Intra- and Intermolecular Disulfide Bonds of theGP2b Glycoprotein of Equine Arteritis Virus: Relevance forVirus Assembly andInfectivity

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV virions contain six different envelope proteins. The glycoprotein GP5 (previously named GL) and the unglycosylated membrane protein M are the major envelope proteins, while the glycoproteins GP2b (previously named GS), GP3, and GP4 are minor structural proteins. The unglycosylated small hydrophobic envelope protein E is present in virus particles in intermediate molar amounts compared to the other transmembrane proteins. The GP5 and M proteins are both essential for particle assembly. They occur as covalently linked heterodimers that constitute the basic protein matrix of the envelope. The GP2b, GP3, and GP4 proteins occur as a heterotrimeric complex in which disulfide bonds play an important role. The function of this complex has not been established yet, but the available data suggest it to be involved in the viral entry process. Here we investigated the role of the four cysteine residues of the mature GP2b protein in the assembly of the GP2b/GP3/GP4 complex. Open reading frames encoding cysteine-to-serine mutants of the GP2b protein were expressed independently or from a full-length infectious EAV cDNA clone. The results of these experiments support a model in which the cysteine residue at position 102 of GP2b forms an intermolecular cystine bridge with one of the cysteines of the GP4 protein, while the cysteine residues at positions 48 and 137 of GP2b are linked by an intrachain disulfide bond. In this model, another cysteine residue in the GP4 protein is responsible for the covalent association of GP3 with the disulfide-linked GP2b/GP4 heterodimer. In addition, our data highlight the importance of the correct association of the minor EAV envelope glycoproteins for their efficient incorporation into viral particles and for virus infectivity.

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Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity

Journal of Virology ◽

10.1128/jvi.77.1.97-104.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 97-104 ◽

Cited By ~ 68

Author(s):

Eric J. Snijder ◽

Jessika C. Dobbe ◽

Willy J. M. Spaan

Keyword(s):

Membrane Protein ◽

Disulfide Bond ◽

Cdna Clone ◽

Virus Assembly ◽

Cysteine Residue ◽

Envelope Proteins ◽

Glycosylation Site ◽

Equine Arteritis Virus ◽

Virus Infectivity ◽

Full Length Clone

ABSTRACT The two major envelope proteins of arteriviruses, the membrane protein (M) and the major glycoprotein (GP5), associate into a disulfide-linked heterodimer that is incorporated into the virion and has been assumed to be a prerequisite for virus assembly. Using an equine arteritis virus (EAV) infectious cDNA clone, we have analyzed the requirement for GP5-M heterodimerization and have identified the Cys residues involved in the formation of the GP5-M disulfide bond. The single Cys residue (Cys-8) in the M ectodomain was crucial for heterodimerization and virus infectivity. Mutagenesis of any of the five Cys residues in the GP5 ectodomain or removal of the single GP5 N-glycosylation site also rendered the full-length clone noninfectious. However, an analysis of revertants yielded an exceptional pseudorevertant in which residues 52 to 79 of the GP5 ectodomain had been deleted and the original Cys-80→Ser mutation had been maintained. Consequently, this revertant lacked the GP5 N-glycosyation site (Asn-56) and retained only a single cysteine residue (Cys-34). By using this GP5 deletion, we confirmed that Cys-34 of GP5 and Cys-8 of M are essential for GP5-M heterodimerization, a key event in the assembly of the EAV envelope.

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Lysosome-Associated Membrane Proteins Support the Furin-Mediated Processing of the Mumps Virus Fusion Protein

Journal of Virology ◽

10.1128/jvi.00050-20 ◽

2020 ◽

Vol 94 (12) ◽

Author(s):

Ayako Ueo ◽

Marie Kubota ◽

Yuta Shirogane ◽

Shinji Ohno ◽

Takao Hashiguchi ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Fusion ◽

Rna Virus ◽

Envelope Proteins ◽

Mumps Virus ◽

Hek293 Cells ◽

Virus Infectivity ◽

Protein Cleavage ◽

F Protein ◽

Target Cell Membrane

ABSTRACT Mumps virus (MuV), an enveloped RNA virus of the Paramyxoviridae family and the causative agent of mumps, affects the salivary glands and other glandular tissues as well as the central nervous system. The virus enters the cell by inducing the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by MuV envelope proteins: the hemagglutinin-neuraminidase and fusion (F) protein. Cleavage of the MuV F protein (MuV-F) into two subunits by the cellular protease furin is a prerequisite for fusion and virus infectivity. Here, we show that 293T (a derivative of HEK293) cells do not produce syncytia upon expression of MuV envelope proteins or MuV infection. This failure is caused by the inefficient MuV-F cleavage despite the presence of functional furin in 293T cells. An expression cloning strategy revealed that overexpression of lysosome-associated membrane proteins (LAMPs) confers on 293T cells the ability to produce syncytia upon expression of MuV envelope proteins. The LAMP family comprises the ubiquitously expressed LAMP1 and LAMP2, the interferon-stimulated gene product LAMP3, and the cell type-specific proteins. The expression level of the LAMP3 gene, but not of LAMP1 and LAMP2 genes, differed markedly between 293T and HEK293 cells. Overexpression of LAMP1, LAMP2, or LAMP3 allowed 293T cells to process MuV-F efficiently. Furthermore, these LAMPs were found to interact with both MuV-F and furin. Our results indicate that LAMPs support the furin-mediated cleavage of MuV-F and that, among them, LAMP3 may be critical for the process, at least in certain cells. IMPORTANCE The cellular protease furin mediates proteolytic cleavage of many host and pathogen proteins and plays an important role in viral envelope glycoprotein maturation. MuV, an enveloped RNA virus of the Paramyxoviridae family and an important human pathogen, enters the cell through the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by the viral attachment protein and the F protein. Cleavage of MuV-F into two subunits by furin is a prerequisite for fusion and virus infectivity. Here, we show that LAMPs support the furin-mediated cleavage of MuV-F. Expression levels of LAMPs affect the processing of MuV-F and MuV-mediated membrane fusion. Among LAMPs, the interferon-stimulated gene product LAMP3 is most critical in certain cells. Our study provides potential targets for anti-MuV therapeutics.

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On the Host Side of the Hepatitis E Virus Life Cycle

Cells ◽

10.3390/cells9051294 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1294 ◽

Cited By ~ 1

Author(s):

Noémie Oechslin ◽

Darius Moradpour ◽

Jérôme Gouttenoire

Keyword(s):

Life Cycle ◽

Viral Entry ◽

Hepatitis E Virus ◽

Molecular Mechanisms ◽

Rna Virus ◽

Hepatitis E ◽

Host Factors ◽

Model Systems ◽

Novel Technologies ◽

Positive Strand Rna

Hepatitis E virus (HEV) infection is one of the most common causes of acute hepatitis in the world. HEV is an enterically transmitted positive-strand RNA virus found as a non-enveloped particle in bile as well as stool and as a quasi-enveloped particle in blood. Current understanding of the molecular mechanisms and host factors involved in productive HEV infection is incomplete, but recently developed model systems have facilitated rapid progress in this area. Here, we provide an overview of the HEV life cycle with a focus on the host factors required for viral entry, RNA replication, assembly and release. Further developments of HEV model systems and novel technologies should yield a broader picture in the future.

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Modification of cysteine residues for mass spectrometry-based proteomic analysis: facts and artifacts

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20206601018 ◽

2020 ◽

Vol 66 (1) ◽

pp. 18-29

Author(s):

K.G. Kuznetsova ◽

E.M. Solovyeva ◽

A.V. Kuzikov ◽

M.V. Gorshkov ◽

S.A. Moshkovskii

Keyword(s):

Sample Preparation ◽

Proteomic Analysis ◽

Disulfide Bonds ◽

Adverse Reactions ◽

Alkylating Agents ◽

Cysteine Residue ◽

Mass Spectrometric ◽

Cysteine Residues ◽

Nucleotide Substitutions ◽

Special Groups

Mass spectrometric proteomic analysis at the sample preparation stage involves the artificial reduction of disulfide bonds in proteins formed between cysteine residues. Such bonds, when preserved in their native state, complicate subsequent enzymatic hydrolysis and interpretation of the research results. To prevent the re-formation of the disulfide bonds, cysteine residues are protected by special groups, most often by alkylation. In this review, we consider the methods used to modify cysteine residues during sample preparation, as well as possible artifacts of this stage. Particularly, adverse reactions of the alkylating agents with other amino acid residues are described. The most common alkylating compound used to protect cysteine residues in mass spectrometric proteomic analysis is iodoacetamide. However, an analysis of the literature in this area indicates that this reagent causes more adverse reactions than other agents used, such as chloroacetamide and acrylamide. The latter can be recommended for wider use. In the review we also discuss the features of the cysteine residue modifications and their influence on the efficiency of the search for post-translational modifications and protein products of single nucleotide substitutions.

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Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00403-4 ◽

2022 ◽

Vol 23 (1) ◽

Author(s):

Shijulal Nelson-Sathi ◽

P. K. Umasankar ◽

E. Sreekumar ◽

R. Radhakrishnan Nair ◽

Iype Joseph ◽

...

Keyword(s):

Amino Acid ◽

Receptor Binding ◽

Viral Entry ◽

In Silico ◽

Rna Virus ◽

Binding Domain ◽

Virus Infectivity ◽

Receptor Binding Domain ◽

Synonymous Mutations ◽

Binding Interface

Abstract Background SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. Results Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. Conclusion Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

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Formation of Disulfide-Linked Complexes between the Three Minor Envelope Glycoproteins (GP2b, GP3, and GP4) of Equine Arteritis Virus

Journal of Virology ◽

10.1128/jvi.77.11.6216-6226.2003 ◽

2003 ◽

Vol 77 (11) ◽

pp. 6216-6226 ◽

Cited By ~ 43

Author(s):

Roeland Wieringa ◽

Antoine A. F. de Vries ◽

Peter J. M. Rottier

Keyword(s):

Noncovalent Interactions ◽

Rna Virus ◽

Noncovalent Interaction ◽

Envelope Proteins ◽

Viral Envelope ◽

Equine Arteritis Virus ◽

Viral Particles ◽

The Family ◽

Infected Cells ◽

Covalently Linked

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP5 (previously named GL), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP2b (formerly designated GS), GP3, and GP4. Analysis of the appearance of the GP2b, GP3, and GP4 proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP2b and GP4 and heterotrimers of GP2b, GP3, and GP4. Shortly after their release from infected cells, virions contained mainly cystine-linked GP2b/GP4 heterodimers, which were subsequently converted into disulfide-bonded GP2b/GP3/GP4 trimers through the covalent recruitment of GP3. This process occurred faster at a higher pH but was arrested at 4°C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP2b/GP3/GP4 trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP3 with the cystine-linked GP2b/GP4 dimer in freshly released virions, nor did we observe higher-order structures of the GP2b/GP4 or GP2b/GP3/GP4 complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP2b/GP3/GP4 heterotrimers at 4°C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP2b/GP4 dimer and GP3 was also inferred from coexpression experiments showing that the presence of GP3 increased the electrophoretic mobility of the disulfide-bonded GP2b/GP4 dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.

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Disulfide Bond Configuration of Human Cytomegalovirus Glycoprotein B

Journal of Virology ◽

10.1128/jvi.76.12.6073-6082.2002 ◽

2002 ◽

Vol 76 (12) ◽

pp. 6073-6082 ◽

Cited By ~ 23

Author(s):

Matthew Lopper ◽

Teresa Compton

Keyword(s):

Human Cytomegalovirus ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Cysteine Residue ◽

Multiple Roles ◽

Glycoprotein B ◽

Cysteine Residues ◽

Amino Terminal ◽

High Degree ◽

Human Herpesviruses

ABSTRACT Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically determining structures even for highly conserved proteins.

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A novel approach for predicting protein S-glutathionylation

BMC Bioinformatics ◽

10.1186/s12859-020-03571-w ◽

2020 ◽

Vol 21 (S11) ◽

Cited By ~ 1

Author(s):

Anastasia A. Anashkina ◽

Yuri M. Poluektov ◽

Vladimir A. Dmitriev ◽

Eugene N. Kuznetsov ◽

Vladimir A. Mitkevich ◽

...

Keyword(s):

Disulfide Bonds ◽

Prediction Method ◽

Protein S ◽

Redox Status ◽

Cysteine Residue ◽

Cysteine Residues ◽

Amino Acid Residues ◽

Novel Approach ◽

Positive Dataset ◽

Unknown Structure

Abstract Background S-glutathionylation is the formation of disulfide bonds between the tripeptide glutathione and cysteine residues of the protein, protecting them from irreversible oxidation and in some cases causing change in their functions. Regulatory glutathionylation of proteins is a controllable and reversible process associated with cell response to the changing redox status. Prediction of cysteine residues that undergo glutathionylation allows us to find new target proteins, which function can be altered in pathologies associated with impaired redox status. We set out to analyze this issue and create new tool for predicting S-glutathionylated cysteine residues. Results One hundred forty proteins with experimentally proven S-glutathionylated cysteine residues were found in the literature and the RedoxDB database. These proteins contain 1018 non-S-glutathionylated cysteines and 235 S-glutathionylated ones. Based on 235 S-glutathionylated cysteines, non-redundant positive dataset of 221 heptapeptide sequences of S-glutathionylated cysteines was made. Based on 221 heptapeptide sequences, a position-specific matrix was created by analyzing the protein sequence near the cysteine residue (three amino acid residues before and three after the cysteine). We propose the method for calculating the glutathionylation propensity score, which utilizes the position-specific matrix and a criterion for predicting glutathionylated peptides. Conclusion Non-S-glutathionylated sites were enriched by cysteines in − 3 and + 3 positions. The proposed prediction method demonstrates 76.6% of correct predictions of S-glutathionylated cysteines. This method can be used for detecting new glutathionylation sites, especially in proteins with an unknown structure.

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The Role of RodA-Conserved Cysteine Residues in the Aspergillus fumigatus Conidial Surface Organization

Journal of Fungi ◽

10.3390/jof6030151 ◽

2020 ◽

Vol 6 (3) ◽

pp. 151

Author(s):

Isabel Valsecchi ◽

Emmanuel Stephen-Victor ◽

Sarah Sze Wah Wong ◽

Anupama Karnam ◽

Margaret Sunde ◽

...

Keyword(s):

Parental Strain ◽

Disulfide Bonds ◽

Deletion Mutant ◽

Cysteine Residue ◽

Cysteine Residues ◽

Fungal Cell ◽

Differential Reactivity ◽

Alternative Approach ◽

Conidial Surface

Immune inertness of Aspergillusfumigatus conidia is attributed to its surface rodlet-layer made up of RodAp, characterized by eight conserved cysteine residues forming four disulfide bonds. Earlier, we showed that the conserved cysteine residue point (ccrp) mutations result in conidia devoid of the rodlet layer. Here, we extended our study comparing the surface organization and immunoreactivity of conidia carrying ccrp-mutations with the RODA deletion mutant (∆rodA). Western blot analysis using anti-RodAp antibodies indicated the absence of RodAp in the cytoplasm of ccrp-mutant conidia. Immunolabeling revealed differential reactivity to conidial surface glucans, the ccrp-mutant conidia preferentially binding to α-(1,3)-glucan, ∆rodA conidia selectively bound to β-(1,3)-glucan; the parental strain conidia showed negative labeling. However, permeability of ccrp-mutants and ∆rodA was similar to the parental strain conidia. Proteomic analyses of the conidial surface exposed proteins of the ccrp-mutants showed more similarities with the parental strain, but were significantly different from the ∆rodA. Ccrp-mutant conidia were less immunostimulatory compared to ∆rodA conidia. Our data suggest that (i) the conserved cysteine residues are essential for the trafficking of RodAp and the organization of the rodlet layer on the conidial surface, and (ii) targeted point mutation could be an alternative approach to study the role of fungal cell-wall genes in host–fungal interaction.

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Characterization of Two New Structural Glycoproteins, GP3 and GP4, of Equine Arteritis Virus

Journal of Virology ◽

10.1128/jvi.76.21.10829-10840.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 10829-10840 ◽

Cited By ~ 34

Author(s):

Roeland Wieringa ◽

Antoine A. F. de Vries ◽

Martin J. B. Raamsman ◽

Peter J. M. Rottier

Keyword(s):

Membrane Protein ◽

Rna Virus ◽

Expression System ◽

Integral Membrane Protein ◽

Envelope Proteins ◽

Equine Arteritis Virus ◽

In Vitro Translation ◽

Infected Cells ◽

Virus Expression

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Four envelope proteins have hitherto been identified in EAV particles: the predominant membrane proteins M and GL, the unglycosylated small envelope protein E, and the nonabundant membrane glycoprotein GS. In this study, we established that the products of EAV open reading frame 3 (ORF3) and ORF4 (designated GP3 and GP4, respectively) are also minor structural glycoproteins. The proteins were first characterized by various analyses after in vitro translation of RNA transcripts in a rabbit reticulocyte lysate in the presence and absence of microsomal membranes. We subsequently expressed ORF3 and -4 in baby hamster kidney cells by using the vaccinia virus expression system and, finally, analyzed the GP3 and GP4 proteins synthesized in EAV-infected cells. The results showed that GP4 is a class I integral membrane protein of 28 kDa with three functional N-glycosylation sites and with little, if any, of its carboxy terminus exposed. Both after independent expression and in EAV-infected cells, the protein localizes in the endoplasmic reticulum (ER), as demonstrated biochemically by analysis of its oligosaccharide side chains and as visualized directly by immunofluorescence studies. GP3, on the other hand, is a heavily glycosylated protein whose hydrophobic amino terminus is not cleaved off. It is an integral membrane protein anchored by either or both of its hydrophobic terminal domains and with no parts detectably exposed cytoplasmically. Also, GP3 localizes in the ER when expressed independently and in the context of an EAV infection. Only a small fraction of the GP3 and GP4 proteins synthesized in infected cells ends up in virions. Most, but not all, of the oligosaccharides of these virion glycoproteins are biochemically mature. Our results bring the number of EAV envelope proteins to six.

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