scholarly journals Palmitoylation, Membrane-Proximal Basic Residues, and Transmembrane Glycine Residues in the Reovirus p10 Protein Are Essential for Syncytium Formation

2003 ◽  
Vol 77 (18) ◽  
pp. 9769-9779 ◽  
Author(s):  
Maya Shmulevitz ◽  
Jayme Salsman ◽  
Roy Duncan
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Nonstructural Protein ◽  
Fusion Reaction ◽  
Specific Interaction ◽  
Syncytium Formation ◽  
Surface Expression ◽  
Fusion Activity ◽  
Basic Region

ABSTRACT Avian reovirus and Nelson Bay reovirus are two unusual nonenveloped viruses that induce extensive cell-cell fusion via expression of a small nonstructural protein, termed p10. We investigated the importance of the transmembrane domain, a conserved membrane-proximal dicysteine motif, and an endodomain basic region in the membrane fusion activity of p10. We now show that the p10 dicysteine motif is palmitoylated and that loss of palmitoylation correlates with a loss of fusion activity. Mutational and functional analyses also revealed that a triglycine motif within the transmembrane domain and the membrane-proximal basic region were essential for p10-mediated membrane fusion. Mutations in any of these three motifs did not influence events upstream of syncytium formation, such as p10 membrane association, protein topology, or surface expression, suggesting that these motifs are more intimately associated with the membrane fusion reaction. These results suggest that the rudimentary p10 fusion protein has evolved a mechanism of inducing membrane merger that is highly dependent on the specific interaction of several different motifs with donor membranes. In addition, cross-linking, coimmunoprecipitation, and complementation assays provided no evidence for p10 homo- or heteromultimer formation, suggesting that p10 may be the first example of a membrane fusion protein that does not form stable, higher-order multimers.

scholarly journals Atypical Fusion Peptide of Nelson Bay Virus Fusion-Associated Small Transmembrane Protein

2005 ◽  
Vol 79 (3) ◽  
pp. 1853-1860 ◽  
Author(s):  
LiTing T. Cheng ◽  
Richard K. Plemper ◽  
Richard W. Compans
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Syncytium Formation ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Cell Surface Expression ◽  
Type I ◽  
Fusion Activity ◽  
Hydrophobic Domain

ABSTRACT A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.

scholarly journals A Functional F Analogue of Autographa californica Nucleopolyhedrovirus GP64 from the Agrotis segetum Granulovirus

2008 ◽  
Vol 82 (17) ◽  
pp. 8922-8926 ◽  
Author(s):  
Feifei Yin ◽  
Manli Wang ◽  
Ying Tan ◽  
Fei Deng ◽  
Just M. Vlak ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Plutella Xylostella ◽  
Syncytium Formation ◽  
Low Ph ◽  
Autographa Californica ◽  
Agrotis Segetum ◽  
Induced Membrane ◽  
F Protein ◽  
Autographa Californica Nucleopolyhedrovirus

ABSTRACT The envelope fusion protein F of Plutella xylostella granulovirus is a computational analogue of the GP64 envelope fusion protein of Autographa californica nucleopolyhedrovirus (AcMNPV). Granulovirus (GV) F proteins were thought to be unable to functionally replace GP64 in the AcMNPV pseudotyping system. In the present study the F protein of Agrotis segetum GV (AgseGV) was identified experimentally as the first functional GP64 analogue from GVs. AgseF can rescue virion propagation and infectivity of gp64-null AcMNPV. The AgseF-pseudotyped AcMNPV also induced syncytium formation as a consequence of low-pH-induced membrane fusion.

scholarly journals Variations in Disparate Regions of the Murine Coronavirus Spike Protein Impact the Initiation of Membrane Fusion

2001 ◽  
Vol 75 (6) ◽  
pp. 2792-2802 ◽  
Author(s):  
Dawn K. Krueger ◽  
Sean M. Kelly ◽  
Daniel N. Lewicki ◽  
Rosanna Ruffolo ◽  
Thomas M. Gallagher
Keyword(s):  
Tissue Culture ◽  
Membrane Fusion ◽  
Cultured Cells ◽  
Hepatitis Virus ◽  
Fusion Reaction ◽  
Spike Protein ◽  
Soluble Form ◽  
Fusion Activity ◽  
Murine Hepatitis Virus

ABSTRACT The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.

scholarly journals Mutations in the Cytoplasmic Domain of a Paramyxovirus Fusion Glycoprotein Rescue Syncytium Formation and Eliminate the Hemagglutinin-Neuraminidase Protein Requirement for Membrane Fusion

2003 ◽  
Vol 77 (1) ◽  
pp. 167-178 ◽  
Author(s):  
Shaguna Seth ◽  
Annelet Vincent ◽  
R. W. Compans
Keyword(s):  
Membrane Fusion ◽  
Critical Role ◽  
Syncytium Formation ◽  
Inhibitory Effect ◽  
Simian Virus ◽  
Dependent Manner ◽  
Fusion Activity ◽  
Double Mutation ◽  
F Protein ◽  
Fusion Glycoprotein

ABSTRACT SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.

scholarly journals Mutations in Pseudorabies Virus Glycoproteins gB, gD, and gH Functionally Compensate for the Absence of gL

2015 ◽  
Vol 90 (5) ◽  
pp. 2264-2272 ◽  
Author(s):  
Christina Schröter ◽  
Melina Vallbracht ◽  
Jan Altenschmidt ◽  
Sabrina Kargoll ◽  
Walter Fuchs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Pseudorabies Virus ◽  
Syncytium Formation ◽  
Single Amino Acid ◽  
Independent Experiment ◽  
Viral Glycoprotein ◽  
Bona Fide ◽  
Infectious Entry

ABSTRACTEntry of herpesviruses depends on the combined action of viral glycoprotein B (gB) and the heterodimeric gH/gL complex, which are activated by binding of the virion to specific cellular receptors. While gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus pseudorabies virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-ΔgL was used for reversion analyses by serial cell culture passages. In a first experiment, an infectious gL-negative mutant in which gL function was replaced by generation of a gD-gH hybrid protein was isolated (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014–3022, 1999). In a second, independent experiment PrV-ΔgLPassB4.1, which also replicated productively without gL, was isolated. Sequence analysis revealed mutations in gH but also in gB and gD. In a transfection-based fusion assay, two amino acid substitutions in the N-terminal part of gHB4.1(L70P and W103R) were found to be sufficient to compensate for lack of gL, while mutations present in gBB4.1enhanced fusogenicity. Coexpression of gBB4.1with the homologous gHB4.1resulted in strongly increased syncytium formation, which was further augmented by truncation of the gBB4.1C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, coexpression of gDB4.1blocked syncytium formation in the fusion assays, which could be attributed to a V106A substitution within the ectodomain of gDB4.1.IMPORTANCEIn contrast to many other enveloped viruses, herpesviruses rely on the concerted action of four viral glycoproteins for membrane fusion during infectious entry. Although the highly conserved gB shows signatures of a fusion protein, for fusion induction it requires the gH/gL complex, whose role is still elusive. Here we demonstrated fusion activation by gH in the absence of gL after reversion analysis of gL-deleted pseudorabies virus. This gL-independent fusion activity depended on single amino acid exchanges affecting the gL-binding domain in gH, increasing fusogenicity in gB and allowing negative fusion regulation by gD. Thus, our results provide novel information on the interplay in the fusion machinery of herpesviruses.

scholarly journals Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

2006 ◽  
Vol 80 (3) ◽  
pp. 1302-1310 ◽  
Author(s):  
Rene Broer ◽  
Bertrand Boson ◽  
Willy Spaan ◽  
François-Loïc Cosset ◽  
Jeroen Corver
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Transmembrane Domains ◽  
Spike Protein ◽  
Fusion Activity ◽  
Wild Type ◽  
Cell Cell

ABSTRACT The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

scholarly journals The Polybasic Juxtamembrane Region of Sso1p Is Required for SNARE Function In Vivo

2005 ◽  
Vol 4 (12) ◽  
pp. 2017-2028 ◽  
Author(s):  
Jeffrey S. Van Komen ◽  
Xiaoyang Bai ◽  
Travis L. Rodkey ◽  
Johanna Schaub ◽  
James A. McNew
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Specific Interaction ◽  
Coiled Coil ◽  
Snare Complex ◽  
Juxtamembrane Region

ABSTRACT Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the“ SNARE domain” or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.

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