scholarly journals Glycosylation of the Severe Acute Respiratory Syndrome Coronavirus Triple-Spanning Membrane Proteins 3a and M

2006 ◽  
Vol 80 (5) ◽  
pp. 2326-2336 ◽  
Author(s):  
M. Oostra ◽  
C. A. M. de Haan ◽  
R. J. de Groot ◽  
P. J. M. Rottier
Keyword(s):  
Membrane Proteins ◽  
Severe Acute Respiratory Syndrome ◽  
Expression System ◽  
M Protein ◽  
Metabolic Labeling ◽  
Structural Protein ◽  
Reading Frame ◽  
M Proteins ◽  
Group 2 ◽  
Membrane Spanning

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.

scholarly journals The Product of the Respiratory Syncytial Virus M2 Gene ORF1 Enhances Readthrough of Intergenic Junctions during Viral Transcription

1998 ◽  
Vol 72 (1) ◽  
pp. 520-526 ◽  
Author(s):  
Richard W. Hardy ◽  
Gail W. Wertz
Keyword(s):  
Expression System ◽  
Rna Replication ◽  
Transcription Unit ◽  
Viral Rna ◽  
Metabolic Labeling ◽  
Structural Protein ◽  
M2 Protein ◽  
Positive Sense ◽  
Syncytial Virus ◽  
Polyadenylated Mrna

ABSTRACT The mRNA encoding the M2 protein of respiratory syncytial (RS) virus contains two open reading frames (ORFs). ORF1 encodes the 22-kDa structural protein, M2, and ORF2 has the potential to encode a 10-kDa protein (90 amino acids). Using a vaccinia virus T7 expression system, we examined the RNA synthetic activities of mono- and dicistronic subgenomic replicons of RS virus by direct metabolic labeling of RNA in the presence and absence of the products of ORF1 and ORF2. In the absence of ORF1 and ORF2, the negative- and positive-sense products of genomic RNA replication and positive-sense polyadenylated mRNA(s) were synthesized. Expression of the whole M2 transcription unit (containing ORF1 and ORF2) or ORF1 alone caused an increase in the synthesis of polyadenylated mRNA, the majority of which was due to a substantial increase in the quantity of polycistronic mRNAs generated by the polymerase failing to terminate at gene end signals. In agreement with previous reports, the ORF2 product was found to inhibit viral RNA replication and mRNA transcription. These data show that the M2 protein functions as a transcriptional antiterminator that enhances the ability of the viral RNA polymerase to read through intergenic junctions. The role of such a function during the viral life cycle is discussed.

scholarly journals OPTIMIZATION OF EXPRESSION CONDITIONS OF GENE ENCODING ANTIGEN M OF PRRSV IN LEAVES OF NICOTIANA BENTHAMIANA BY AGROINFILTRATION METHOD

2018 ◽  
Vol 16 (2) ◽  
pp. 293-300
Author(s):  
Nguyen Thi Minh Hang ◽  
Ho Thi Thuong ◽  
Nguyen Thu Giang ◽  
Pham Bich Ngoc ◽  
Nguyen Trung Nam ◽  
...  
Keyword(s):  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
M Protein ◽  
Physiological Age ◽  
Structural Protein ◽  
Gene Encoding ◽  
Tobacco Plants ◽  
Reading Frame ◽  
Prrs Virus ◽  
A Subunit

Reproductive disorders and respiratory swine (Porcine Reproductive and Respiratory Syndrome - PRRS) is an infectious disease caused by the PRRS virus (PRRSV), spread fast speeds, causing mass death when infected pigs. M protein is one of the major structural protein of the PRRSV, has a molecular weight of about 19 kDa, encoded by the open reading frame 6 (ORF6), which is used to design a subunit vaccine against back PRRSV. In this study, conditions for transient expression of the gene encoded the M protein of PRRSV in leaves of the Nicotiana benthamiana by agroinfiltration method were determined. The results show that optimal conditions for transient expression of protein M in the leaf are used the simultaneous of the A. tumefaciens strain containing vector carrying the gene encoding the protein M and A. tumefaciens containing vector carrying gene encoding supported protein HC-Pro PVY, concentration of acetosyringone with 450 µM, the bacterial cell density used to infect into leaf with OD600 is 0.5, The physiological age of the leaf suitable for infecting bacteria was young leaf and mature leaf of tobacco plants 4 to 6 weeks of age, and the transient expression time of gene encoding protein M of PRRSV in tobacco leaves is most effective 6 days after infecting. This agroinfiltration method can be used to express large amounts of protein - antigen M of PRRSV in tobacco plants to produce vaccines against this virus.

scholarly journals Genetic Evidence for a Structural Interaction between the Carboxy Termini of the Membrane and Nucleocapsid Proteins of Mouse Hepatitis Virus

2002 ◽  
Vol 76 (10) ◽  
pp. 4987-4999 ◽  
Author(s):  
Lili Kuo ◽  
Paul S. Masters
Keyword(s):  
Hepatitis Virus ◽  
Expression System ◽  
Mouse Hepatitis Virus ◽  
Viral Envelope ◽  
Genetic Evidence ◽  
M Protein ◽  
Structural Protein ◽  
Virion Protein ◽  
Structural Interaction ◽  
Intact Virion

ABSTRACT The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, MΔ2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the MΔ2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.

scholarly journals Multiple, heart-cross-reactive epitopes of streptococcal M proteins.

1985 ◽  
Vol 161 (1) ◽  
pp. 113-122 ◽  
Author(s):  
J B Dale ◽  
E H Beachey
Keyword(s):  
Membrane Proteins ◽  
Heart Tissue ◽  
Human Myocardium ◽  
M Protein ◽  
Antigenic Determinants ◽  
Serotype Distribution ◽  
Present Evidence ◽  
Sarcolemmal Membrane ◽  
Streptococcal M Protein ◽  
M Proteins

We present evidence that a highly purified pepsin extract of type 5 streptococcal M protein (pep M5) contains at least three epitopes that are cross-reactive with sarcolemmal membrane proteins of human myocardium. The tissue-cross-reactive determinants of pep M5 are also partially shared with pep M6 and pep M19. Three rabbits immunized with a single 300 micrograms dose of pep M5 developed significant levels of heart-cross-reactive antibodies, as determined by indirect immunofluorescence tests. All three sera also contained antibodies that cross-reacted with pep M6 and pep M19. The heart tissue--specific antibodies that were eluted from sarcolemmal membranes opsonized types 5, 6, and 19 streptococci, indicating that they were directed against protective M protein epitopes on the surface of virulent organisms. Immunofluorescence inhibition tests, using purified M proteins as soluble inhibitors of heart-cross-reactive antibodies, revealed the number and M protein serotype distribution of the tissue-cross-reactive epitopes. Immunoblot analyses demonstrated the sarcolemmal membrane proteins containing the various cross-reactive antigenic determinants.

scholarly journals Assembly of the Coronavirus Envelope: Homotypic Interactions between the M Proteins

2000 ◽  
Vol 74 (11) ◽  
pp. 4967-4978 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Harry Vennema ◽  
Peter J. M. Rottier
Keyword(s):  
Membrane Proteins ◽  
Equine Arteritis Virus ◽  
M Protein ◽  
M Proteins ◽  
Foreign Proteins ◽  
Sorting Process ◽  
Vesicular Stomatitis ◽  
Viruslike Particles ◽  
Carboxy Terminal ◽  
Envelope Incorporation

ABSTRACT The viral membrane proteins M and E are the minimal requirements for the budding of coronavirus particles. Since the E protein occurs in particles only in trace amounts, the lateral interactions between the M proteins apparently generate the major driving force for envelope formation. By using coimmunoprecipitation and envelope incorporation assays, we provide extensive evidence for the existence of such M-M interactions. In addition, we determined which domains of the M protein are involved in this homotypic association, using a mutagenetic approach. Mutant M proteins which were not able to assemble into viruslike particles (VLPs) by themselves (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838–6850, 1998) were tested for the ability to associate with other M proteins and to be rescued into VLPs formed by assembly-competent M proteins. We found that M proteins lacking parts of the transmembrane cluster, of the amphipathic domain, or of the hydrophilic carboxy-terminal tail, or M proteins that had their luminal domain replaced by heterologous ectodomains, were still able to associate with assembly-competent M proteins, resulting in their coincorporation into VLPs. Only a mutant M protein in which all three transmembrane domains had been replaced lost this ability. The results indicate that M protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. Finally, we tested the stringency with which membrane proteins are selected for incorporation into the coronavirus envelope by probing the coassembly of some foreign proteins. The observed efficient exclusion from budding of the vesicular stomatitis virus G protein and the equine arteritis virus M protein indicates that envelope assembly is indeed a highly selective sorting process. The low but detectable incorporation of CD8 molecules, however, demonstrated that this process is not perfect.

scholarly journals The 29-Nucleotide Deletion Present in Human but Not in Animal Severe Acute Respiratory Syndrome Coronaviruses Disrupts the Functional Expression of Open Reading Frame 8

2007 ◽  
Vol 81 (24) ◽  
pp. 13876-13888 ◽  
Author(s):  
Monique Oostra ◽  
Cornelis A. M. de Haan ◽  
Peter J. M. Rottier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Signal Sequence ◽  
Expression System ◽  
Functional Expression ◽  
Open Reading Frame ◽  
T7 Promoter ◽  
Reading Frame ◽  
Genomic Changes ◽  
Nucleotide Deletion ◽  
Infected Cells

ABSTRACT One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab+), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.

scholarly journals Role of Small Envelope Protein in Sustaining the Intracellular and Extracellular Levels of Hepatitis B Virus Large and Middle Envelope Proteins

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 613
Author(s):  
Jing Zhang ◽  
Yongxiang Wang ◽  
Shuwen Fu ◽  
Quan Yuan ◽  
Qianru Wang ◽  
...  
Keyword(s):  
Envelope Proteins ◽  
Full Length ◽  
M Protein ◽  
Intracellular Level ◽  
S Protein ◽  
L Protein ◽  
M Proteins ◽  
B Virus ◽  
Subviral Particle ◽  
Genotype A

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins. S protein drives virion and subviral particle secretion, whereas L protein inhibits subviral particle secretion but coordinates virion morphogenesis. We previously found that preventing S protein expression from a subgenomic construct eliminated M protein. The present study further examined impact of S protein on L and M proteins. Mutations were introduced to subgenomic construct of genotype A or 1.1mer replication construct of genotype A or D, and viral proteins were analyzed from transfected Huh7 cells. Mutating S gene ATG to prevent expression of full-length S protein eliminated M protein, reduced intracellular level of L protein despite its blocked secretion, and generated a truncated S protein through translation initiation from a downstream ATG. Truncated S protein was secretion deficient and could inhibit secretion of L, M, S proteins from wild-type constructs. Providing full-length S protein in trans rescued L protein secretion and increased its intracellular level from mutants of lost S gene ATG. Lost core protein expression reduced all the three envelope proteins. In conclusion, full-length S protein could sustain intracellular and extracellular L and M proteins, while truncated S protein could block subviral particle secretion.

scholarly journals GIT1, a Gene Encoding a Novel Transporter for Glycerophosphoinositol in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1707-1715 ◽  
Author(s):  
J L Patton-Vogt ◽  
S A Henry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Gene ◽  
Sugar Transport ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Membrane Spanning ◽  
Membrane Spanning Domains ◽  
Uptake And Metabolism ◽  
Cells Cultured

Abstract Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git−) defective in the uptake and metabolism of GroPIns. One mutant was found to be affected in the gene encoding the transcription factor, SPT7. Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git− phenotype. Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol). This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD. The predicted Git1p has similarity to a variety of S. cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p). Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains. Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

Specificity and promiscuity in membrane helix interactions

1994 ◽  
Vol 27 (2) ◽  
pp. 157-218 ◽  
Author(s):  
Mark A. Lemmon ◽  
Donald M. Engelman
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Helix ◽  
Integral Membrane Proteins ◽  
Lipid Packing ◽  
Membrane Protein Folding ◽  
Membrane Spanning ◽  
Α Helix ◽  
Prosthetic Groups ◽  
Packing Effects ◽  
Helix Interactions

The membrane-spanning portions of many integral membrane proteins consist of one or a number of transmembrane α-helices, which are expected to be independently stable on thermodynamic grounds. Side-by-side interactions between these transmembrane α-helices are important in the folding and assembly of such integral membrane proteins and their complexes. In considering the contribution of these helix–helix interactions to membrane protein folding and oligomerization, a distinction between the energetics and specificity should be recognized. A number of contributions to the energetics of transmembrane helix association within the lipid bilayer will be relatively non-specific, including those resulting from charge–charge interactions and lipid–packing effects. Specificity (and part of the energy) in transmembrane α-helix association, however, appears to rely mainly upon a detailed stereochemical fit between sets of dynamically accessible states of particular helices. In some cases, these interactions are mediated in part by prosthetic groups.

scholarly journals Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways

2010 ◽  
Vol 84 (19) ◽  
pp. 9897-9906 ◽  
Author(s):  
Florence Larrous ◽  
Alireza Gholami ◽  
Shahul Mouhamad ◽  
Jérôme Estaquier ◽  
Hervé Bourhy
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Matrix Protein ◽  
Full Length ◽  
M Protein ◽  
Genotype 3 ◽  
Cell Death Pathways ◽  
Acid Fragment ◽  
M Proteins ◽  
Cellular Apoptosis

ABSTRACT The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

Sign in / Sign up

Export Citation Format

Share Document