scholarly journals Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion

2018 ◽  
Vol 92 (12) ◽  
Author(s):  
Melina Vallbracht ◽  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Essential Role ◽  
Pseudorabies Virus ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Class Iii ◽  
Complex Needs ◽  
Hsv 1

ABSTRACTHerpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation.IMPORTANCEEnveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.

The Role of the Transmembrane and of the Intraviral Domain of Glycoproteins in Membrane Fusion of Enveloped Viruses

2000 ◽  
Vol 20 (6) ◽  
pp. 571-595 ◽  
Author(s):  
Britta Schroth-Diez ◽  
Kai Ludwig ◽  
Bolormaa Baljinnyam ◽  
Christine Kozerski ◽  
Qiang Huang ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Fusion Process ◽  
Fusion Pore ◽  
Minimum Length ◽  
Viral Fusion ◽  
Fusion Activity ◽  
Enveloped Viruses

Fusion of enveloped viruses with their target membrane is mediated by viral integral glycoproteins. A conformational change of their ectodomain triggers membrane fusion. Several studies suggest that an extended, triple-stranded rod-shaped α-helical coiled coil resembles a common structural and functional motif of the ectodomain of fusion proteins. From that, it is believed that essential features of the fusion process are conserved among the various enveloped viruses. However, this has not been established so far for the highly conserved transmembrane and intraviral sequences of fusion proteins. The article will focus on the role of both sequences in the fusion process. Recent studies from various enveloped viruses strongly imply that a transmembrane domain with a minimum length is required for later steps of membrane fusion, i.e., the formation and enlargement of the aqueous fusion pore. Although no specific sequence of the TM is necessary for pore formation, distinct properties and motifs of the domain may be obligatory to ascertain full fusion activity. However, with some exceptions, the intraviral domain seems to be not required for fusion activity of viral fusion proteins.

scholarly journals Functional Role of the Cytoplasmic Tail Domain of the Major Envelope Fusion Protein of Group II Baculoviruses

2006 ◽  
Vol 80 (22) ◽  
pp. 11226-11234 ◽  
Author(s):  
Gang Long ◽  
Xiaoyu Pan ◽  
Marcel Westenberg ◽  
Just M. Vlak
Keyword(s):  
Fusion Proteins ◽  
Functional Role ◽  
Cytoplasmic Tail ◽  
Viral Envelope ◽  
Repair System ◽  
Dependent Manner ◽  
Tail Domain ◽  
F Protein ◽  
Group Ii

ABSTRACT F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD), ranging from 48 (Spodoptera litura multicapsid NPV [MNPV]) to 78 (Adoxophyes honmai NPV) amino acid (aa) residues, with a nonassigned function. This CTD is much longer than the CTD of GP64-like envelope fusion proteins (7 aa), which appear to be nonessential for BV infectivity. Here we have investigated the functional role of the CTD of Helicoverpa armigera single-capsid NPV (HearNPV), a group II NPV. We combined a newly constructed HearNPV f-null bacmid knockout-repair system and an Autographa californica MNPV (AcMNPV) gp64-null bacmid knockout-pseudotype system with mutation and rescue experiments to study the functional role of the baculovirus F protein CTD. We show that except for the 16 C-terminal aa, the HearNPV F CTD is essential for virus spread from cell to cell. In addition, the CTD of HearNPV F is involved in BV production in a length-dependent manner and is essential for BV infectivity. The tyrosine residue Y658, located 16 aa from the C terminus, seems to be critical. However, HearNPV F without a CTD still rescues the infectivity of gp64-null AcMNPV BV, indicating that the CTD is not involved in processing and fusogenicity. Altogether, our results indicate that the F protein is essential for baculovirus BV infectivity and that the CTD is important for F protein incorporation into BV.

scholarly journals Effect of Cytoplasmic Tail Truncations on the Activity of the M2 Ion Channel of Influenza A Virus

1999 ◽  
Vol 73 (12) ◽  
pp. 9695-9701 ◽  
Author(s):  
Kurt Tobler ◽  
Marie L. Kelly ◽  
Lawrence H. Pinto ◽  
Robert A. Lamb
Keyword(s):  
Ion Channel ◽  
Influenza A Virus ◽  
Influenza A ◽  
Transmembrane Domain ◽  
Stop Codon ◽  
Specific Inhibitor ◽  
Cytoplasmic Tail ◽  
Proton Channel ◽  
Tail Region

ABSTRACT The M2 protein of influenza A virus forms a proton channel that is required for viral replication. The M2 ion channel is a homotetramer and has a 24-residue N-terminal extracellular domain, a 19-residue transmembrane domain, and a 54-residue cytoplasmic tail. We show here that the N-terminal methionine residue is cleaved from the mature protein. Translational stop codons were introduced into the M2 cDNA at residues 46, 52, 62, 72, 77, 82, 87, and 92. The deletion mutants were designated truncx, according to the amino acid position that was changed to a stop codon. We studied the role of the cytoplasmic tail by measuring the ion channel activity (the current sensitive to the M2-specific inhibitor amantadine) of the cytoplasmic tail truncation mutants expressed in oocytes of Xenopus laevis. When their conductance was measured over time, mutants trunc72, trunc77, and trunc92 behaved comparably to wild-type M2 protein (a decrease of only 4% over 30 min). In contrast, conductance decreased by 28% for trunc82, 27% for trunc62, and 81% for trunc52 channels. Complete closure of the channel could be observed in some cells for trunc62 and trunc52 within 30 min. These data suggest that a role of the cytoplasmic tail region of the M2 ion channel is to stabilize the pore against premature closure while the ectodomain is exposed to low pH.

scholarly journals Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

IUCrJ ◽  
2014 ◽  
Vol 1 (6) ◽  
pp. 505-513 ◽  
Author(s):  
Asma Rehman ◽  
Julia K. Archbold ◽  
Shu-Hong Hu ◽  
Suzanne J. Norwood ◽  
Brett M. Collins ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Blood Glucose Control ◽  
Vital Role ◽  
Snare Complex ◽  
Snare Proteins ◽  
Regulatory Role ◽  
Sm Proteins ◽  
Protein Receptor

Membrane fusion is essential for human health, playing a vital role in processes as diverse as neurotransmission and blood glucose control. Two protein families are key: (1) the Sec1p/Munc18 (SM) and (2) the solubleN-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins. Whilst the essential nature of these proteins is irrefutable, their exact regulatory roles in membrane fusion remain controversial. In particular, whether SM proteins promote and/or inhibit the SNARE-complex formation required for membrane fusion is not resolved. Crystal structures of SM proteins alone and in complex with their cognate SNARE proteins have provided some insight, however, these structures lack the transmembrane spanning regions of the SNARE proteins and may not accurately reflect the native state. Here, we review the literature surrounding the regulatory role of mammalian Munc18 SM proteins required for exocytosis in eukaryotes. Our analysis suggests that the conflicting roles reported for these SM proteins may reflect differences in experimental design. SNARE proteins appear to require C-terminal immobilization or anchoring, for example through a transmembrane domain, to form a functional fusion complex in the presence of Munc18 proteins.

scholarly journals Viral Membrane Fusion and the Transmembrane Domain

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 693 ◽  
Author(s):  
Chelsea T. Barrett ◽  
Rebecca Ellis Dutch
Keyword(s):  
Membrane Fusion ◽  
Host Cell ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Viral Fusion ◽  
Transmembrane Region ◽  
Host Cell Membrane ◽  
The Past ◽  
Viral Fusion Proteins

Initiation of host cell infection by an enveloped virus requires a viral-to-host cell membrane fusion event. This event is mediated by at least one viral transmembrane glycoprotein, termed the fusion protein, which is a key therapeutic target. Viral fusion proteins have been studied for decades, and numerous critical insights into their function have been elucidated. However, the transmembrane region remains one of the most poorly understood facets of these proteins. In the past ten years, the field has made significant advances in understanding the role of the membrane-spanning region of viral fusion proteins. We summarize developments made in the past decade that have contributed to the understanding of the transmembrane region of viral fusion proteins, highlighting not only their critical role in the membrane fusion process, but further demonstrating their involvement in several aspects of the viral lifecycle.

scholarly journals The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex.

1994 ◽  
Vol 127 (6) ◽  
pp. 1843-1857 ◽  
Author(s):  
K C Hart ◽  
Y F Xu ◽  
A N Meyer ◽  
B A Lee ◽  
D J Donoghue
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Proteolytic Processing ◽  
Nih3t3 Cells ◽  
Transforming Activity ◽  
Retention Signal

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.

scholarly journals Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

2000 ◽  
Vol 74 (15) ◽  
pp. 6760-6768 ◽  
Author(s):  
Barbara G. Klupp ◽  
Ralf Nixdorf ◽  
Thomas C. Mettenleiter
Keyword(s):  
Amino Acids ◽  
Membrane Fusion ◽  
Pseudorabies Virus ◽  
Inhibitory Effect ◽  
Rabbit Kidney ◽  
General Mechanism ◽  
Homologous Proteins ◽  
Infectious Laryngotracheitis Virus ◽  
Syncytial Virus ◽  
Hsv 1

ABSTRACT A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.

scholarly journals Interaction of the Dengue Virus Fusion Peptide with Membranes Assessed by NMR: The Essential Role of the Envelope Protein Trp101 for Membrane Fusion

2009 ◽  
Vol 392 (3) ◽  
pp. 736-746 ◽  
Author(s):  
Manuel Nuno Melo ◽  
Francisco J.R. Sousa ◽  
Fabiana A. Carneiro ◽  
Miguel A.R.B. Castanho ◽  
Ana Paula Valente ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Membrane Fusion ◽  
Envelope Protein ◽  
Essential Role ◽  
Fusion Peptide ◽  
Virus Fusion

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.

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