scholarly journals Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding

2008 ◽  
Vol 82 (22) ◽  
pp. 11454-11460 ◽  
Author(s):  
Maximiliano Wilda ◽  
Nora Lopez ◽  
Juan Cruz Casabona ◽  
Maria T. Franze-Fernandez
Keyword(s):  
Binding Sites ◽  
Reverse Genetics ◽  
Point Mutations ◽  
Ring Finger ◽  
Protein Z ◽  
Glycoprotein Precursor ◽  
Polymerase Domain ◽  
N Terminus ◽  
Domain Iii ◽  
Nucleoprotein N

ABSTRACT Tacaribe virus (TacV) is the prototype of the New World group of arenaviruses. The TacV genome encodes four proteins: the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a RING finger protein (Z). Using a reverse genetics system, we demonstrated that TacV N and L are sufficient to drive transcription and replication mediated by TacV-like RNAs and that Z is a powerful inhibitor of these processes (Lopez et al., J. Virol. 65:12241-12251, 2001). More recently, we provided the first evidence of an interaction between Z and L and showed that Z's inhibitory activity was dependent on its ability to bind to L (Jácamo et al., J. Virol. 77:10383-10393, 2003). In the present study, we mapped the TacV Z-binding sites on the 2,210-amino-acid L polymerase. To that end, we performed deletion analysis and point mutations of L and studied the Z-L interaction by coimmunoprecipitation with specific sera. We found that the C-terminal region of L was not essential for the interaction and identified two noncontiguous regions that were critical for binding: one at the N-terminus of L between residues 156 and 292 and a second one in the polymerase domain (domain III). The importance of domain III in binding was revealed by substitutions in D1188 and H1189 within motif A and in each residue of the conserved SDD sequence (residues 1328, 1329, and 1330) within motif C. Our results showed that of the substituted residues, only H1189 and D1329 appeared to be critically involved in binding Z.

scholarly journals Tacaribe Virus Z Protein Interacts with the L Polymerase Protein To Inhibit Viral RNA Synthesis

2003 ◽  
Vol 77 (19) ◽  
pp. 10383-10393 ◽  
Author(s):  
Rodrigo Jácamo ◽  
Nora López ◽  
Maximiliano Wilda ◽  
María T. Franze-Fernández
Keyword(s):  
Inhibitory Activity ◽  
Rna Synthesis ◽  
Ring Finger ◽  
Direct Interaction ◽  
Viral Rna ◽  
Reverse Genetic System ◽  
Small Ring ◽  
Protein Z ◽  
Glycoprotein Precursor ◽  
Tacaribe Virus

ABSTRACT Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. The TV genome encodes four proteins, the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a small RING finger protein (Z). Using a reverse genetic system, we recently demonstrated that TV N and L are sufficient to drive transcription and full-cycle RNA replication mediated by TV-like RNAs and that Z is a powerful inhibitor of these processes (N. López, R. Jácamo, and M. T. Franze-Fernández, J. Virol. 65:12241-12251, 2001). In the present study we investigated whether Z might interact with either of the proteins, N and L, required for RNA synthesis. To that end, we used coimmunoprecipitation with monospecific antibodies against the viral proteins and coimmunoprecipitation with serum against glutathione S-transferase (GST) and binding to glutathione-Sepharose beads when Z was expressed as a fusion protein with GST. We demonstrated that Z interacted with L but not with N and that Z inhibitory activity was dependent on its ability to bind to L. We also evaluated the contribution of different Z regions to its binding ability and functional activity. We found that integrity of the RING structure is essential for Z binding to L and for Z inhibitory activity. Mutants with deletions at the N and C termini of Z showed that amino acids within the C-terminal region and immediately adjacent to the RING domain N terminus contribute to efficient Z-L interaction and are required for inhibitory activity. The data presented here provide the first evidence of an interaction between Z and L, suggesting that Z interferes with viral RNA synthesis by direct interaction with L. In addition, coimmunoprecipitation studies revealed a previously unreported interaction between N and L.

scholarly journals Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity

2007 ◽  
Vol 88 (8) ◽  
pp. 2320-2328 ◽  
Author(s):  
Christian Klewitz ◽  
Hans-Dieter Klenk ◽  
Jan ter Meulen
Keyword(s):  
Amino Acids ◽  
Structural Changes ◽  
Point Mutations ◽  
Lassa Virus ◽  
Virus Envelope ◽  
Glycoprotein Precursor ◽  
Alanine Scan ◽  
N Terminus ◽  
Hydrophobic Amino Acids ◽  
Ph Dependent

Lassa virus glycoprotein 2 (LASV GP-2) belongs to the class I fusion protein family. Its N terminus contains two stretches of highly conserved hydrophobic amino acids (residues 260–266 and 276–298) that have been proposed as N-terminal or internal fusion peptide segments (N-FPS, I-FPS) by analogy with similar sequences of other viral glycoproteins or based on experimental data obtained with synthetic peptides, respectively. By using a pH-dependent, recombinant LASV glycoprotein mediated cell–cell fusion assay and a retroviral pseudotype infectivity assay, an alanine scan of all hydrophobic amino acids within both proposed FPSs was performed. Fusogenicity and infectivity were correlated, both requiring correct processing of the glycoprotein precursor. Most point mutations in either FPS accounted for reduced or abolished fusion or infection, respectively. Some mutations also had an effect on pre-fusion steps of virus entry, possibly by inducing structural changes in the glycoprotein. The data demonstrate that several amino acids from both hydrophobic regions of the N terminus, some of which (W264, G277, Y278 and L280) are 100 % conserved in all arenaviruses, are involved in fusogenicity and infectivity of LASV GP-2.

scholarly journals Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin

1995 ◽  
Vol 309 (1) ◽  
pp. 355-359 ◽  
Author(s):  
G E Morris ◽  
C Nguyen ◽  
Nguyen thi Man
Keyword(s):  
Monoclonal Antibodies ◽  
Point Mutations ◽  
Hypervariable Region ◽  
Variable Region ◽  
Binding Studies ◽  
Cdna Sequences ◽  
N Terminus ◽  
Random Library ◽  
Blast Cells

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first three amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest that this hydrophobic N-terminus of dystrophin is accessible to antibody in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable region (VH) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing. After elimination of PCR errors, the two cDNA sequences were found to be identical except for five somatic mutations which resulted in three amino acid changes in the second hypervariable region (CDR2). The results suggest that the two hybridomas originated from the same lymphocyte clone in a germinal centre of the spleen, but underwent different point mutations and subtype switches during clonal expansion to form blast cells.

scholarly journals The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

2009 ◽  
Vol 83 (18) ◽  
pp. 9449-9463 ◽  
Author(s):  
Jun Han ◽  
Mark S. Rutherford ◽  
Kay S. Faaberg
Keyword(s):  
Cysteine Protease ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Respiratory Syndrome Virus ◽  
Protease Domain ◽  
Nonstructural Protein 2 ◽  
Cleavage Activity ◽  
Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

scholarly journals Evolution of mouse circadian enhancers from transposable elements

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Julius Judd ◽  
Hayley Sanderson ◽  
Cédric Feschotte
Keyword(s):  
Gene Expression ◽  
Transposable Elements ◽  
Binding Sites ◽  
Mouse Liver ◽  
Integration Site ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Circadian Gene ◽  
Binding Motifs ◽  
Reporter Assays

Abstract Background Transposable elements are increasingly recognized as a source of cis-regulatory variation. Previous studies have revealed that transposons are often bound by transcription factors and some have been co-opted into functional enhancers regulating host gene expression. However, the process by which transposons mature into complex regulatory elements, like enhancers, remains poorly understood. To investigate this process, we examined the contribution of transposons to the cis-regulatory network controlling circadian gene expression in the mouse liver, a well-characterized network serving an important physiological function. Results ChIP-seq analyses reveal that transposons and other repeats contribute ~ 14% of the binding sites for core circadian regulators (CRs) including BMAL1, CLOCK, PER1/2, and CRY1/2, in the mouse liver. RSINE1, an abundant murine-specific SINE, is the only transposon family enriched for CR binding sites across all datasets. Sequence analyses and reporter assays reveal that the circadian regulatory activity of RSINE1 stems from the presence of imperfect CR binding motifs in the ancestral RSINE1 sequence. These motifs matured into canonical motifs through point mutations after transposition. Furthermore, maturation occurred preferentially within elements inserted in the proximity of ancestral CR binding sites. RSINE1 also acquired motifs that recruit nuclear receptors known to cooperate with CRs to regulate circadian gene expression specifically in the liver. Conclusions Our results suggest that the birth of enhancers from transposons is predicated both by the sequence of the transposon and by the cis-regulatory landscape surrounding their genomic integration site.

scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. Stedman
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Capsid Protein Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene vp3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. STEDMAN
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1 ◽  
Virion Formation

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein VP3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals The phosphoprotein (P) and L binding sites reside in the N-terminus of the L subunit of the measles virus RNA polymerase

Virology ◽  
2004 ◽  
Vol 327 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Bayram Cevik ◽  
David E. Holmes ◽  
Emmanuel Vrotsos ◽  
Joyce A. Feller ◽  
Sherin Smallwood ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Sites ◽  
Measles Virus ◽  
Virus Rna ◽  
N Terminus

Abstract MP20: Optogenetic Control Of Pip2 To Assess Mechanisms Regulating The Epithelial Sodium Channel

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Crystal R Archer ◽  
Amanpreet Kaur ◽  
Tarek Mohamed ◽  
James D Stockand
Keyword(s):  
Binding Sites ◽  
Hek293 Cells ◽  
Proper Function ◽  
Small Decrease ◽  
Kidney Tubules ◽  
Wild Type ◽  
N Terminus ◽  
Patch Clamp Electrophysiology ◽  
G Protein Coupled

The epithelial Na + channel (ENaC) plays a key role in Na + transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na + reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP 2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP 2 binding sites, by controlled depletion of PIP 2 . We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP 2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. A fluorescent Na + indicator, was used to monitor ENaC activity by tracking the relative intracellular Na + levels. Upon optogenetic-controlled depletion of PIP 2 , Na + levels decreased in cells expressing wt ENaC. Mutations to the PIP 2 sites of ENaC were expected to have no change in Na + levels upon PIP 2 depletion due to the disruption of PIP 2 binding. As a control, mutations to non-PIP 2 binding sites were included, and were expected to have decreased Na + levels similar to wt ENaC. Interestingly, mutation of each independent PIP 2 site resulted in only a small decrease of intracellular Na + , compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP 2 binding site, resulted in a significant increase of Na + upon PIP 2 depletion. We performed patch clamp electrophysiology and found that the ENaC recordings corresponded to the Na + fluctuations. These data suggest that the residues surrounding the PIP 2 binding sites play a significant role in the affinity of PIP 2 for ENaC. The role of these other domains in PIP 2 binding is still under investigation.

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