scholarly journals Identification and functional analysis of inter-subunit disulfide bonds of the F protein of Helicoverpa armigera nucleopolyhedrovirus

2014 ◽  
Vol 95 (12) ◽  
pp. 2820-2830 ◽  
Author(s):  
Feifei Yin ◽  
Manli Wang ◽  
Ying Tan ◽  
Fei Deng ◽  
Just M. Vlak ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Viral Entry ◽  
Helicoverpa Armigera ◽  
Disulfide Bonds ◽  
Site Directed Mutagenesis ◽  
Disulfonic Acid ◽  
Viral Infectivity ◽  
F Protein ◽  
Free Thiol ◽  
Helicoverpa Armígera

The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4′-maleimidylstilbene 2,2′-disulfonic acid (AMS) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.

scholarly journals Functional studies of per os infectivity factor 3 of Helicoverpa armigera nucleopolyhedrovirus

2012 ◽  
Vol 93 (2) ◽  
pp. 374-382 ◽  
Author(s):  
Jingjiao Song ◽  
Manli Wang ◽  
Huachao Huang ◽  
Xin Luo ◽  
Fei Deng ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Helicoverpa Armigera ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
Terminal Region ◽  
Disulfide Bond Formation ◽  
Functional Studies ◽  
Nuclear Ring ◽  
Helicoverpa Armígera ◽  
And Function

PIF3 is one of the six conserved per os infectivity factors (PIFs) of baculoviruses. In this study, PIF3 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was analysed by infectivity bioassays using a series of recombinant viruses harbouring various PIF3 truncation/substitution mutants. The results demonstrated that the N-terminal region (L26–Y45) and C-terminal region (T160–Q199) are essential for HearNPV oral infectivity. In the C-terminal T160–Q199 region, there are three conserved cysteines (C162, C164 and C185). Our results showed that substitutions of C162 or C164, predicted to be involved in disulfide-bond formation, led to a severe decrease in HearNPV per os infectivity. Mutation of C185, predicted not to be involved in disulfide-bond formation, did not affect the per os infectivity. The data suggest that disulfide bonds are important for PIF3 conformation and function. Immunofluorescence assays showed that none of the mutations affected the subcellular localization of PIF3 to the nuclear ring zone region of infected cells. Western blot results showed that all mutants except C162G and C185G failed to incorporate PIF3 into occlusion-derived viruses, which resulted in impaired oral infectivity of the latter. The data provide insights for future study of PIF3 function.

scholarly journals The F protein of Helicoverpa armigera single nucleopolyhedrovirus can be substituted functionally with its homologue from Spodoptera exigua multiple nucleopolyhedrovirus

2008 ◽  
Vol 89 (3) ◽  
pp. 791-798 ◽  
Author(s):  
Manli Wang ◽  
Ying Tan ◽  
Feifei Yin ◽  
Fei Deng ◽  
Just M. Vlak ◽  
...  
Keyword(s):  
Helicoverpa Armigera ◽  
Spodoptera Exigua ◽  
Late Phase ◽  
Positive Control ◽  
Host Proteins ◽  
F Protein ◽  
Helicoverpa Armígera ◽  
Group Ii ◽  
Budded Virus ◽  
Species Specific

F proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacΔF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When HaBacΔF was pseudotyped with the homologous F protein (HaBacΔF-HaF, positive control) or with the heterologous F protein from Spodoptera exigua multinucleocapsid NPV (SeMNPV) (HaBacΔF-SeF), infectious BVs were produced with similar kinetics. In the late phase of infection, the BV titre of HaBacΔF-SeF virus was about ten times lower than that of HaBacΔF-HaF virus. Both pseudotyped viruses were able to fuse HzAM1 cells in a similar fashion. The F proteins of both HearNPV and SeMNPV were completely cleaved into F1 and F2 in the BVs of vHaBacΔF-HaF and vHaBacΔF-SeF, respectively, but the cleavage of SeF in vHaBacΔF-SeF-infected HzAM1 cells was incomplete, explaining the lower BV titre of vHaBacΔF-SeF. Polyclonal antisera against HaF1 and SeF1 specifically neutralized the infection of vHaBacΔF-HaF and vHaBacΔF-SeF, respectively. HaF1 antiserum showed some cross-neutralization with vHaBacΔF-SeF. These results demonstrate that group II NPV F proteins can be functionally replaced with a homologue of other group II NPVs, suggesting that the interaction of F with other viral or host proteins is not absolutely species-specific.

scholarly journals Roles of the Intramolecular Disulfide Bridge in MotX and MotY, the Specific Proteins for Sodium-Driven Motors in Vibrio spp

2006 ◽  
Vol 188 (14) ◽  
pp. 5308-5314 ◽  
Author(s):  
Jin Yagasaki ◽  
Mayuko Okabe ◽  
Rie Kurebayashi ◽  
Toshiharu Yakushi ◽  
Michio Homma
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Disulfide Bridge ◽  
Site Directed Mutagenesis ◽  
Proper Function ◽  
Intramolecular Disulfide Bond ◽  
Specific Proteins ◽  
The Stability ◽  
Vibrio Spp ◽  
Lines Of Evidence

ABSTRACT The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.

scholarly journals Function, oligomerization and N-linked glycosylation of the Helicoverpa armigera single nucleopolyhedrovirus envelope fusion protein

2006 ◽  
Vol 87 (4) ◽  
pp. 839-846 ◽  
Author(s):  
Gang Long ◽  
Marcel Westenberg ◽  
Hualin Wang ◽  
Just M. Vlak ◽  
Zhihong Hu
Keyword(s):  
Fusion Protein ◽  
Helicoverpa Armigera ◽  
Disulfide Bonds ◽  
Attractive Alternative ◽  
Present Observation ◽  
Reading Frame ◽  
Group I ◽  
The Family ◽  
Helicoverpa Armígera ◽  
Group Ii

In the family Baculoviridae, two distinct envelope fusion proteins are identified in budded virions (BVs). GP64 is the major envelope fusion protein of group I nucleopolyhedrovirus (NPV) BVs. An unrelated type of envelope fusion protein, named F, is encoded by group II NPVs. The genome of Helicoverpa armigera (Hear) NPV, a group II NPV of the single nucleocapsid or S type, also encodes an F-like protein: open reading frame 133 (Ha133). It was demonstrated by N-terminal sequencing of the major 59 kDa protein present in HearNPV BV that this protein is one of the two F subunits: F1 (transmembrane subunit of 59 kDa) and F2 (surface subunit of 20 kDa), both the result of cleavage by a proprotein convertase and disulfide-linked. The HearNPV F protein proved to be a functional analogue of GP64, as the infectivity of an AcMNPV gp64-deletion mutant was rescued by the introduction of the HearNPV F gene. It was also demonstrated by chemical cross-linking that HearNPV F is present in BVs as an oligomer whereby, unlike GP64, disulfide bonds are not involved. Deglycosylation assays indicated that both F1 and F2 possess N-linked glycans. However, when F was made in Hz2E5 cells, these glycans did not have an α-1-3 core fucose modification that usually occurs in insect cells. As α-1-3 core fucose is a major inducer of an allergic response in humans, the present observation makes the HearNPV–Hz2E5 system an attractive alternative for the production of recombinant glycoproteins for therapeutic use in humans.

scholarly journals Mutagenesis and Nuclear Magnetic Resonance Analyses of the Fusion Peptide of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus F Protein

2008 ◽  
Vol 82 (16) ◽  
pp. 8138-8148 ◽  
Author(s):  
Ying Tan ◽  
Ling Jiang ◽  
Manli Wang ◽  
Feifei Yin ◽  
Fei Deng ◽  
...  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Helicoverpa Armigera ◽  
Sodium Dodecyl ◽  
Fusion Peptide ◽  
Structure And Function ◽  
F Protein ◽  
Helicoverpa Armígera ◽  
And Function ◽  
Nuclear Magnetic

ABSTRACT The entry of enveloped viruses into cells is normally mediated by fusion between viral and cellular membranes, in which the fusion peptide plays a crucial role. The fusion peptides of group II nucleopolyhedrovirus (NPV) F proteins are quite conserved, with a hydrophobic region located at the N terminal of the F1 fragment. For this report, we used mutagenesis and nuclear magnetic resonance (NMR) to study the structure and function of the fusion peptide of the Helicoverpa armigera single-nucleocapsid NPV (HearNPV) F protein (HaF). Five mutations in the fusion peptide of HaF, N1G, N1L, I2N, G3L, and D11L, were generated separately, and the mutated f genes were transformed into the f-null HearNPV bacmid. The mutations N1L, I2N, and D11L were found to completely abolish the ability of the recombinant bacmids to produce infectious budded virus, while the mutations N1G and G3L did not. The low-pH-induced envelope fusion assay demonstrated that the N1G substitution increased the fusogenicity of HaF, while the G3L substitution reduced its fusogenicity. NMR spectroscopy was used to determine the structure of a synthetic fusion peptide of HaF in the presence of sodium dodecyl sulfate micelles at pH 5.0. The fusion peptide appeared to be an amphiphilic structure composed of a flexible coil in the N terminus from N1 to N5, a 310-helix from F6 to G8, a turn at S9, and a regular α-helix from V10 to D19. The data provide the first NMR structure of a baculovirus fusion peptide and allow us to further understand the relationship of structure and function of the fusion peptide.

Regulation of Tissue Factor Activity: Conformational Changes in Tissue Factor Associated with Cleavage of the Cysteine 186-Cysteine 209 Disulfide Bond.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 270-270 ◽  
Author(s):  
Jacky Chi Ki Ngo ◽  
Margaret Jacobs ◽  
Barbara C. Furie ◽  
Bruce Furie
Keyword(s):  
Tissue Factor ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Conformational Transition ◽  
Active Form ◽  
Structural Difference ◽  
Partial Reduction ◽  
Difference Spectroscopy ◽  
Free Thiol ◽  
Tryptophan Residues

Abstract Tissue factor (TF) exists in an encrypted, inactive form and an active, procoagulant form. The extracellular domain of TF contains two disulfide bonds. The crystal structure of the active form of TF reveals Cys186 and Cys209 in an unusual solvent-exposed disulfide bond. It was recently proposed that encrypted TF contains two free thiols at Cys186 and Cys209, and formation of a disulfide bond between Cys186 and Cys209 is associated with the decryption of TF (Chen et al, Biochemistry 2006). To characterize the structural differences between the putative encrypted TF and active TF, containing one and two disulfide bonds respectively, we developed an assay to quantitate the free thiols and disulfide bonds in recombinant TF and examined the solvent accessibility of the two disulfide bonds under native and denatured conditions. The perturbations of chromophores of TF by UV difference spectroscopy during specific reduction of Cys186–Cys209 were then examined. In order to quantitate the free thiols and disulfide bonds, TF was subjected to Alexa 488-maleimide (green) labeling to identify any free thiols and, after reduction with TCEP, a second labeling with Alexa 647-maleimide (red) to identify newly exposed free thiols formerly involved in disulfide bonds. Alexa-labeled TF was subjected to SDS gel electrophoresis and the gel was developed on a fluorescence imager to provide quantitative analysis of the integrated fluorescence intensity for each Alexa dye separately. The molar concentrations of free thiol in TF before TCEP treatment and thiols exposed after reduction were determined by comparison to calibration curves that were generated using Alexa maleimide-albumin conjugates. Recombinant TF in solution contains 0.2 moles of free thiol. Upon treatment with 10 mM TCEP under native conditions, TF is partially reduced and contains 1.97 moles of half cystine thiols. TF is fully reduced by 10 mM TCEP under denaturing conditions in the presence of 4 M urea (4.29 moles of thiols, theoretical=4.0 moles). This is consistent with the observation that only one disulfide bond is solvent-exposed in native TF and is accessible for reduction. We then performed ultraviolet absorption difference spectroscopy using the partitioned cell technique to monitor any structural transition during partial reduction of TF. The UV difference spectrum comparing the TCEP-treated TF and unreduced TF showed three peaks, one at 278 nm, a major peak at 285 nm and a small shoulder at 292 nm, that is characteristic of red-shifts in absorption of both tyrosine and tryptophan. Under the conditions employed, this conformational transition was completed in 60 minutes. These results comparing fully oxidized, active TF and partially reduced, encrypted TF indicate that tyrosine and tryptophan residues in TF become protected from solvent upon reduction of the Cys186–Cys209 disulfide bond. In conclusion, we have demonstrated by a new assay that only one disulfide bond within active TF is solvent-exposed and accessible for reduction. The partial reduction of TF at this disulfide bond results in a conformational transition that is associated with the protection of tyrosine and tryptophan residues, indicating structural difference between active TF and encrypted, inactive TF.

scholarly journals Role of Intermolecular Disulfide Bonds of the Organic Solvent-Stable PST-01 Protease in Its Organic Solvent Stability

2001 ◽  
Vol 67 (2) ◽  
pp. 942-947 ◽  
Author(s):  
Hiroyasu Ogino ◽  
Takeshi Uchiho ◽  
Jyunko Yokoo ◽  
Reina Kobayashi ◽  
Rikiya Ichise ◽  
...  
Keyword(s):  
Organic Solvent ◽  
Organic Solvents ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Site Directed Mutagenesis ◽  
Solvent Stability ◽  
Organic Solvent Tolerant ◽  
Intermolecular Disulfide Bonds ◽  
Solvent Tolerant ◽  
Solvent Stable

ABSTRACT The PST-01 protease is secreted by the organic solvent-tolerant microorganism Pseudomonas aeruginosa PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one or both of the disulfide bonds were deleted were constructed by site-directed mutagenesis, and the effect of the disulfide bonds on the activity and the various stabilities was investigated. The disulfide bond between Cys-270 and Cys-297 in the PST-01 protease was found to be essential for its activity. The disulfide bond between Cys-30 and Cys-58 played an important role in the organic solvent stability of the PST-01 protease.

scholarly journals Partial Functional Rescue of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Infectivity by Replacement of F Protein with GP64 from Autographa californica Multicapsid Nucleopolyhedrovirus

2010 ◽  
Vol 84 (21) ◽  
pp. 11505-11514 ◽  
Author(s):  
Manli Wang ◽  
Feifei Yin ◽  
Shu Shen ◽  
Ying Tan ◽  
Fei Deng ◽  
...  
Keyword(s):  
Helicoverpa Armigera ◽  
Culture Supernatant ◽  
Selective Advantage ◽  
Autographa Californica ◽  
Cell Culture Supernatant ◽  
F Protein ◽  
Group I ◽  
Control Virus ◽  
Helicoverpa Armígera ◽  
Group Ii

ABSTRACT Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64-null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64-rescued group II NPV of Helicoverpa armigera (vHaBacΔF-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBacΔF-gp64 compared to the HaF rescue control virus vHaBacΔF-HaF. Electron microscopy further showed that most vHaBacΔF-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBacΔF-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.

scholarly journals Engineered Disulfide Bonds in Herpes Simplex Virus Type 1 gD Separate Receptor Binding from Fusion Initiation and Viral Entry

2007 ◽  
Vol 82 (2) ◽  
pp. 700-709 ◽  
Author(s):  
Eric Lazear ◽  
Andrea Carfi ◽  
J. Charles Whitbeck ◽  
Tina M. Cairns ◽  
Claude Krummenacher ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Disulfide Bond ◽  
Viral Entry ◽  
Disulfide Bonds ◽  
C Terminus ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT Glycoprotein D (gD) is the receptor binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, herpesvirus entry mediator (HVEM) and nectin-1. While both receptor binding regions are found within the first 234 amino acids, a crystal structure shows that the C terminus of the gD ectodomain normally occludes the receptor binding sites. Receptor binding must therefore displace the C terminus, and this conformational change is postulated to be required for inducing fusion via gB and gH/gL. When cysteine residues are introduced at positions 37 and 302 of gD, a disulfide bond is formed that stabilizes the C terminus and prevents binding to either receptor. We speculated that if disulfide bonds were engineered further upstream, receptor binding might be separated from the induction of fusion. To test this, we made five additional double cysteine mutants, each potentially introducing a disulfide bond between the ectodomain C terminus and the core of the gD ectodomain. The two mutants predicted to impose the greatest constraint were unable to bind receptors or mediate cell-cell fusion. However, the three mutants with the most flexible C terminus bound well to both HVEM and nectin-1. Two of these mutants were impaired in cell-cell fusion and null-virus complementation. Importantly, a third mutant in this group was nonfunctional in both assays. This mutant clearly separates the role of gD in triggering fusion from its role in receptor binding. Based upon the properties of the panel of mutants we conclude that fusion requires greater flexibility of the gD ectodomain C terminus than does receptor binding.

scholarly journals Incorporation of GP64 into Helicoverpa armigera nucleopolyhedrovirus enhances virus infectivity in vivo and in vitro

2012 ◽  
Vol 93 (12) ◽  
pp. 2705-2711 ◽  
Author(s):  
Shu Shen ◽  
Yinyin Gan ◽  
Manli Wang ◽  
Zhihong Hu ◽  
Hualin Wang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Helicoverpa Armigera ◽  
Fusion Proteins ◽  
Virus Infectivity ◽  
F Protein ◽  
Recombinant Viruses ◽  
Group I ◽  
Helicoverpa Armígera

The envelope fusion proteins of baculoviruses, glycoprotein GP64 from group I nucleopolyhedrovirus (NPV) or the F protein from group II NPV and granulovirus, are essential for baculovirus morphogenesis and infectivity. The F protein is considered the ancestral baculovirus envelope fusion protein, while GP64 is a more recent evolutionary introduction into baculoviruses and exhibits higher fusogenic activity than the F protein. Each of the fusion proteins is required by the respective virus to spread infection within larval tissues. A recombinant Helicoverpa armigera NPV (HearNPV) expressing GP64 from Autographa californica multiple nucleopolyhedrovirus, vHaBac-gp64-egfp, was constructed, which still retained the native F protein, and its infectivity was assayed in vivo and in vitro. Analyses by one-step growth curve to determine viral titre and by quantitative PCR to determine viral DNA copy number showed that vHaBac-gp64-egfp was more infectious in vitro than the control, vHaBac-egfp. The polyhedrin gene (polh) was reintroduced into the recombinant viruses and bioassays showed that vHaBac-gp64-polh accelerated the mortality of infected larvae compared with the vHaBac-egfp-polh control, and the LC50 (median lethal concentration) of vHaBac-gp64-polh was reduced to approximately 20 % of that of vHaBac-egfp-polh. Therefore, incorporation of GP64 into HearNPV budded virions improved virus infectivity both in vivo and in vitro. The construction of this bivalent virus with a more efficient fusion protein could improve the use of baculoviruses in different areas such as gene therapy and biocontrol.

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