scholarly journals Structural Protein Requirements in Equine Arteritis Virus Assembly

2004 ◽  
Vol 78 (23) ◽  
pp. 13019-13027 ◽  
Author(s):  
Roeland Wieringa ◽  
Antoine A. F. de Vries ◽  
Jannes van der Meulen ◽  
Gert-Jan Godeke ◽  
Jos J. M. Onderwater ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Rna Virus ◽  
Buoyant Density ◽  
Protein Composition ◽  
Equine Arteritis Virus ◽  
Structural Proteins ◽  
Structural Protein ◽  
Electron Microscopic ◽  
Microscopic Appearance ◽  
Virus Particles

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP2b (previously named GS), GP3, GP4, and GP5 (previously named GL). Proteins N, M, and GP5 are major virion components, E occurs in virus particles in intermediate amounts, and GP4, GP3, and GP2b are minor structural proteins. The M and GP5 proteins occur in virus particles as disulfide-linked heterodimers while the GP4, GP3, and GP2b proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP2b, GP3, or GP4 proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP2b, GP3, and GP4 proteins into viral particles. EAV particles lacking GP2b, GP3, GP4, and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP2b/GP3/GP4 heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.

scholarly journals Microvilli of the human term placenta Isolation and subfractionation by centrifugation in sucrose density gradients

1981 ◽  
Vol 196 (1) ◽  
pp. 121-132 ◽  
Author(s):  
P Truman ◽  
J S Wakefield ◽  
H C Ford
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Density Gradients ◽  
Electron Microscopic ◽  
Microscopic Appearance ◽  
Human Term Placenta ◽  
Morphology And Morphometry ◽  
Microscopic Morphology

Human placental microvilli were isolated and separated into two fractions by centrifugation in sucrose density gradients. Electron-microscopic morphology and morphometry, the distribution of enzymic activities and the results of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of proteins were used to assess the purity of the final preparations and to define their properties. The combined evidence strongly suggested that the preparations contained negligible material that was not plasma membrane. The two fractions of microvilli differed in buoyant density, protein composition, enzyme specific activities and microscopic appearance. Some of these differences were explained by the absence of internal structure in the microvilli of the lighter fraction.

Molecular biology of rubella virus structural proteins

1994 ◽  
Vol 72 (9-10) ◽  
pp. 349-356 ◽  
Author(s):  
Shirley Gillam
Keyword(s):  
Molecular Biology ◽  
Rubella Virus ◽  
Cell Biology ◽  
Intracellular Transport ◽  
Rna Virus ◽  
Viral Gene Expression ◽  
Proteolytic Processing ◽  
Structural Proteins ◽  
Viral Gene ◽  
Structural Protein

Rubella virus is a small, enveloped, positive-stranded RNA virus in the Togaviridae family and bears similarities to the prototype alphaviruses in terms of its genome organization and strategy for viral gene expression. Despite being an important human pathogen, the cell biology of rubella virus remains poorly characterized. This review focuses on the molecular biology of rubella virus structural proteins, with emphasis on the proteolytic processing and maturation of virus structural proteins, the glycosylation requirement for intracellular transport and function of glycoproteins, and the localization of hemagglutinin- and virus-neutralizing epitopes. A number of significant differences between rubella virus and alphavirus structural protein expression and maturation were discovered.Key words: rubella virus, N-linked glycosylation, epitope mapping, proteolytic processing.

scholarly journals Serologic Response of Horses to the Structural Proteins of Equine Arteritis Virus

1998 ◽  
Vol 10 (3) ◽  
pp. 229-236 ◽  
Author(s):  
N. James MacLachlan ◽  
Udeni B. R. Balasuriya ◽  
Jodi F. Hedges ◽  
Therese M. Schweidler ◽  
William H. McCollum ◽  
...  
Keyword(s):  
Humoral Response ◽  
Equine Arteritis Virus ◽  
Structural Proteins ◽  
Structural Protein ◽  
Serum Samples ◽  
Equine Viral Arteritis ◽  
Serologic Response ◽  
Immunoblotting Assay ◽  
Duration Of Infection ◽  
Standard Serum

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, an apparently emerging disease of equids. In this study, the antibody response of horses to the structural proteins of EAV was evaluated using gradient-purified EAV virions and baculovirus-expressed recombinant EAV structural proteins (GL, GS, M, N) as antigens in a Western immunoblotting assay. Thirty-three sera from horses that previously had been naturally or experimentally infected with EAV were evaluated, including samples from mares, geldings, and both persistently and nonpersistently infected stallions. Sera also were evaluated from 4 horses that had been vaccinated with the commercial modified live EAV vaccine. The data suggest that the serologic response of individual horses to EAV may vary with the infecting virus strain and duration of infection. The M protein was most consistently recognized by the various serum samples, whereas the response to the N and GL proteins was variable and the GS protein was bound by only 1 serum sample. The immunoblotting assay definitively established the protein specificity of the humoral response of horses to EAV; however, it clearly is less sensitive than the standard serum neutralization (SN) test—2 of the 37 sera that were serpositive by th SN test failed to react in the immunoblot assay with any EAV structural protein. Furthermore, the GL protein expresses the known neutralization determinants of EAV, yet only 22 of the 37 sera that had SN antibodies bound the GL protein in the immunoblotting assay. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection of horses.

scholarly journals Genetic Manipulation of Arterivirus Alternative mRNA Leader-Body Junction Sites Reveals Tight Regulation of Structural Protein Expression

2000 ◽  
Vol 74 (24) ◽  
pp. 11642-11653 ◽  
Author(s):  
Alexander O. Pasternak ◽  
Alexander P. Gultyaev ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Structure ◽  
The Body ◽  
Equine Arteritis Virus ◽  
Site Directed Mutagenesis ◽  
Structural Proteins ◽  
Limiting Factors ◽  
Progeny Virus ◽  
Structural Protein ◽  
Sequence Element ◽  
Conserved Sequence

ABSTRACT To express its structural proteins, the arterivirus Equine arteritis virus (EAV) produces a nested set of six subgenomic (sg) RNA species. These RNA molecules are generated by a mechanism of discontinuous transcription, during which a common leader sequence, representing the 5′ end of the genomic RNA, is attached to the bodies of the sg RNAs. The connection between the leader and body parts of an mRNA is formed by a short, conserved sequence element termed the transcription-regulating sequence (TRS), which is present at the 3′ end of the leader as well as upstream of each of the structural protein genes. With the exception of RNA3, only one body TRS was previously assumed to be used to join the leader and body of each EAV sg RNA. Here we show that for the synthesis of two other sg RNAs, RNA4 and RNA5, alternative leader-body junction sites that differ substantially in transcriptional activity are used. By site-directed mutagenesis of an EAV infectious cDNA clone, the alternative TRSs used to generate RNA3, -4, and -5 were inactivated, which strongly influenced the corresponding RNA levels and the production of infectious progeny virus. The relative amounts of RNA produced from alternative TRSs differed significantly and corresponded to the relative infectivities of the virus mutants. This strongly suggested that the structural proteins that are expressed from these RNAs are limiting factors during the viral life cycle and that the discontinuous step in sg RNA synthesis is crucial for the regulation of their expression. On the basis of a theoretical analysis of the predicted RNA structure of the 3′ end of the EAV genome, we propose that the local secondary RNA structure of the body TRS regions is an important factor in the regulation of the discontinuous step in EAV sg mRNA synthesis.

scholarly journals Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids

2016 ◽  
Vol 90 (16) ◽  
pp. 7196-7204 ◽  
Author(s):  
Fikret Aydemir ◽  
Maxim Salganik ◽  
Justyna Resztak ◽  
Jasbir Singh ◽  
Antonette Bennett ◽  
...  
Keyword(s):  
Viral Genome ◽  
Structural Proteins ◽  
Structural Protein ◽  
Wild Type ◽  
Mrna Accumulation ◽  
Functional Region ◽  
Adeno Associated Virus ◽  
Virus Particles ◽  
Capsid Maturation

ABSTRACTWe previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071–1079, 2013, doi:http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus.IMPORTANCEMany viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to be the first example of a nonenveloped viral capsid that appears to have a role in promoting transcription. A total of six mutants at the AAV capsid 2-fold interface were shown to have a severe defect in expressing their genomes, and the defect was at the level of mRNA accumulation. This suggests that AAV capsids have a novel role in promoting the transcription of the genomes that they have packaged. Since wt virions could not complement the mutant viruses, and the mutant viruses did not effectively inhibit wt gene expression, our results suggest that the capsid exerts its effect on transcription incis.

scholarly journals Autonomously Replicating RNAs of Bungowannah Pestivirus: ERNS Is Not Essential for the Generation of Infectious Particles

2020 ◽  
Vol 94 (14) ◽  
Author(s):  
Anja Dalmann ◽  
Ilona Reimann ◽  
Kerstin Wernike ◽  
Martin Beer
Keyword(s):  
Virus Assembly ◽  
Host Cells ◽  
Structural Proteins ◽  
Progeny Virus ◽  
Human Populations ◽  
Structural Protein ◽  
Vaccine Platform ◽  
Susceptible Host ◽  
Infectious Progeny Virus

ABSTRACT Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These “defective-in-third-cycle” BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors. IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.

scholarly journals Generation of Filamentous Instead of Icosahedral Particles by Repression of African Swine Fever Virus Structural Protein pB438L

2006 ◽  
Vol 80 (23) ◽  
pp. 11456-11466 ◽  
Author(s):  
Carolina Epifano ◽  
Jacomine Krijnse-Locker ◽  
María L. Salas ◽  
José Salas ◽  
Javier M. Rodríguez
Keyword(s):  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Integral Membrane Protein ◽  
Fever Virus ◽  
Icosahedral Symmetry ◽  
Structural Protein ◽  
Viral Origin ◽  
Virus Particles ◽  
Capsid Formation

ABSTRACT The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.

scholarly journals Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus

2002 ◽  
Vol 83 (4) ◽  
pp. 795-800 ◽  
Author(s):  
Marieke A. Tijms ◽  
Yvonne van der Meer ◽  
Eric J. Snijder
Keyword(s):  
Nuclear Localization ◽  
Nuclear Export ◽  
Virus Assembly ◽  
Equine Arteritis Virus ◽  
Genome Replication ◽  
Structural Protein ◽  
Leptomycin B ◽  
Nuclear Retention ◽  
N Protein ◽  
Cytoplasmic Membranes

RNA synthesis (genome replication and subgenomic mRNA transcription) directed by equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) occurs on modified cytoplasmic membranes to which most viral replicase subunits localize. Remarkably, a fraction of non-structural protein 1 (nsp1), a protein essential for transcription but dispensable for genome replication, is present in the host cell nucleus, in particular during the earlier stages of infection. Expression of GFP-tagged fusion proteins revealed that nsp1 is actively imported into the nucleus. Although the signals responsible for nsp1 transport could not be identified, our studies revealed that another EAV protein with a partially nuclear localization, the nucleocapsid (N) protein, utilizes the CRM1-mediated nuclear export pathway. Inactivation of this pathway with the drug leptomycin B resulted in the unexpected and immediate nuclear retention of all N protein molecules, thus revealing that the protein shuttles between cytoplasm and nucleus before playing its role in cytoplasmic virus assembly.

scholarly journals Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

2009 ◽  
Vol 83 (22) ◽  
pp. 11477-11490 ◽  
Author(s):  
Desyreé Murta Jesus ◽  
Lilian T. Costa ◽  
Daniela L. Gonçalves ◽  
Carlos Alberto Achete ◽  
Marcia Attias ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Detailed Analysis ◽  
Virus Assembly ◽  
Microscopic Analysis ◽  
Progeny Virus ◽  
Late Gene Expression ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Core Proteins ◽  
Genome Encapsidation

ABSTRACT Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

scholarly journals Evolutionary Insights into the Envelope Protein of SARS-CoV-2

2020 ◽  
Author(s):  
M. Shaminur Rahman ◽  
M. Nazmul Hoque ◽  
M. Rafiul Islam ◽  
Israt Islam ◽  
Israt Dilruba Mishu ◽  
...  
Keyword(s):  
Continuous Monitoring ◽  
Transmembrane Domain ◽  
Virus Assembly ◽  
Structural Proteins ◽  
Structural Protein ◽  
Cysteine Motif ◽  
E Protein ◽  
Key Factor ◽  
And Control ◽  
Major Impediment

The ongoing mutations in the structural proteins of SARS-CoV-2 is the major impediment for prevention and control of the COVID-19 disease. The envelope (E) protein of SARS-CoV-2 is a structural protein existing in both monomeric and homopentameric forms, associated with a multitude of functions including virus assembly, replication, dissemination, release of virions, infection, pathogenesis, and immune response stimulation. In the present study, 81,818 high quality E protein sequences retrieving from the GISAID were subjected to mutational analyses. Our analysis revealed that only 0.012 % (982/81818) stains possessed amino acid (aa) substitutions in 63 sites of the genome while 58.77% mutations in the primary structure of nucleotides in 134 sites. We found the V25A mutation in the transmembrane domain which is a key factor for the homopentameric conformation of E protein. We also observed a triple cysteine motif harboring mutations (L39M, A41S, A41V, C43F, C43R, C43S, C44Y, N45R) which may hinder the binding of E protein with spike glycoprotein. These results therefore suggest the continuous monitoring of each structural protein of SARS-CoV-2 since the number of genome sequences from across the world are continuously increasing.

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