scholarly journals Expression and Self-Assembly of Grimsby Virus: Antigenic Distinction from Norwalk and Mexico Viruses

1999 ◽  
Vol 6 (1) ◽  
pp. 142-145 ◽  
Author(s):  
Antony D. Hale ◽  
Sue E. Crawford ◽  
Max Ciarlet ◽  
Jonathan Green ◽  
Christopher Gallimore ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Self Assembly ◽  
Recombinant Baculovirus ◽  
Apparent Molecular Weight ◽  
Antigenic Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Norwalk Virus ◽  
Reading Frame ◽  
The United Kingdom ◽  
Hyperimmune Antiserum

ABSTRACT A cDNA obtained from Grimsby virus (GRV), a Norwalk-like virus, purified from a stool sample of a symptomatic adult associated with a gastroenteritis outbreak in the United Kingdom, was used to obtain the complete nucleotide sequence of the second open reading frame (ORF2). The ORF2 sequence of GRV predicts a capsid of 539 amino acids (aa) which exhibits aa identities of 96% to Lordsdale virus, 67% to Mexico virus (MXV), and 43% to Norwalk virus (NV). The GRV capsid protein was expressed in insects cells by using a recombinant baculovirus, and the resulting virus-like particles (VLPs) possessed a protein with an apparent molecular weight of 58,000. Hyperimmune antisera raised against purified GRV, MXV, and NV VLPs were tested in an indirect enzyme-linked immunosorbent assay (ELISA) against GRV, NV, and MXV VLPs, revealing that GRV is antigenically distinct from both NV and MXV. The antigenic specificity of the GRV-hyperimmune antiserum was confirmed in an antigen capture ELISA using GRV-, NV-, or MXV-containing fecal specimens. The expression of the GRV capsid protein has, for the first time, allowed the antigenic comparison of three distinct recombinant Norwalk-like viruses.

scholarly journals Essential Elements of the Capsid Protein for Self-Assembly into Empty Virus-Like Particles of Hepatitis E Virus

2005 ◽  
Vol 79 (20) ◽  
pp. 12999-13006 ◽  
Author(s):  
Tian-Cheng Li ◽  
Naokazu Takeda ◽  
Tatsuo Miyamura ◽  
Yoshiharu Matsuura ◽  
Joseph C. Y. Wang ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Self Assembly ◽  
Hepatitis E Virus ◽  
Recombinant Baculovirus ◽  
Hepatitis E ◽  
Essential Elements ◽  
Mucosal Vaccine ◽  
Sf9 Cells ◽  
Reading Frame ◽  
Virus Like Particles

ABSTRACT Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.

scholarly journals Expression and Self-Assembly of Norwalk Virus Capsid Protein from Venezuelan Equine Encephalitis Virus Replicons

2002 ◽  
Vol 76 (6) ◽  
pp. 3023-3030 ◽  
Author(s):  
Ralph S. Baric ◽  
Boyd Yount ◽  
Lisa Lindesmith ◽  
Patrick R. Harrington ◽  
Shermalyn R. Greene ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Norwalk Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Large Numbers ◽  
Virus Capsid Protein

ABSTRACT The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.

scholarly journals Two Nonoverlapping Domains on the Norwalk Virus Open Reading Frame 3 (ORF3) Protein Are Involved in the Formation of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions

2003 ◽  
Vol 77 (6) ◽  
pp. 3569-3577 ◽  
Author(s):  
Pamela J. Glass ◽  
Carl Q. Zeng ◽  
Mary K. Estes
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Capsid Protein ◽  
Full Length ◽  
Open Reading Frame ◽  
Norwalk Virus ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Orf3 Protein ◽  
Orf2 Protein

ABSTRACT Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF31-179, ORF31-152, and ORF31-107) no longer produced the 35K form. An N-terminal truncation mutant (ORF351-212) and a site-directed mutant (ORF3T201V) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3FL), ORF31-179, ORF31-152, and ORF351-212 interacted with the ORF2 protein, while an ORF31-107 protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.

scholarly journals Identification of Genogroup I and Genogroup II Broadly Reactive Epitopes on the Norovirus Capsid

2005 ◽  
Vol 79 (12) ◽  
pp. 7402-7409 ◽  
Author(s):  
Tracy Dewese Parker ◽  
Noritoshi Kitamoto ◽  
Tomoyuki Tanaka ◽  
Anne M. Hutson ◽  
Mary K. Estes
Keyword(s):  
Monoclonal Antibody ◽  
Immune Response ◽  
Monoclonal Antibodies ◽  
Capsid Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Linear Epitope ◽  
Norwalk Virus ◽  
Diagnostic Assays ◽  
Separate Genus ◽  
The Family

ABSTRACT Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.

scholarly journals Presence of a Surface-Exposed Loop Facilitates Trypsinization of Particles of Sinsiro Virus, a Genogroup II.3 Norovirus

2006 ◽  
Vol 81 (3) ◽  
pp. 1119-1128 ◽  
Author(s):  
Shantanu Kumar ◽  
Wendy Ochoa ◽  
Shinichi Kobayashi ◽  
Vijay S. Reddy
Keyword(s):  
Capsid Protein ◽  
Receptor Binding ◽  
Hot Spot ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Recombinant Baculovirus ◽  
Immunological Response ◽  
Dimensional Structure ◽  
Norwalk Virus ◽  
Capsid Protein Gene

ABSTRACT Noroviruses (NoVs) are the causative agents of nonbacterial acute gastroenteritis in humans. NoVs that belong to genogroup II (GII) are quite prevalent and prone to undergo recombination, and their three-dimensional structure is not yet known. Protein homology modeling of Sinsiro virus (SV), a member of the GII.3 NoVs, revealed the presence of a surface-exposed 20-amino-acid (aa) insertion in the P2 domain of the capsid protein (CP) relative to the Norwalk virus (NV) CP, which is a well known hot spot for mutations to counter the host immunological response. To further characterize the role of the long insertion in SV, the capsid protein gene was expressed using the recombinant baculovirus system. Trypsinization of the resultant virus-like particles yielded two predominant bands (31.7 and 26.1 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. N-terminal sequencing and analysis of the mass spectroscopic data indicated that these fragments correspond to residues 1 to 292 (26.1 kDa) and 307 to 544 (31.7 kDa). In addition, the above data taken together with the comparative modeling studies indicated that the trypsin cleavage sites of the Sinsiro virus CP, Arg292 and Arg307, are located at the beginning of and within the 20-aa insertion in the P2 domain, respectively. This study demonstrates that the presence of the surface-exposed loop in the GII.3 NoVs facilitates the trypsinization of the capsid protein in the assembled form. The SV particles remain intact even after trypsin digestion and retain the suggested receptor binding linear epitope of residues 325 to 334. The above results are distinct from those obtained from the trypsinization studies performed earlier on the NV (GI) and VA387 (GII) viruses, both of which lack the large surface insertion and associated basic residues. These new observations may have implications for host receptor binding, cell entry, and norovirus infection in general.

scholarly journals Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein.

1992 ◽  
Vol 66 (11) ◽  
pp. 6527-6532 ◽  
Author(s):  
X Jiang ◽  
M Wang ◽  
D Y Graham ◽  
M K Estes
Keyword(s):  
Capsid Protein ◽  
Self Assembly ◽  
Norwalk Virus ◽  
Virus Capsid ◽  
Virus Capsid Protein

scholarly journals Heterologous RNA Encapsidated in Pariacoto Virus-Like Particles Forms a Dodecahedral Cage Similar to Genomic RNA in Wild-Type Virions

2004 ◽  
Vol 78 (20) ◽  
pp. 11371-11378 ◽  
Author(s):  
Karyn N. Johnson ◽  
Liang Tang ◽  
John E. Johnson ◽  
L. Andrew Ball
Keyword(s):  
Capsid Protein ◽  
Rna Structure ◽  
Recombinant Baculovirus ◽  
Wild Type ◽  
Reading Frame ◽  
Virus Like Particles ◽  
Virus Particles ◽  
X Ray Crystallography ◽  
Specific Nucleotide Sequence ◽  
Specific Determinant

ABSTRACT The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-Å resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.

scholarly journals The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein

2003 ◽  
Vol 77 (21) ◽  
pp. 11603-11615 ◽  
Author(s):  
Andrea Bertolotti-Ciarlet ◽  
Sue E. Crawford ◽  
Anne M. Hutson ◽  
Mary K. Estes
Keyword(s):  
Capsid Protein ◽  
Mammalian Cells ◽  
Recombinant Baculovirus ◽  
Prototype Strain ◽  
Open Reading Frames ◽  
Norwalk Virus ◽  
Subgenomic Rna ◽  
Expression Levels ◽  
Capsid Protein Vp1 ◽  
Protease Degradation

ABSTRACT Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3′ untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3′ terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3′ elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3′UTR (ORF2+3+3′UTR). In contrast, expression of VP1 from constructs that lacked the 3′UTR (ORF2+3), ORF3 (ORF2+3′UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3′UTR revealed that the 3′UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.

scholarly journals DESIGN OF HEPATITIS E VIRUS GENOTYPE 1 RECOMBINANT CAPSID PROTEIN: CLONING, EXPRESSION, PURIFICATION, EVALUATION OF THE ANTIGENIC PROPERTIES

2017 ◽  
pp. 72-80 ◽  
Author(s):  
G. I. Alatortseva ◽  
A. V. Sidorov ◽  
L. N. Nesterenko ◽  
L. N. Luhverchik ◽  
V. V. Dotsenko ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Size Exclusion ◽  
Antigenic Specificity ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Genotype 1 ◽  
Recombinant Polypeptide ◽  
Antigenic Properties

Aim. The development of the hepatitis E virus (HEV) genotype 1 recombinant capsid protein. Materials and methods. Escherichia coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods. Results. Using HEV genotype 1 DNA copy of subgenomic virus RNA we made E.coli strains producing recombinabt capsid protein, containing C-terminal fragment of ORF2 protein fused to E.coli beta-galactosidase. Recombinant protein ORF2 had been isolated from the inclusion bodies of the E.coli biomass and purified by size exclusion chromatography. By Western blotting it had been shown specific interaction of the recombinant polypeptide with anti-HEV IgG from pool of positive sera. Antigenic specificity of the recombinant polypeptide had been confirmed by enzyme-linked immunosorbent assay with sera of hepatitis E patients and reference groups: healthy donors, patients with hepatitis А, В, C, infectious mononucleosis and cytomegalovirus infection, HIV-infected patients. Conclusion. HEV genotype 1 ORF2 recombinant antigen had been developed, and its possible use in diagnostic tests had been experimentally shown.

scholarly journals Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System

2021 ◽  
Vol 22 (6) ◽  
pp. 3098
Author(s):  
Aleksander Strugała ◽  
Jakub Jagielski ◽  
Karol Kamel ◽  
Grzegorz Nowaczyk ◽  
Marcin Radom ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Mosaic Virus ◽  
Capsid Protein ◽  
Self Assembly ◽  
Environmental Changes ◽  
Expression System ◽  
Brome Mosaic Virus ◽  
Virus Like Particles ◽  
Assembly Principles ◽  
The Impact

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Sign in / Sign up

Export Citation Format

Share Document