scholarly journals The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein

2020 ◽  
Author(s):  
Bertrand Boson ◽  
Vincent Legros ◽  
Bingjie Zhou ◽  
Cyrille Mathieu ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Causative Agent ◽  
Secretory Pathway ◽  
Retention Mechanism ◽  
Structural Proteins ◽  
Imaging Data ◽  
Viral Particles ◽  
Infected Cells ◽  
Nucleoprotein N ◽  
N Proteins

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely β-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses.Author SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses.

scholarly journals The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing assembly of virus-like particles

2020 ◽  
pp. jbc.RA120.016175
Author(s):  
Bertrand Boson ◽  
Vincent Legros ◽  
Bingjie Zhou ◽  
Eglantine Siret ◽  
Cyrille Mathieu ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Structural Proteins ◽  
Imaging Data ◽  
Virus Like Particles ◽  
Viral Particles ◽  
M Proteins ◽  
Infected Cells ◽  
Intermediate Compartment ◽  
Nucleoprotein N ◽  
Syncytia Formation

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: Spike (S), Envelope (E), Membrane (M) and Nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of β-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected vs. transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is re-localized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon co-expression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M and N are required for optimal production of virus- like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.

scholarly journals A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, Is Transported to the Cell Surface and Undergoes Endocytosis

2004 ◽  
Vol 78 (13) ◽  
pp. 6723-6734 ◽  
Author(s):  
Yee-Joo Tan ◽  
Eileen Teng ◽  
Shuo Shen ◽  
Timothy H. P. Tan ◽  
Phuay-Yee Goh ◽  
...  
Keyword(s):  
Amino Acids ◽  
Severe Acute Respiratory Syndrome ◽  
Cell Surface ◽  
Open Reading Frames ◽  
Structural Proteins ◽  
Replicase Gene ◽  
Subgenomic Rna ◽  
C Terminus ◽  
Infected Cells ◽  
Potential Orfs

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxxφ and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.

scholarly journals Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ricardo S. Cardoso ◽  
Lucas Alves Tavares ◽  
Bruna Lais S. Jesus ◽  
Miria F. Criado ◽  
Andreia Nogueira de Carvalho ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Secretory Pathway ◽  
Human Respiratory Syncytial Virus ◽  
Structural Proteins ◽  
Sorting Nexin ◽  
Syncytial Virus ◽  
N Proteins

ABSTRACT Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites. IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

scholarly journals Role of PACS-1 in Trafficking of Human Cytomegalovirus Glycoprotein B and Virus Production

2003 ◽  
Vol 77 (20) ◽  
pp. 11105-11113 ◽  
Author(s):  
Colin M. Crump ◽  
Chien-Hui Hung ◽  
Laurel Thomas ◽  
Lei Wan ◽  
Gary Thomas
Keyword(s):  
Envelope Glycoprotein ◽  
Secretory Pathway ◽  
Virus Production ◽  
Viral Proteins ◽  
Glycoprotein B ◽  
Efficient Production ◽  
Viral Particles ◽  
Infected Cells ◽  
Golgi Network

ABSTRACT The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.

scholarly journals Intracellular Trafficking of HBV Particles

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2023 ◽  
Author(s):  
Bingfu Jiang ◽  
Eberhard Hildt
Keyword(s):  
Intracellular Trafficking ◽  
Secretory Pathway ◽  
Surface Proteins ◽  
Nuclear Pore ◽  
Enveloped Virus ◽  
Double Stranded Dna ◽  
Viral Particles ◽  
B Virus ◽  
Infected Cells ◽  
Covalently Linked

The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.

scholarly journals Key Golgi Factors for Structural and Functional Maturation of Bunyamwera Virus

2005 ◽  
Vol 79 (17) ◽  
pp. 10852-10863 ◽  
Author(s):  
Reyes R. Novoa ◽  
Gloria Calderita ◽  
Pilar Cabezas ◽  
Richard M. Elliott ◽  
Cristina Risco
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Dependent Manner ◽  
Sugar Composition ◽  
Enveloped Viruses ◽  
Functional Maturation ◽  
Viral Particles ◽  
Viral Glycoproteins ◽  
Infected Cells ◽  
Viral Form

ABSTRACT Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a trans-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-β-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.

Electron Microscopy of a Herpesvirus Isolated from Marek's Disease in Duck and Chicken Embryo Fibroblast Cultures

1968 ◽  
Vol 26 ◽  
pp. 222-223 ◽  
Author(s):  
Keyvan Nazerian
Keyword(s):  
Inclusion Bodies ◽  
Chicken Embryo Fibroblast ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Duck Embryo Fibroblast ◽  
Multinucleated Cells ◽  
Viral Particles ◽  
Infected Cells ◽  
And Control ◽  
Do So

A herpes-like virus has been isolated from duck embryo fibroblast (DEF) cultures inoculated with blood from Marek's disease (MD) infected birds. Cultures which contained this virus produced MD in susceptible chickens while virus negative cultures and control cultures failed to do so. This and other circumstantial evidence including similarities in properties of the virus and the MD agent implicate this virus in the etiology of MD.Histochemical studies demonstrated the presence of DNA-staining intranuclear inclusion bodies in polykarocytes in infected cultures. Distinct nucleo-plasmic aggregates were also seen in sections of similar multinucleated cells examined with the electron microscope. These aggregates are probably the same as the inclusion bodies seen with the light microscope. Naked viral particles were observed in the nucleus of infected cells within or on the edges of the nucleoplasmic aggregates. These particles measured 95-100mμ, in diameter and rarely escaped into the cytoplasm or nuclear vesicles by budding through the nuclear membrane (Fig. 1). The enveloped particles (Fig. 2) formed in this manner measured 150-170mμ in diameter and always had a densely stained nucleoid. The virus in supernatant fluids consisted of naked capsids with 162 hollow, cylindrical capsomeres (Fig. 3). Enveloped particles were not seen in such preparations.

Covid 19 – Structural proteins and prospective drug candidates (Preprint)

2020 ◽  
Author(s):  
A.N Anoopkumar ◽  
Sharrel Rebello ◽  
Embalil Mathachan Aneesh
Keyword(s):  
Causative Agent ◽  
Protein Level ◽  
Drug Targets ◽  
Current Paper ◽  
Structural Proteins ◽  
Drug Candidates ◽  
Propagation Properties

UNSTRUCTURED Covid 19 the causative agent of the current devastating pandemic has turned out to be a notorious virus to all men-irrespective of either common to scientific calibre. Attempts to combat this deadly virus are the need of the hour and quite often the best way to defeat an opponent is to keenly study about its structural and propagation properties. The current paper describes briefly Covid 19 at the genomic, structural and protein level to the best of our knowledge. Furthermore, the prospects of possible drug targets that could aid in the control of this virus are also discussed.

scholarly journals Immunoinformatics-guided design of a multi-epitope vaccine based on the structural proteins of severe acute respiratory syndrome coronavirus 2

RSC Advances ◽  
2021 ◽  
Vol 11 (29) ◽  
pp. 18103-18121
Author(s):  
Ahmad J. Obaidullah ◽  
Mohammed M. Alanazi ◽  
Nawaf A. Alsaif ◽  
Hussam Albassam ◽  
Abdulrahman A. Almehizia ◽  
...  
Keyword(s):  
Tract Infection ◽  
Severe Acute Respiratory Syndrome ◽  
Respiratory Tract ◽  
Respiratory Tract Infection ◽  
Structural Proteins ◽  
Epitope Vaccine

COVID-19 is caused by SARS-CoV-2, resulting in a contagious respiratory tract infection. For designing a multi-epitope vaccine, we utilized the four structural proteins from the SARS-CoV-2 by using bioinformatics and immunoinformatics analysis.

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