scholarly journals Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein

2003 ◽  
Vol 77 (14) ◽  
pp. 7779-7785 ◽  
Author(s):  
Chisu Song ◽  
Eric Hunter
Keyword(s):  
Membrane Fusion ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Relative Sensitivity ◽  
Heptad Repeat ◽  
Single Amino Acid ◽  
Virus Infectivity ◽  
Single Amino Acid Change ◽  
Variable Sensitivity ◽  
Heptad Repeats

ABSTRACT The transmembrane protein of Mason-Pfizer monkey virus contains two heptad repeats that are predicted to form amphipathic α-helices that mediate the conformational change necessary for membrane fusion. To analyze the relative sensitivity of the predicted hydrophobic face of the N-terminal heptad repeat to the insertion of uncharged, polar, and charged substitutions, mutations that introduced alanine, serine, or glutamic acid into positions 436, 443, 450, and 457 of the envelope protein were examined. Novel systems using Tat protein and the GHOST cell line were developed to test and quantitate the effects of the mutations on Env-mediated fusion and infectivity of the virus. While no single amino acid change at any of the positions interfered significantly with the synthesis, processing, or transport to the plasma membrane of glycoprotein complexes, 9 of the 12 nonconservative mutations in these residues completely abolished fusion activity and virus infectivity. Mutations in the central positions (443 and 450) of the heptad repeat region were the most detrimental to Env function, and even single alanine substitutions in these positions dramatically altered the fusogenicity of the protein. These results demonstrate that this N-terminal heptad repeat plays a critical role in Env-mediated membrane fusion and highlight the key function of central hydrophobic residues in this process and the sensitivity of all positions to charge substitutions.

scholarly journals A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

2007 ◽  
Vol 81 (15) ◽  
pp. 8303-8314 ◽  
Author(s):  
Amanda E. Gardner ◽  
Rebecca E. Dutch
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Critical Role ◽  
Simian Virus ◽  
Hendra Virus ◽  
Heptad Repeat ◽  
F Protein ◽  
Conserved Region ◽  
Heptad Repeats ◽  
Cell Cell

ABSTRACT Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.

scholarly journals A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

1999 ◽  
Vol 10 (12) ◽  
pp. 4191-4200 ◽  
Author(s):  
David H. Kingsley ◽  
Ali Behbahani ◽  
Afshin Rashtian ◽  
Gary W. Blissard ◽  
Joshua Zimmerberg
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Conformational Changes ◽  
Fusion Proteins ◽  
Critical Role ◽  
Low Ph ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Heptad Repeats

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

scholarly journals Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions

2005 ◽  
Vol 79 (18) ◽  
pp. 11559-11568 ◽  
Author(s):  
Chisu Song ◽  
Keith Micoli ◽  
Helena Bauerova ◽  
Iva Pichova ◽  
Eric Hunter
Keyword(s):  
Amino Acid ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Cytoplasmic Domain ◽  
Virus Infectivity ◽  
Amino Acid Residues ◽  
Glycoprotein Complex ◽  
Uptake Studies ◽  
Domain Block ◽  
And Function

ABSTRACT Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.

scholarly journals A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

1998 ◽  
Vol 140 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Malini Vashishtha ◽  
Thomas Phalen ◽  
Marianne T. Marquardt ◽  
Jae S. Ryu ◽  
Alice C. Ng ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Single Point ◽  
Cellular Membrane ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Protein Subunit ◽  
Single Amino Acid Change

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

scholarly journals Identification of Ebola Virus Inhibitors Targeting GP2 Using Principles of Molecular Mimicry

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Courtney D. Singleton ◽  
Monica S. Humby ◽  
Hyun Ah Yi ◽  
Robert C. Rizzo ◽  
Amy Jacobs
Keyword(s):  
Membrane Fusion ◽  
Conformational Changes ◽  
Ebola Virus ◽  
Molecular Mimicry ◽  
Virus Disease ◽  
Experimental Testing ◽  
Critical Role ◽  
Heptad Repeat ◽  
Viral Glycoprotein ◽  
Host Membrane

ABSTRACTA key step in the Ebola virus (EBOV) replication cycle involves conformational changes in viral glycoprotein 2 (GP2) which facilitate host-viral membrane fusion and subsequent release of the viral genome. Ebola GP2 plays a critical role in virus entry and has similarities in mechanism and structure to the HIV gp41 protein for which inhibitors have been successfully developed. In this work, a putative binding pocket for the C-terminal heptad repeat in the N-terminal heptad repeat trimer was targeted for identification of small molecules that arrest EBOV-host membrane fusion. Two computational structure-based virtual screens of ∼1.7 M compounds were performed (DOCK program) against a GP2 five-helix bundle, resulting in 165 commercially available compounds purchased for experimental testing. Based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with 50% inhibitory concentration values in the 3 to 26 μM range. Molecular dynamics simulations of the two most potent candidates in their DOCK-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues that can be used to guide further inhibitor development and refinement targeting EBOV.IMPORTANCEThe most recent Ebola virus disease outbreak, from 2014 to 2016, resulted in approximately 28,000 individuals becoming infected, which led to over 12,000 causalities worldwide. The particularly high pathogenicity of the virus makes paramount the identification and development of promising lead compounds to serve as inhibitors of Ebola infection. To limit viral load, the virus-host membrane fusion event can be targeted through the inhibition of the class I fusion glycoprotein ofEbolavirus. In the current work, several promising small-molecule inhibitors that target the glycoprotein GP2 were identified through systematic application of structure-based computational and experimental drug design procedures.

scholarly journals Analysis of Murine Hepatitis Virus Strain A59 Temperature-Sensitive Mutant TS-LA6 Suggests that nsp10 Plays a Critical Role in Polyprotein Processing

2007 ◽  
Vol 81 (13) ◽  
pp. 7086-7098 ◽  
Author(s):  
Eric F. Donaldson ◽  
Rachel L. Graham ◽  
Amy C. Sims ◽  
Mark R. Denison ◽  
Ralph S. Baric
Keyword(s):  
Hepatitis Virus ◽  
Infectious Clone ◽  
Critical Role ◽  
Critical Factor ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Murine Hepatitis Virus ◽  
Negative Strand ◽  
New Findings ◽  
Single Amino Acid Change

ABSTRACT Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 (∼150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.

scholarly journals Characterization of a Novel Rice Dynamic Narrow-Rolled Leaf Mutant with Deficiencies in Aromatic Amino Acids

2020 ◽  
Vol 21 (4) ◽  
pp. 1521
Author(s):  
Huimei Wang ◽  
Yongfeng Shi ◽  
Xiaobo Zhang ◽  
Xia Xu ◽  
Jian-Li Wu
Keyword(s):  
Amino Acids ◽  
Expression Patterns ◽  
Critical Role ◽  
Aromatic Amino Acids ◽  
Single Amino Acid ◽  
Wild Type ◽  
Leaf Morphogenesis ◽  
Leaf Phenotype ◽  
Single Amino Acid Change ◽  
Rolled Leaf

The leaf blade is the main photosynthetic organ and its morphology is related to light energy capture and conversion efficiency. We isolated a novel rice Dynamic Narrow-Rolled Leaf 1 (dnrl1) mutant showing reduced width of leaf blades, rolled leaves and lower chlorophyll content. The narrow-rolled leaf phenotype resulted from the reduced number of small longitudinal veins per leaf, smaller size and irregular arrangement of bulliform cells compared with the wild-type. DNRL1 was mapped to chromosome 7 and encoded a putative 3-deoxy-7-phosphoheptulonate synthase (DAHPS) which catalyzes the conversion of phosphoenolpyruvate and D-erythrose 4-phosphate to DAHP and phosphate. Sequence analysis revealed that a single base substitution (T–A) was detected in dnrl1, leading to a single amino acid change (L376H) in the coding protein. The mutation led to a lower expression level of DNRL1 as well as the lower activity of DAHPS in the mutant compared with the wild type. Genetic complementation and over-expression of DNRL1 could rescue the narrow-rolled phenotype. DNRL1 was constitutively expressed in all tested organs and exhibited different expression patterns from other narrow-rolled leaf genes. DNRL1-GFP located to chloroplasts. The lower level of chlorophyll in dnrl1 was associated with the downregulation of the genes responsible for chlorophyll biosynthesis and photosynthesis. Furthermore, dnrl1 showed significantly reduced levels of aromatic amino acids including Trp, Phe and Tyr. We conclude that OsDAHPS, encoded by DNRL1, plays a critical role in leaf morphogenesis by mediating the biosynthesis of amino acids in rice.

scholarly journals Heptad Repeat 2-Based Peptides Inhibit Avian Sarcoma and Leukosis Virus Subgroup A Infection and Identify a Fusion Intermediate

2004 ◽  
Vol 78 (24) ◽  
pp. 13430-13439 ◽  
Author(s):  
Robert C. Netter ◽  
Sean M. Amberg ◽  
John W. Balliet ◽  
Mark J. Biscone ◽  
Arwen Vermeulen ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heptad Repeat ◽  
Single Amino Acid ◽  
Receptor Interaction ◽  
Envelope Conformation ◽  
Single Amino Acid Change ◽  
Avian Sarcoma

ABSTRACT Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4°C became sensitive to peptide within the first 5 min of elevating the temperature to 37°C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.

scholarly journals Bitopic Membrane Topology of the Stable Signal Peptide in the Tripartite Junín Virus GP-C Envelope Glycoprotein Complex

2007 ◽  
Vol 81 (8) ◽  
pp. 4331-4337 ◽  
Author(s):  
Sudhakar S. Agnihothram ◽  
Joanne York ◽  
Meg Trahey ◽  
Jack H. Nunberg
Keyword(s):  
Membrane Fusion ◽  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Lipid Membrane ◽  
Critical Role ◽  
Membrane Topology ◽  
Single Amino Acid ◽  
Fusion Activity ◽  
C Complex ◽  
Stable Signal

ABSTRACT The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hφ1 and hφ2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hφ2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.

scholarly journals MCD4Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

1999 ◽  
Vol 10 (3) ◽  
pp. 627-648 ◽  
Author(s):  
Erin C. Gaynor ◽  
Guillaume Mondésert ◽  
Stephen J. Grimme ◽  
Steve I. Reed ◽  
Peter Orlean ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Essential Gene ◽  
Critical Role ◽  
Single Amino Acid ◽  
Endoplasmic Reticulum Membrane ◽  
Gpi Anchor ◽  
Bud Emergence ◽  
Single Amino Acid Change ◽  
Lumenal Domain

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However,mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.

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