ◽

Franz X. Heinz

Keyword(s):

Fusion Protein ◽

Fusion Proteins ◽

Membrane Binding ◽

Class Ii ◽

Hydroxyl Group ◽

Viral Fusion ◽

Promoting Effect ◽

Protein Dimers ◽

Target Membrane ◽

Tick Borne Encephalitis Virus

ABSTRACT Flavivirus membrane fusion is triggered by acidic pH and mediated by the major envelope protein E. A structurally very similar fusion protein is found in alphaviruses, and these molecules are designated class II viral fusion proteins. In contrast to that of flaviviruses, however, alphavirus fusion has been shown to be absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane, suggesting significant differences in the fusion protein-membrane interactions that lead to fusion. With the flavivirus tick-borne encephalitis virus (TBEV), we have therefore conducted a study on the lipid requirements of viral fusion with liposomes and on the processes preceding fusion, specifically, the membrane-binding step and the fusion-associated oligomeric switch from E protein dimers to trimers. As with alphaviruses, cholesterol had a strong promoting effect on membrane binding and trimerization of the fusion protein, and—as shown by the use of cholesterol analogs—the underlying interactions involve the 3β-hydroxyl group at C-3 in both viral systems. In contrast to alphaviruses, however, these effects are much less pronounced with respect to the overall fusion of TBEV and can only be demonstrated when fusion is slowed down by lowering the temperature. The data presented thus suggest the existence of structurally related interactions of the flavivirus and alphavirus fusion proteins with cholesterol in the molecular processes required for fusion but, at the same time, point to significant differences between the class II fusion machineries of these viruses.

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The Postfusion Structure of the Heartland Virus Gc Glycoprotein Supports Taxonomic Separation of the Bunyaviral Families Phenuiviridae and Hantaviridae

Journal of Virology ◽

10.1128/jvi.01558-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 7

Author(s):

Yaohua Zhu ◽

Yan Wu ◽

Yan Chai ◽

Jianxun Qi ◽

Ruchao Peng ◽

...

Keyword(s):

Crystal Structure ◽

Fusion Protein ◽

Protein Conformation ◽

Fusion Proteins ◽

Taxonomic Revision ◽

Class Ii ◽

Viral Envelope ◽

Host Cells ◽

Human Pathogens ◽

Content Type

ABSTRACT Heartland virus (HRTV) is an emerging human pathogen that belongs to the newly defined family Phenuiviridae, order Bunyavirales. Gn and Gc are two viral surface glycoproteins encoded by the M segment and are required for early events during infection. HRTV delivers its genome into the cytoplasm by fusion of the viral envelope and endosomal membranes under low-pH conditions. Here, we describe the crystal structure of HRTV Gc in its postfusion conformation. The structure shows that Gc displays a typical class II fusion protein conformation, and the overall structure is identical to severe fever with thrombocytopenia syndrome virus (SFTSV) Gc, which also belongs to the Phenuiviridae family. However, our structural analysis indicates that the hantavirus Gc presents distinct features in the aspects of subdomain orientation, N-linked glycosylation, the interaction pattern between protomers, and the fusion loop conformation. This suggests their family-specific subunit arrangement during the fusogenic process and supports the recent taxonomic revision of bunyaviruses. Our results provide insights into the comprehensive comparison of class II membrane fusion proteins in two bunyavirus families, yielding valuable information for treatments against these human pathogens. IMPORTANCE HRTV is an insect-borne virus found in America that can infect humans. It belongs to the newly defined family Phenuiviridae, order Bunyavirales. HRTV contains three single-stranded RNA segments (L, M, and S). The M segment of the virus encodes a polyprotein precursor that is cleaved into two glycoproteins, Gn and Gc. Gc is a fusion protein facilitating virus entry into host cells. Here, we report the crystal structure of the HRTV Gc protein. The structure displays a typical class II fusion protein conformation. Comparison of HRTV Gc with a recently solved structure of another bunyavirus Gc revealed that these Gc structures display a newly defined family specificity, supporting the recent International Committee on Taxonomy of Viruses reclassification of the bunyaviruses. Our results expand the knowledge of bunyavirus fusion proteins and help us to understand bunyavirus characterizations. This study provides useful information to improve protection against and therapies for bunyavirus infections.

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Faculty Opinions recommendation of Autoregulation of Class II Alpha PI3K Activity by Its Lipid-Binding PX-C2 Domain Module.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733671904.793548692 ◽

2018 ◽

Author(s):

Brett Collins

Keyword(s):

Class Ii ◽

Lipid Binding ◽

C2 Domain ◽

Pi3k Activity ◽

Class Ii Alpha

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RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480

BMC Cancer ◽

10.1186/s12885-021-08056-4 ◽

2021 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Chen-Chen Huang ◽

Fang-Rui Liu ◽

Qiang Feng ◽

Xin-Yan Pan ◽

Shu-Ling Song ◽

...

Keyword(s):

Antitumor Activity ◽

Cell Membrane ◽

Fusion Protein ◽

Tumor Cell ◽

Tumor Cells ◽

Fusion Proteins ◽

Inhibitory Effect ◽

Endosomal Escape ◽

Antitumor Effects ◽

Sw480 Cells

Abstract Background We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. Methods RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. Results RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. Conclusion The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

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Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.

The Journal of Cell Biology ◽

10.1083/jcb.123.1.119 ◽

1993 ◽

Vol 123 (1) ◽

pp. 119-126 ◽

Cited By ~ 111

Author(s):

W Voos ◽

B D Gambill ◽

B Guiard ◽

N Pfanner ◽

E A Craig

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Fusion Protein ◽

Fusion Proteins ◽

Dual Role ◽

Intermembrane Space ◽

Heme Binding ◽

Membrane Translocation ◽

Amino Terminal ◽

The Matrix

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

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Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

Journal of Virology ◽

10.1128/jvi.73.3.2263-2269.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2263-2269 ◽

Cited By ~ 31

Author(s):

Pascal Cherpillod ◽

Karin Beck ◽

Andreas Zurbriggen ◽

Riccardo Wittek

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Translation Initiation ◽

Fusion Proteins ◽

Canine Distemper Virus ◽

Protein A ◽

Biological Properties ◽

Canine Distemper ◽

Wild Type ◽

Attachment Protein

ABSTRACT The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

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