scholarly journals Mutational Analysis, Using a Full-Length Rubella Virus cDNA Clone, of Rubella Virus E1 Transmembrane and Cytoplasmic Domains Required for Virus Release

1999 ◽  
Vol 73 (6) ◽  
pp. 4622-4630 ◽  
Author(s):  
Jiansheng Yao ◽  
Shirley Gillam
Keyword(s):  
Cdna Clone ◽  
Rubella Virus ◽  
Transmembrane Domain ◽  
Virus Assembly ◽  
Cytoplasmic Domain ◽  
Full Length ◽  
Virus Yield ◽  
Virus Release ◽  
Cytoplasmic Domains ◽  
Virus Budding

ABSTRACT We report on the construction of a full-length cDNA clone, pBRM33, derived from wild-type rubella virus M33 strain. The RNA transcripts synthesized in vitro from pBRM33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental M33 virus in growth rate and plaque size. This cDNA clone was used to study the role of E1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. Three different alanine substitutions were introduced in the transmembrane domain of E1. These included substitution of leucine 464, cysteine 466, cysteine 467, and both cysteines 466 and 467 to alanine. In the E1 cytoplasmic domain, cysteine 470 and leucine 471 were altered to alanine. We found that these mutations did not significantly affect viral RNA replication, viral structural protein synthesis and transport, or E2/E1 heterodimer formation. Except for the substitution of cysteine 470, these mutations did, however, lead to a reduction in virus release. Substitution of cysteine 467 in the transmembrane region and of leucine 471 in the cytoplasmic domain dramatically reduced virus yield, resulting in the production of only 1 and 10% of the parental virus yield, respectively, in a parallel infection. These data show that E1 transmembrane and cytoplasmic domains play an important role in late stages of virus assembly, possibly during virus budding, consistent with earlier studies indicating that the E1 cytoplasmic domain may interact with nucleocapsids and that this interaction drives virus budding.

scholarly journals Role of Rubella Virus Glycoprotein Domains in Assembly of Virus-Like Particles

1999 ◽  
Vol 73 (5) ◽  
pp. 3524-3533 ◽  
Author(s):  
Mike Garbutt ◽  
Lok Man J. Law ◽  
Honey Chan ◽  
Tom C. Hobman
Keyword(s):  
Golgi Complex ◽  
Rubella Virus ◽  
Transmembrane Domain ◽  
Viral Envelope ◽  
Structural Proteins ◽  
Type I ◽  
Cytoplasmic Domains ◽  
Virus Like Particles ◽  
Positive Strand Rna

ABSTRACT Rubella virus is a small enveloped positive-strand RNA virus that assembles on intracellular membranes in a variety of cell types. The virus structural proteins contain all of the information necessary to mediate the assembly of virus-like particles in the Golgi complex. We have recently identified intracellular retention signals within the two viral envelope glycoproteins. E2 contains a Golgi retention signal in its transmembrane domain, whereas a signal for retention in the endoplasmic reticulum has been localized to the transmembrane and cytoplasmic domains of E1 (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7–20, 1995; T. C. Hobman, H. F. Lemon, and K. Jewell, J. Virol. 71:7670–7680, 1997). In the present study, we have analyzed the role of these retention signals in the assembly of rubella virus-like particles. Deletion or replacement of these domains with analogous regions from other type I membrane glycoproteins resulted in failure of rubella virus-like particles to be secreted from transfected cells. The E1 transmembrane and cytoplasmic domains were not required for targeting of the structural proteins to the Golgi complex and, surprisingly, assembly and budding of virus particles into the lumen of this organelle; however, the resultant particles were not secreted. In contrast, replacement or alteration of the E2 transmembrane or cytoplasmic domain, respectively, abrogated the targeting of the structural proteins to the budding site, and consequently, no virion formation was observed. These results indicate that the transmembrane and cytoplasmic domains of E2 and E1 are required for early and late steps respectively in the viral assembly pathway and that rubella virus morphogenesis is very different from that of the structurally similar alphaviruses.

Point mutation of C-terminal region of CD20 molecule predicts rituximab-induced complement-dependent cytotoxicity and clinical response to rituximab in non-Hodgkin’s lymphoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7563-7563 ◽  
Author(s):  
Y. Terui ◽  
Y. Mishima ◽  
Y. Mishima ◽  
M. Yokoyama ◽  
K. Hatake ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Laser Scanning ◽  
Transmembrane Domain ◽  
Point Mutations ◽  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Laser Scanning Confocal Microscopy ◽  
Cytoplasmic Domains ◽  
Isolated Cells ◽  
Cd20 Expression

7563 Background: Although rituximab is commonly used as induction and maintenance therapy for malignant lymphoma, some patients become refractory to treatment and the mechanism of resistance is unclear. The aim of this study was to investigate the relationship between CD20 mutations and rituximab resistance. Methods: To investigate whether CD20 mutations affect response to rituximab, fresh CD19+ lymphoma cells were isolated from the lymph nodes, or bone marrow of 68 patients with NHL. The cells were subsequently sorted by flow cytometry. RNA was prepared from the isolated cells and RT-PCR was performed. The PCR products were sequenced, subcloned into an expression vector pTARGET, transfected into K562 cells. CD20 expression was examined by flow cytometry and laser scanning confocal microscopy. Results: In all 68 patients, overall response rate (CR+CRu+PR) to rituximab was 91.2% (62/68), but t four cases became PD after PR. DNA sequence analysis revealed that point mutations were mostly observed in three CD20 domains - extracellular/cytoplasmic domains, the third transmembrane domain and the C-terminal cytoplasmic domain. Two cases had point mutations in extracellular/cytoplasmic domains, one patient had point mutations in the transmembrane domain, four cases showed point mutations in the C-terminal cytoplasmic domain and six cases had non-specific CD20 mutations, which did not affect CD20 expression. 56 patients showed no mutations of CD20 gene. CD20 expression was very weak in patients with point mutations in the C-terminal cytoplasmic domain, whereas expression was increased in patients with point mutations in the transmembrane domain. Conclusions: Point mutations in CD20 may cause rituximab resistance and identification of CD20 mutations upon diagnosis may help to predict a patient’s response to rituximab. No significant financial relationships to disclose.

scholarly journals Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain.

1996 ◽  
Vol 134 (6) ◽  
pp. 1531-1541 ◽  
Author(s):  
M H Wong ◽  
M T Filbin
Keyword(s):  
Cho Cells ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Effect ◽  
Cytoplasmic Domains ◽  
Aggregation Assay ◽  
Negative Effect ◽  
Po Protein ◽  
Homophilic Adhesion

The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its dominant-negative effect by preventing clustering of full-length Po. We also show that colchicine, which disrupts microtubules, prevents adhesion of cells expressing only the full-length Po. This strengthens our suggestion that an interaction of Po with the cytoskeleton, either directly or indirectly, is required for adhesion to take place.

scholarly journals The Membrane-Proximal Stem Region of Vesicular Stomatitis Virus G Protein Confers Efficient Virus Assembly

2000 ◽  
Vol 74 (5) ◽  
pp. 2239-2246 ◽  
Author(s):  
Clinton S. Robison ◽  
Michael A. Whitt
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Virus Assembly ◽  
Membrane Curvature ◽  
Virus Release ◽  
Wild Type ◽  
Virus Budding ◽  
Recombinant Viruses ◽  
Vesicular Stomatitis ◽  
High Level

ABSTRACT In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant ΔG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced ∼10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.

scholarly journals A Single-Amino-Acid Substitution of a Tyrosine Residue in the Rubella Virus E1 Cytoplasmic Domain Blocks Virus Release

2000 ◽  
Vol 74 (7) ◽  
pp. 3029-3036 ◽  
Author(s):  
Jiansheng Yao ◽  
Shirley Gillam
Keyword(s):  
Amino Acid ◽  
Rubella Virus ◽  
Cytoplasmic Domain ◽  
Terminal Region ◽  
Single Amino Acid ◽  
Sequencing Analysis ◽  
Dependent Manner ◽  
Tyrosine Residue ◽  
Virus Release ◽  
Amino Terminal

ABSTRACT Rubella virus particles, consisting of a nucleocapsid surrounded by a lipid envelope in which two virus-encoded glycoproteins E1 and E2 are embedded, assemble on intracellular membranes and are secreted from cells, possibly via the cellular secretory pathway. We have recently demonstrated that the cytoplasmic domain of E1 (residues 469 to 481, KCLYYLRGAIAPR) is required for virus release. Alteration of cysteine 470 to alanine did not affect virus release, whereas mutation of leucine 471 to alanine reduced virus production by 90%. In the present study, substitutions of remaining amino acids in the E1 cytoplasmic domain were made in order to investigate the role of each amino acid in regulating rubella virus release. Generated mutants were analyzed in the context of infectious full-length cDNA clone and virus-like particles using combined genetic, biochemical, and electron microscopic approaches. Substitution of a single residue of tyrosine 472 to alanine or tyrosine 473 to serine resulted in a block in virus release without affecting protein transport and virus budding into the lumen of the Golgi complexes. Infectious RNA transcripts bearing these mutations were incapable of forming plaques. Mutants with substitutions at the amino-terminal region (leucine 474, arginine 475, and glycine 476) in the E1 cytoplasmic domain had reduced virus release and small-plaque phenotype, while mutants with substitutions at the carboxy-terminal region (alanine 477, isoleucine 478, alanine 479, proline 480, and arginine 481) had only marginal defects in virus release. Plaque-forming revertants could be isolated from mutants Y472A and Y473S. Sequencing analysis revealed that the substituted serine residue in mutant Y473S reverted to the original tyrosine residue, whereas the substituted alanine residue in mutant Y472A was retained. These results indicate that the E1 cytoplasmic domain modulates virus release in a sequence-dependent manner and that the tyrosine residues are critical for this function. We postulate that residues YYLRG constitute a domain in the E1 tail that may interact with other proteins and this interaction is involved in regulating virus release.

Isolated integrin β3 subunit cytoplasmic domains require membrane anchorage and the NPXY motif to recruit to adhesion complexes but do not discriminate between β1– and β3-positive complexes

2005 ◽  
Vol 94 (07) ◽  
pp. 155-166 ◽  
Author(s):  
Lionel Ponsonnet ◽  
Alessandro Foletti ◽  
Gian Carlo Alghisi ◽  
Curzio Rüegg
Keyword(s):  
Endothelial Cells ◽  
Transmembrane Domain ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Β Subunit ◽  
Cytoplasmic Domains ◽  
Npxy Motif ◽  
Covalently Linked ◽  
Focal Structures ◽  
Adhesion Complex

SummaryIntegrin adhesion receptors consist of non-covalently linked α and β subunits each of which contains a large extracellular domain, a single transmembrane domain and a short cytoplasmic tail. Engaged integrins recruit to focal structures globally termed adhesion complexes. The cytoplasmic domain of the β subunit is essential for this clustering. β1 and β3 integrins can recruit at distinct cellular locations (i.e. fibrillar adhesions vs focal adhesions, respectively) but it is not clear whether individual β subunit cytoplasmic and transmembrane domains are by themselves sufficient to drive orthotopic targeting to the cognate adhesion complex. To address this question, we expressed fulllength β3 transmembrane anchored cytoplasmic domains and truncated β3 cytoplasmic domains as GFP-fusion constructs and monitored their localization in endothelial cells. Membrane-anchored full-length β3 cytoplasmic domain and a β3 mutant lacking the NXXY motif recruited to adhesion complexes, while β3 mutants lacking the NPXY and NXXY motifs or the transmembrane domain did not. Replacing the natural β subunit transmembrane domain with an unrelated (i.e. HLA-A2 α chain) transmembrane domain significantly reduced recruitment to adhesion complexes. Transmembrane anchored β3 and cytoplasmic domain constructs, however, recruited without discrimination to β1– and β3-rich adhesions complexes. These findings demonstrate that membrane anchorage and the NPXY (but not the NXXY) motif are necessary for β3 cytoplasmic domain recruitment to adhesion complexes and that the natural transmembrane domain actively contributes to this recruitment. The β3 transmembrane and cytoplasmic domains alone are insufficient for orthotopic recruitment to cognate adhesion complexes.

scholarly journals A Proline-Rich Motif (PPPY) in the Gag Polyprotein of Mason-Pfizer Monkey Virus Plays a Maturation-Independent Role in Virion Release

1998 ◽  
Vol 72 (5) ◽  
pp. 4095-4103 ◽  
Author(s):  
Jiro Yasuda ◽  
Eric Hunter
Keyword(s):  
Virus Assembly ◽  
Single Amino Acid ◽  
Distinct Region ◽  
Virus Release ◽  
Coding Region ◽  
Virus Budding ◽  
Fusion Event ◽  
Gag Polyprotein ◽  
Type D ◽  
Virion Release

ABSTRACT Virus assembly represents one of the last steps in the retrovirus life cycle. During this process, Gag polyproteins assemble at specific sites within the cell to form viral capsids and induce membrane extrusion (viral budding) either as assembly progresses (type C virus) or following formation of a complete capsid (type B and type D viruses). Finally, the membrane must undergo a fusion event to pinch off the particle in order to release a complete enveloped virion. Structural elements within the MA region of the Gag polyprotein define the route taken to the plasma membrane and direct the process of virus budding. Results presented here suggest that a distinct region of Gag is necessary for virus release. The pp24 and pp16 proteins of the type D retrovirus Mason-Pfizer monkey virus (M-PMV) are phosphoproteins that are encoded in the gag gene of the virus. The pp16 protein is a C-terminally located cleavage product of pp24 and contains a proline-rich motif (PPPY) that is conserved among the Gag proteins of a wide variety of retroviruses. By performing a functional analysis of this coding region with deletion mutants, we have shown that the pp16 protein is dispensable for capsid assembly but essential for virion release. Moreover, additional experiments indicated that the virus release function of pp16 was abolished by the deletion of only the PPPY motif and could be restored when this motif alone was reinserted into a Gag polyprotein lacking the entire pp16 domain. Single-amino-acid substitutions for any of the residues within this motif confer a similar virion release-defective phenotype. It is unlikely that the function of the proline-rich motif is simply to inhibit premature activation of protease, since the PPPY deletion blocked virion release in the context of a protease-defective provirus. These results demonstrate that in type D retroviruses a PPPY motif plays a key role in a late stage of virus budding that is independent of and occurs prior to virion maturation.

scholarly journals Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein

2020 ◽  
Author(s):  
Lucas Kuhlen ◽  
Steven Johnson ◽  
Jerry Y. Cao ◽  
Justin C. Deme ◽  
Susan M. Lea
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Cytoplasmic Domain ◽  
Secretion System ◽  
Full Length ◽  
Cytoplasmic Domains ◽  
Bacterial Flagella ◽  
Type Iii ◽  
Closed State ◽  
N Terminus

AbstractType three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, FlhA/SctV is most often studied as a monomer and only a single structure of an SctV homologue assembled into the biologically relevant nonameric ring is available. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in the assembled ring structure SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer’s cytoplasmic domain, which stabilises the nonameric ring assembly. This represents the first structure of SctV in the open state, the first observation of the SctVL interaction with the adjacent protomer and confirms the importance of FlhAL for the stability of the FlhA nonameric ring.ImportanceBacterial flagella are assembled from proteins secreted through a type III secretion system. A related type III secretion system is found in injectisomes, molecular syringes that bridge three membranes to secrete proteins directly from the bacterial cytoplasm into eukaryotic host cells. The major protein of the export apparatus of type III secretion is made up of a membrane and a cytoplasmic domain, which in the flagellar system can adopt an open or a closed state, is known to form a nonameric ring in vivo. We produced the full-length proteins from both injectisome and flagellar systems in the assembled state. The structures of the cytoplasmic domains demonstrate the conserved principle of the N-terminus of one subunit binding the membrane proximal face of the adjacent subunit to stabilise the assembled ring. Our structure of the homologue from the injectisome also demonstrates that the open state of the cytoplasmic domain is not unique to flagella.

scholarly journals Role of the Cytoplasmic Domain of the NewcastleDisease Virus Fusion Protein in Association with LipidRafts

2003 ◽  
Vol 77 (24) ◽  
pp. 12968-12979 ◽  
Author(s):  
V. Dolganiuc ◽  
L. McGinnes ◽  
E. J. Luna ◽  
T. G. Morrison
Keyword(s):  
Cell Fusion ◽  
Transmembrane Domain ◽  
Virus Assembly ◽  
Cytoplasmic Domain ◽  
Membrane Domains ◽  
Fusion Activity ◽  
F Protein ◽  
Specific Localization ◽  
L 14 ◽  
Triton X

ABSTRACT To explore the association of the Newcastle disease virus (NDV) fusion (F) protein with cholesterol-rich membrane domains, its localization in detergent-resistant membranes (DRMs) in transfected cells was characterized. After solubilization of cells expressing the F protein with 1% Triton X-100 at 4°C, ca. 40% of total, cell-associated F protein fractionated with classical DRMs with densities of 1.07 to l.14 as defined by flotation into sucrose density gradients. Association of the F protein with this cell fraction was unaffected by the cleavage of F0 to F1 and F2 or by coexpression of the NDV attachment protein, the hemagglutinin-neuraminidase protein (HN). Furthermore, elimination by mutation, of potential palmitate addition sites in and near the F-protein transmembrane domain had no effect on F-protein association with DRMs. Rather, specific deletions of the cytoplasmic domain of the F protein eliminated association with classical DRMs. Comparisons of deletions that affected fusion activity of the protein and deletions that affected DRM association suggested that there is no direct link between the cell-cell fusion activity of the F protein and DRM association. Furthermore, depletion of cholesterol from cells expressing F and HN protein, while eliminating DRM association, had no effect on the ability of these cells to fuse with avian red blood cells. These results suggest that specific localization of the F protein in cholesterol-rich membrane domains is not required for cell-to-cell fusion. Paramyxovirus F-protein cytoplasmic domains have been implicated in virus assembly. The results presented here raise the possibility that the cytoplasmic domain is important in virus assembly at least in part because it directs the protein to cholesterol-rich membrane domains.

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