scholarly journals Spring-Loaded Heptad Repeat Residues Regulate the Expression and Activation of Paramyxovirus Fusion Protein

2007 ◽  
Vol 81 (7) ◽  
pp. 3130-3141 ◽  
Author(s):  
Laura E. Luque ◽  
Charles J. Russell
Keyword(s):  
Protein Expression ◽  
Membrane Fusion ◽  
Conformational Changes ◽  
Structural Changes ◽  
Tertiary Structure ◽  
Sendai Virus ◽  
Coiled Coil ◽  
Viral Envelope ◽  
Heptad Repeat ◽  
F Protein

ABSTRACT During viral entry, the paramyxovirus fusion (F) protein fuses the viral envelope to a cellular membrane. Similar to other class I viral fusion glycoproteins, the F protein has two heptad repeat regions (HRA and HRB) that are important in membrane fusion and can be targeted by antiviral inhibitors. Upon activation of the F protein, HRA refolds from a spring-loaded, crumpled structure into a coiled coil that inserts a hydrophobic fusion peptide into the target membrane and binds to the HRB helices to form a fusogenic hairpin. To investigate how F protein conformational changes are regulated, we mutated in the Sendai virus F protein a highly conserved 10-residue sequence in HRA that undergoes major structural changes during protein refolding. Nine of the 15 mutations studied caused significant defects in F protein expression, processing, and fusogenicity. Conversely, the remaining six mutations enhanced the fusogenicity of the F protein, most likely by helping spring the HRA coil. Two of the residues that were neither located at “a” or “d” positions in the heptad repeat nor conserved among the paramyxoviruses were key regulators of the folding and fusion activity of the F protein, showing that residues not expected to be important in coiled-coil formation may play important roles in regulating membrane fusion. Overall, the data support the hypothesis that regions in the F protein that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations regulate F protein expression and activation.

scholarly journals Residues in the Heptad Repeat A Region of the Fusion Protein Modulate the Virulence of Sendai Virus in Mice

2009 ◽  
Vol 84 (2) ◽  
pp. 810-821 ◽  
Author(s):  
Laura E. Luque ◽  
Olga A. Bridges ◽  
John N. Mason ◽  
Kelli L. Boyd ◽  
Allen Portner ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Sendai Virus ◽  
Syncytium Formation ◽  
Heptad Repeat ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
F Protein

ABSTRACT While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.

scholarly journals Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

2005 ◽  
Vol 79 (3) ◽  
pp. 1543-1551 ◽  
Author(s):  
Dava S. West ◽  
Michael S. Sheehan ◽  
Patrick K. Segeleon ◽  
Rebecca Ellis Dutch
Keyword(s):  
Membrane Fusion ◽  
Intracellular Transport ◽  
Coiled Coil ◽  
Syncytium Formation ◽  
Simian Virus ◽  
Heptad Repeat ◽  
F Protein ◽  
Helix Bundle ◽  
Fusogenic Activity ◽  
Simian Virus 5

ABSTRACT Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion.

scholarly journals Coiled-Coil Domains in Glycoproteins B and H Are Involved in Human Cytomegalovirus Membrane Fusion

2004 ◽  
Vol 78 (15) ◽  
pp. 8333-8341 ◽  
Author(s):  
Matthew Lopper ◽  
Teresa Compton
Keyword(s):  
Membrane Fusion ◽  
Human Cytomegalovirus ◽  
Virus Entry ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Viral Envelope ◽  
Heptad Repeat ◽  
Repeat Sequences ◽  
Human Cmv ◽  
Hsv 1

ABSTRACT Human cytomegalovirus (CMV) utilizes a complex route of entry into cells that involves multiple interactions between viral envelope proteins and cellular receptors. Three conserved viral glycoproteins, gB, gH, and gL, are required for CMV-mediated membrane fusion, but little is known of how these proteins cooperate during entry (E. R. Kinzler and T. Compton, submitted for publication). The goal of this study was to begin defining the molecular mechanisms that underlie membrane fusion mediated by herpesviruses. We identified heptad repeat sequences predicted to form alpha-helical coiled coils in two glycoproteins required for fusion, gB and gH. Peptides derived from gB and gH containing the heptad repeat sequences inhibited virus entry when introduced coincident with virus inoculation onto cells or when mixed with virus prior to inoculation. Neither peptide affected binding of CMV to fibroblasts, suggesting that the peptides inhibit membrane fusion. Both gB and gH coiled-coil peptides blocked entry of several laboratory-adapted and clinical strains of human CMV, but neither peptide affected entry of murine CMV or herpes simplex virus type 1 (HSV-1). Although murine CMV and HSV-1 gB and gH have heptad repeat regions, the ability of human CMV gB and gH peptides to inhibit virus entry correlates with the specific residues that comprise the heptad repeat region. The ability of gB and gH coiled-coil peptides to inhibit virus entry independently of cell contact suggests that the coiled-coil regions of gB and gH function differently from those of class I, single-component fusion proteins. Taken together, these data support a critical role for alpha-helical coiled coils in gB and gH in the entry pathway of CMV.

scholarly journals Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

2004 ◽  
Vol 78 (6) ◽  
pp. 2994-3002 ◽  
Author(s):  
Anne Op De Beeck ◽  
Cécile Voisset ◽  
Birke Bartosch ◽  
Yann Ciczora ◽  
Laurence Cocquerel ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Conformational Changes ◽  
Structural Changes ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Functional Envelope

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

scholarly journals Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes

2017 ◽  
Vol 28 (26) ◽  
pp. 3801-3814 ◽  
Author(s):  
Sunandini Chandra ◽  
Raju Kalaivani ◽  
Manoj Kumar ◽  
Narayanaswamy Srinivasan ◽  
Debi P. Sarkar
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Sendai Virus ◽  
Viral Envelope ◽  
Bioactive Molecules ◽  
Host Cells ◽  
Actin Dynamics ◽  
Envelope Glycoproteins ◽  
Animal Cells ◽  
Viral Envelopes

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

Studies of membrane fusion. VI. Mechanism of the membrane fusion and cell swelling stages of Sendai virus-mediated cell fusion

1980 ◽  
Vol 43 (1) ◽  
pp. 103-118
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Freeze Fracture ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Fusion Event ◽  
Virus Envelope ◽  
Membrane Rupture ◽  
Membrane Tubules

The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane rupture occurs and the tubules appear to break down as small vesicles. When previously observed in cross-sectioned cells these membrane tubules were interpreted as sites of direct membrane fusion. The present study indicates that this interpretation is incorrect and shows that the tubules are generated subsequent to membrane fusion when 2 cells connected by a virus bridge are induced to swell. A mechanism to explain the formation of this system of membrane tubules is proposed.

Studies of membrane fusion. V. Fusion of erythrocytes with non-haemolytic Sendai virus

1979 ◽  
Vol 36 (1) ◽  
pp. 85-96
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Erythrocyte Membrane ◽  
Sendai Virus ◽  
Lateral Diffusion ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Viral Membrane ◽  
Cytoplasmic Bridges ◽  
Viral Envelopes

The fusion of human erythrocytes with non-haemolytic ‘1-day’ Sendai virus has been studied by electron microscopy. The mechanism of viral envelope-cell fusion is the same as that described previously for haemolytic ‘3-day’ Sendai virus except that fusion is frequently arrested at an initial stage when 2 segments of smooth linear viral membrane fuse and become incorporated into the erythrocyte membrane. After longer periods of incubation at 37 degrees C, in addition to many partly fused virus particles, long (up to 4 micrometer) lengths of smooth linear viral membrane are seen within the erythrocyte membrane which arise by linear aggregation of shorter (approximately 0.25 micrometer long) segments of smooth linear membrane derived from individual fused viral envelopes. Cell-Cell fusion, as a result of the fusion of a viral envelope with 2 adjacent erythrocytes also occurs but, in the absence of cell swelling, fusion is arrested at this stage with cells joined by one (or more) small cytoplasmic bridges. Typical fused cells are produced if such cells are swollen with hypotonic buffer. These observations provide further evidence that membrane fusion and cell swelling are distinct events in cell fusion and that cell swelling is the driving force both for completing the incorporation of the viral envelope into the cell membrane and for expanding cells connected by small cytoplasmic bridges to form spherical fused cells. Little lateral diffusion of viral envelope components occurs in the absence of cell swelling; in fact, some aggregation of components occurs. Comparison with previous studies using haemolytic ‘3-day’ Sendai virus suggests that virally induced cell swelling perturbs membrane structure so as to allow the rapid lateral diffusion of integrated viral envelope components.

scholarly journals A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

2008 ◽  
Vol 83 (4) ◽  
pp. 1727-1741 ◽  
Author(s):  
Anuja Krishnan ◽  
Santosh K. Verma ◽  
Prashant Mani ◽  
Rahul Gupta ◽  
Suman Kundu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Sendai Virus ◽  
Biological Activities ◽  
Viral Envelope ◽  
Attachment Protein ◽  
Histidine Residues ◽  
Human Parainfluenza Virus ◽  
Mimicking Peptides

ABSTRACT Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.

scholarly journals New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 413 ◽  
Author(s):  
Mark A. Benhaim ◽  
Kelly K. Lee
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Viral Entry ◽  
Fusion Proteins ◽  
Structural Changes ◽  
Fusion Reaction ◽  
Type I ◽  
Viral Fusion ◽  
Technological Advances ◽  
Viral Fusion Proteins

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.

Sign in / Sign up

Export Citation Format

Share Document