scholarly journals An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1466
Author(s):  
Jobin Varkey ◽  
Jiantao Zhang ◽  
Junghyun Kim ◽  
Gincy George ◽  
Guijuan He ◽  
...  
Keyword(s):  
Viral Replication ◽  
Phospholipid Composition ◽  
Alpha Helix ◽  
Lipid Vesicles ◽  
Replication Proteins ◽  
Membrane Remodeling ◽  
Alpha Helices ◽  
Α Helix ◽  
Positive Strand Rna ◽  
Amphipathic Alpha Helix

Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic α-helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes.

scholarly journals Conserved Determinants for Membrane Association of Nonstructural Protein 5A fromHepatitis C Virus and Related Viruses

2006 ◽  
Vol 81 (6) ◽  
pp. 2745-2757 ◽  
Author(s):  
Volker Brass ◽  
Zsuzsanna Pal ◽  
Nicolas Sapay ◽  
Gilbert Deléage ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nonstructural Protein ◽  
Alpha Helix ◽  
Laser Scanning Microscopy ◽  
Membrane Association ◽  
Alpha Helices ◽  
Replication Complex ◽  
Diarrhea Virus ◽  
Amphipathic Alpha Helix ◽  
Nonstructural Protein 5A

ABSTRACT Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation.

scholarly journals An Arginine-Faced Amphipathic Alpha Helix Is Required for Adenovirus Type 5 E4orf6 Protein Function

1999 ◽  
Vol 73 (6) ◽  
pp. 4600-4610 ◽  
Author(s):  
Joseph S. Orlando ◽  
David A. Ornelles
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Protein Function ◽  
Alpha Helix ◽  
Adenovirus Type ◽  
Wild Type ◽  
Reading Frame ◽  
Α Helix ◽  
Amphipathic Alpha Helix ◽  
Adenovirus Type 5

ABSTRACT A region in the carboxy terminus of the protein encoded by open reading frame 6 in early region 4 (E4orf6) of adenovirus type 5 was determined to be required for directing nuclear localization of the E1B 55-kDa protein and for efficient virus replication. A peptide encompassing this region, corresponding to amino acids 239 through 255 of the E4orf6 protein, was analyzed by circular dichroism spectroscopy. The peptide showed evidence of self-interaction and displayed the characteristic spectra of an amphipathic α helix in the helix-stabilizing solvent trifluoroethanol. Disrupting the integrity of this α helix in the E4orf6 protein by proline substitutions or by removing amino acids 241 through 250 abolished its ability to direct the E1B 55-kDa protein to the nucleus when both proteins were transiently expressed in HeLa cells. Expression of E4orf6 variants that failed to direct nuclear localization of the E1B 55-kDa protein failed to enhance replication of the E4 mutant virus, dl1014, whereas expression of the wild-type E4orf6 protein restored growth of dl1014 to near-wild-type levels. These results suggest that the E4orf6 protein contains an arginine-faced, amphipathic α helix that is critical for a functional interaction with the E1B 55-kDa protein in the cell and for the function of the E4orf6 protein during a lytic infection.

scholarly journals A conserved viral amphipathic helix governs the replication site-specific membrane association

2021 ◽  
Author(s):  
Preethi Sathanantham ◽  
Xiaofeng Wang
Keyword(s):  
Viral Replication ◽  
Fluorescent Proteins ◽  
Brome Mosaic Virus ◽  
Replication Proteins ◽  
Amphipathic Helix ◽  
Golgi Membranes ◽  
Positive Strand Rna ◽  
Specific Membrane ◽  
Replication Site ◽  
Er Membrane

Positive-strand RNA viruses assemble their viral replication complexes (VRCs) on specific host organelle membranes, yet it is unclear how viral replication proteins recognize and what motifs or domains in viral replication proteins determine their localizations. We show here that an amphipathic helix, helix B in replication protein 1a of brome mosaic virus (BMV), is necessary for 1a's localization to the nuclear endoplasmic reticulum (ER) membrane where BMV assembles its VRCs. Helix B is also sufficient to target soluble proteins to the nuclear ER membrane in yeast and plant cells. We further show that an equivalent helix in several plant- and human-infecting viruses of the alphavirus-like superfamily targets fluorescent proteins to the organelle membranes where they form their VRCs, including ER, vacuole, and Golgi membranes. Our work reveals a conserved helix that governs the localization of VRCs among a group of viruses and points to a possible target for developing broad-spectrum antiviral strategies.

ALPHA-HELIX FORMATION IN C-PEPTIDE RNASE-A INVESTIGATED BY PARALLEL TEMPERING SIMULATIONS

2007 ◽  
Vol 18 (01) ◽  
pp. 91-98 ◽  
Author(s):  
GÖKHAN GÖKOĞLU ◽  
TARIK ÇELİK
Keyword(s):  
Energy State ◽  
Minimum Energy ◽  
Alpha Helix ◽  
Salt Bridge ◽  
Rnase A ◽  
Parallel Tempering ◽  
Implicit Solvent ◽  
C Peptide ◽  
Helix Formation ◽  
Α Helix

We have performed parallel tempering simulations of a 13-residue peptide fragment of ribonuclease-A, c-peptide, in implicit solvent with constant dielectric permittivity. This peptide has a strong tendency to form α-helical conformations in solvent as suggested by circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Our results demonstrate that 5th and 8–12 residues are in the α-helical region of the Ramachandran map for global minimum energy state in solvent environment. Effects of salt bridge formation on stability of α-helix structure are discussed.

scholarly journals Serum amyloid A forms stable oligomers that disrupt vesicles at lysosomal pH and contribute to the pathogenesis of reactive amyloidosis

2017 ◽  
Vol 114 (32) ◽  
pp. E6507-E6515 ◽  
Author(s):  
Shobini Jayaraman ◽  
Donald L. Gantz ◽  
Christian Haupt ◽  
Olga Gursky
Keyword(s):  
Serum Amyloid A ◽  
Major Complication ◽  
Lipid Vesicles ◽  
Serum Amyloid ◽  
High Density Lipoproteins ◽  
Aa Amyloidosis ◽  
Intrinsically Disordered ◽  
Amyloid A ◽  
Α Helix ◽  
Structural Methods

Serum amyloid A (SAA) is an acute-phase plasma protein that functions in innate immunity and lipid homeostasis. SAA is a protein precursor of reactive AA amyloidosis, the major complication of chronic inflammation and one of the most common human systemic amyloid diseases worldwide. Most circulating SAA is protected from proteolysis and misfolding by binding to plasma high-density lipoproteins. However, unbound soluble SAA is intrinsically disordered and is either rapidly degraded or forms amyloid in a lysosome-initiated process. Although acidic pH promotes amyloid fibril formation by this and many other proteins, the molecular underpinnings are unclear. We used an array of spectroscopic, biochemical, and structural methods to uncover that at pH 3.5–4.5, murine SAA1 forms stable soluble oligomers that are maximally folded at pH 4.3 with ∼35% α-helix and are unusually resistant to proteolysis. In solution, these oligomers neither readily convert into mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a manner typical of apolipoproteins. Rather, these oligomers undergo an α-helix to β-sheet conversion catalyzed by lipid vesicles and disrupt these vesicles, suggesting a membranolytic potential. Our results provide an explanation for the lysosomal origin of AA amyloidosis. They suggest that high structural stability and resistance to proteolysis of SAA oligomers at pH 3.5–4.5 help them escape lysosomal degradation, promote SAA accumulation in lysosomes, and ultimately damage cellular membranes and liberate intracellular amyloid. We posit that these soluble prefibrillar oligomers provide a missing link in our understanding of the development of AA amyloidosis.

Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein

1992 ◽  
Vol 12 (12) ◽  
pp. 5673-5682 ◽  
Author(s):  
A D Bergemann ◽  
Z W Ma ◽  
E M Johnson
Keyword(s):  
Amino Acids ◽  
Alpha Helix ◽  
Element Analysis ◽  
Sequence Element ◽  
Single Stranded Dna ◽  
Reading Frame ◽  
Consensus Motif ◽  
Rich Domain ◽  
Amphipathic Alpha Helix ◽  
Dna Binding Properties

The human Pur factor binds strongly to a sequence element repeated within zones of initiation of DNA replication in several eukaryotic cells. The protein binds preferentially to the purine-rich single strand of this element, PUR. We report here the cloning and sequencing of a cDNA encoding a protein with strong affinity for the PUR element. Analysis with a series of mutated oligonucleotides defines a minimal single-stranded DNA Pur-binding element. The expressed Pur open reading frame encodes a protein of 322 amino acids. This protein, Pur alpha, contains three repeats of a consensus motif of 23 amino acids and two repeats of a second consensus motif of 26 amino acids. Near its carboxy terminus, the protein possesses an amphipathic alpha-helix and a glutamine-rich domain. The repeat region of Pur cDNA is homologous to multiple mRNA species in each of several human cell lines and tissues. The HeLa cDNA library also includes a clone encoding a related gene, Pur beta, containing a version of the 23-amino-acid consensus motif similar, but not identical, to those in Pur alpha. Results indicate a novel type of modular protein with capacity to bind repeated elements in single-stranded DNA.

The Drosophila gene Bearded encodes a novel small protein and shares 3′ UTR sequence motifs with multiple Enhancer of split complex genes

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4039-4051 ◽  
Author(s):  
M.W. Leviten ◽  
E.C. Lai ◽  
J.W. Posakony
Keyword(s):  
Sequence Similarity ◽  
Notch Pathway ◽  
Alpha Helix ◽  
Sequence Motifs ◽  
Wild Type ◽  
Gain Of Function ◽  
Small Protein ◽  
Drosophila Gene ◽  
Amphipathic Alpha Helix ◽  
Enhancer Of Split

Gain-of-function alleles of the Drosophila gene Bearded (Brd) cause sensory organ multiplication and loss phenotypes indistinguishable at the cellular level from those caused by loss-of-function mutations in the genes of the Notch pathway (Leviten, M. W. and Posakony, J. W. (1996). Dev. Biol. 176, 264–283). We have carried out a molecular analysis of the structure and expression of both wild-type and mutant Brd transcription units. We find that the Brd transcript is truncated and accumulates to substantially higher levels in the gain-of-function mutants, due to the insertion of a transposable element of the blood family in the Brd 3′ untranslated region (UTR). The wild-type Brd 3′ UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that we refer to as the ‘Brd box’. Moreover, the 3′ UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif we refer to as the ‘GY box’ (GTCTTCC). We find that both the Brd box and the GY box are also present in the 3′ UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and two of the Brd boxes present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them. Finally, we find that Brd encodes a novel small protein of only 81 amino acids that is predicted to include a basic amphipathic alpha-helix. The deduced Brd protein shows sequence similarity to the E(spl)m4 protein, which is likewise expected to include a basic amphipathic alpha-helix, suggesting that the two proteins have related biochemical functions.

scholarly journals A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

2020 ◽  
Author(s):  
Felix Pahmeier ◽  
Christoper J Neufeldt ◽  
Berati Cerikan ◽  
Vibhu Prasad ◽  
Costantin Pape ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Reporter System ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACTPositive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

Protein Sequence and Structure of N-terminal Amino Acids of Subunit Delta of Spinach Photosynthetic ATP-Synthase CF1

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1231-1238 ◽  
Author(s):  
Richard J. Berzborn ◽  
Werner Finke ◽  
Joachim Otto ◽  
Helmut E . Meyer
Keyword(s):  
Secondary Structure ◽  
Atp Synthase ◽  
Protein Sequence ◽  
Alpha Helix ◽  
Amino Acid Residues ◽  
E Coli ◽  
Helical Wheel ◽  
Terminal Amino ◽  
Amphipathic Alpha Helix ◽  
Structure Calculations

Chloroplast ATP-synthase (CF1) subunit delta (δ) has been isolated from spinach thylakoids in the presence of SDS. By automated Edman degradation and online analysis of PTH derivatives the 35 N-terminal amino acid residues were sequenced. The mature protein starts with: NH2-Val-Asp-Ser-Thr-Ala-Ser-Arg-Tyr-Ala-. This protein sequence allows alignment of spinach δ with the sequences of Z. mays 25 kDa polypeptide, the δ subunit of Rps. blastica, Rsp. rubrum and E. coli F1, and of bovine OSCP, but not with mitochondrial δ. Secondary structure calculations and helical wheel plots reveal a conserved secondary structure. The analyzed N-terminal sequences probably build a short amphipathic alpha helix with two adjacent turns. The such aligned polar residues around Tyr8 of subunit δ are suitable to channel protons.

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