scholarly journals Alphavirus Nucleocapsid Protein Contains a Putative Coiled Coil α-Helix Important for Core Assembly

2001 ◽  
Vol 75 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rushika Perera ◽  
Katherine E. Owen ◽  
Timothy L. Tellinghuisen ◽  
Alexander E. Gorbalenya ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Coiled Coil ◽  
Terminal Region ◽  
Wild Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
In Vitro Assembly ◽  
Α Helix

ABSTRACT The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an α-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with β-branched residues and conserved leucine residues occupying the a andd positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.

A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions

2002 ◽  
Vol 115 (14) ◽  
pp. 2867-2879 ◽  
Author(s):  
Valérie Derrien ◽  
Carole Couillault ◽  
Michel Franco ◽  
Stéphanie Martineau ◽  
Philippe Montcourrier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Coiled Coil ◽  
Terminal Region ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Sec7 Domain

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.

scholarly journals Amino Acids in the Capsid Protein of Tomato Yellow Leaf Curl Virus That Are Crucial for Systemic Infection, Particle Formation, and Insect Transmission

1998 ◽  
Vol 72 (12) ◽  
pp. 10050-10057 ◽  
Author(s):  
E. Noris ◽  
A. M. Vaira ◽  
P. Caciagli ◽  
V. Masenga ◽  
B. Gronenborn ◽  
...  
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Tomato Yellow Leaf ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Yellow Leaf ◽  
Wild Type ◽  
Insect Transmission ◽  
Leaf Curl Virus ◽  
Leaf Curl

ABSTRACT A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sic, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sic, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.

scholarly journals A Heterologous Coiled Coil Can Substitute for Helix I of the Sindbis Virus Capsid Protein

2003 ◽  
Vol 77 (15) ◽  
pp. 8345-8353 ◽  
Author(s):  
Rushika Perera ◽  
Chanakha Navaratnarajah ◽  
Richard J. Kuhn
Keyword(s):  
Capsid Protein ◽  
Sindbis Virus ◽  
Coiled Coil ◽  
Wild Type ◽  
Transcription Activator ◽  
Particle Assembly ◽  
Virus Capsid ◽  
Assembly Pathway ◽  
Trimer Formation ◽  
Virus Capsid Protein

ABSTRACT Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate. Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I. This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid. The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil. Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis. However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited ∼40-fold reduction in virus replication compared to the wild-type Sindbis virus. Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication. Peptides corresponding to helix I inhibited core-like particle assembly in vitro. On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway.

scholarly journals Alphavirus Capsid Protein Helix I Controls a Checkpoint in Nucleocapsid Core Assembly

2006 ◽  
Vol 80 (18) ◽  
pp. 8848-8855 ◽  
Author(s):  
Eunmee M. Hong ◽  
Rushika Perera ◽  
Richard J. Kuhn
Keyword(s):  
Capsid Protein ◽  
Sindbis Virus ◽  
Coiled Coil ◽  
Assembly System ◽  
Equine Encephalitis Virus ◽  
In Vitro Assembly ◽  
Western Equine Encephalitis Virus ◽  
Phenotypic Mixing ◽  
Western Equine Encephalitis

ABSTRACT The assembly of the alphavirus nucleocapsid core has been investigated using an in vitro assembly system. The C-terminal two-thirds of capsid protein (CP), residues 81 to 264 in Sindbis virus (SINV), have been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly in vitro. Helix I, which is located in the N-terminal dispensable region of the CP, has been proposed to stabilize the core by forming a coiled coil in the CP dimer formed by the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed core-like particles (CLPs) using an in vitro assembly system. The CPs of SINV and Ross River virus (RRV) do not form phenotypically mixed CLPs, but SINV and Western equine encephalitis virus CPs do form mixed cores. In addition, CP dimers do not form between SINV and RRV in these assembly reactions. In contrast, an N-terminal truncated SINV CP (residues 81 to 264) forms phenotypically mixed CLPs when it is assembled with full-length heterologous CPs, suggesting that the region that controls the mixing is present in the N-terminal 80 residues. Furthermore, this result suggests that the dimeric interaction, which was absent between SINV and RRV CPs, can be restored by the removal of the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto helix I by using domain swapping experiments. Thus, discrimination of the CP partner in alphavirus core assembly appears to be dependent on helix I sequence compatibility. These results suggest that helix I provides one of the important interactions during nucleocapsid core formation and may play a regulatory role during the early steps of the assembly process.

scholarly journals A Conserved Capsid Protein Surface Domain of Cucumber Mosaic Virus Is Essential for Efficient Aphid Vector Transmission

2002 ◽  
Vol 76 (19) ◽  
pp. 9756-9762 ◽  
Author(s):  
Sijun Liu ◽  
Xiaohua He ◽  
Gyungsoon Park ◽  
Caroline Josefsson ◽  
Keith L. Perry
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Capsid Protein ◽  
Wild Type Virus ◽  
Cdna Clones ◽  
Wild Type ◽  
Vector Transmission ◽  
Aphid Vector

ABSTRACT A prominent feature on the surfaces of virions of Cucumber mosaic virus (CMV) is a negatively charged loop structure (the βH-βI loop). Six of 8 amino acids in this capsid protein loop are highly conserved among strains of CMV and other cucumoviruses. Five of these amino acids were individually changed to alanine or lysine (an amino acid of opposite charge) to create nine mutants (the D191A, D191K, D192A, D192K, L194A, E195A, E195K, D197A, and D197K mutants). Transcripts of cDNA clones were infectious when they were mechanically inoculated onto tobacco, giving rise to symptoms of a mottle-mosaic typical of the wild-type virus (the D191A, D191K, D192A, E195A, E195K, and D197A mutants), a systemic necrosis (the D192K mutant), or an atypical chlorosis with necrotic flecking (the L194A mutant). The mutants formed virions and accumulated to wild-type levels, but eight of the nine mutants were defective in aphid vector transmission. The aspartate-to-lysine mutation at position 197 interfered with infection; the only recovered progeny (the D197K∗ mutant) harbored a second-site mutation (denoted by the asterisk) of alanine to glutamate at position 193, a proximal site in the βH-βI loop. Since the disruption of charged amino acid residues in the βH-βI loop reduces or eliminates vector transmissibility without grossly affecting infectivity or virion formation, we hypothesize that this sequence or structure has been conserved to facilitate aphid vector transmission.

scholarly journals Conserved hydrophobic amino acid residues in the N-terminal region of DnaA protein are involved in DnaA–DnaA interaction

2002 ◽  
Vol 365 (3) ◽  
pp. 881-887 ◽  
Author(s):  
Shinji MIMA ◽  
Masaki MAKISE ◽  
Motohiro KOTERASAWA ◽  
Tomofusa TSUCHIYA ◽  
Tohru MIZUSHIMA
Keyword(s):  
Amino Acid ◽  
Leucine Zipper ◽  
Terminal Region ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Hydrophobic Amino Acid ◽  
Dnaa Protein

We previously reported that a leucine-zipper-like structure (I26, L33 and L40) located in the N-terminal region of DnaA is essential for the duplex opening at oriC by DnaA. In this study, we focused on three other conserved hydrophobic amino acid residues, L3, L10 and L17, and examined the function of DnaA proteins mutated in these amino acid residues. DnaA427 (L17S) and DnaA413 (L3S, L10S and L17S) were inactive for oriC DNA replication both in vitro and in vivo. Although these mutant DnaA proteins maintained their binding activities for both ATP and oriC, they were unable to induce the opening of duplex DNA at oriC. Glutathione-S-transferase (GST)-fused wild-type DnaA interacted with wild-type DnaA but not with DnaA427 and DnaA413. Based on these results, we propose that conserved hydrophobic amino acid residues in the N-terminal region of DnaA are involved in DnaA oligomerization, in which DnaA—DnaA interaction is required.

scholarly journals Positive and Negative Modulation of Virus Infectivity and Envelope Glycoprotein Incorporation into Virions by Amino Acid Substitutions at the N Terminus of the Simian Immunodeficiency Virus Matrix Protein

2003 ◽  
Vol 77 (20) ◽  
pp. 10881-10888 ◽  
Author(s):  
Julieta M. Manrique ◽  
Cristina C. P. Celma ◽  
Eric Hunter ◽  
José L. Affranchino ◽  
Silvia A. González
Keyword(s):  
Amino Acid ◽  
Matrix Protein ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Amino Acid Substitutions ◽  
Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
Amino Terminal ◽  
Env Glycoprotein

ABSTRACT The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal α-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within α-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the β-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.

scholarly journals Development of an intracellularly acting inhibitory peptide selective for PKN

2009 ◽  
Vol 425 (2) ◽  
pp. 445-543 ◽  
Author(s):  
Kazuhiro Shiga ◽  
Kentaro Takayama ◽  
Shiroh Futaki ◽  
Jessica E. Hutti ◽  
Lewis C. Cantley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Phosphorylation Site ◽  
Coiled Coil ◽  
Terminal Region ◽  
Inhibitory Peptide ◽  
Amino Acid Peptide ◽  
Amino Acid Stretch

PKNs form a subfamily of the AGC serine/threonine protein kinases, and have a catalytic domain homologous with that of PKC (protein kinase C) in the C-terminal region and three characteristic ACC (antiparallel coiled-coil) domain repeats in the N-terminal region. The preferred peptide phosphorylation motif for PKNs determined by a combinatorial peptide library method was highly similar to that of PKCs within a 10-amino-acid stretch. Previously reported PKN inhibitory compounds also inhibit PKCs to a similar extent, and no PKN selective inhibitors have been commercially available. We have identified a 15-amino-acid peptide inhibitor of PKNs based on amino acids 485–499 of the C-terminal region of the C2-like domain of PKN1. This peptide, designated as PRL, selectively inhibits the kinase activity of all isoforms of PKN (Ki=0.7 μM) towards a peptide substrate, as well as autophosphorylation activity of PKN in vitro, in contrast with PKC. Reversible conjugation by a disulfide bond of a carrier peptide bearing a penetration accelerating sequence to PRL, facilitated the cellular uptake of this peptide and significantly inhibited phosphorylation of tau by PKN1 at the PKN1-specific phosphorylation site in vivo. This peptide may serve as a valuable tool for investigating PKN activation and PKN-mediated responses.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

2000 ◽  
Vol 74 (7) ◽  
pp. 3353-3365 ◽  
Author(s):  
Chi-Long Lin ◽  
Che-Sheng Chung ◽  
Hans G. Heine ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

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