scholarly journals Severe Acute Respiratory Syndrome Coronavirus Accessory Protein 6 Is a Virion-Associated Protein and Is Released from 6 Protein-Expressing Cells

2007 ◽  
Vol 81 (10) ◽  
pp. 5423-5426 ◽  
Author(s):  
Cheng Huang ◽  
C. J. Peters ◽  
Shinji Makino
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Sucrose Gradient ◽  
Accessory Protein ◽  
Accessory Proteins ◽  
S Protein ◽  
Virus Like Particles ◽  
Virus Particles ◽  
Capture Assay ◽  
Infected Cells ◽  
Equilibrium Centrifugation

ABSTRACT Analysis of severe acute respiratory syndrome coronavirus (SCoV) by either sucrose gradient equilibrium centrifugation or a virus capture assay using an anti-SCoV S protein antibody demonstrated that the SCoV 6 protein, which is one of the accessory proteins of SCoV, was incorporated into virus particles. Coexpression of the SCoV S, M, E, and 6 proteins was sufficient for incorporation of the 6 protein into virus-like particles. Cells transfected with plasmid expressing the 6 protein released SCoV 6 protein; however, infected cells released SCoV 6 protein only in association with SCoV particles.

scholarly journals SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Amin Addetia ◽  
Nicole A. P. Lieberman ◽  
Quynh Phung ◽  
Tien-Ying Hsiang ◽  
Hong Xie ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nuclear Import ◽  
Mrna Export ◽  
Nucleocytoplasmic Transport ◽  
Single Amino Acid ◽  
Accessory Protein ◽  
Accessory Proteins ◽  
Reporter Expression ◽  
Infected Cells ◽  
Reporter Proteins

ABSTRACT RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6’s nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6’s interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6’s disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.

An examination of Penicillium notatum for the presence of Penicillium chrysogenum-type virus particles

1972 ◽  
Vol 18 (8) ◽  
pp. 1352-1353 ◽  
Author(s):  
Olga Volkoff ◽  
Teresa Walters ◽  
Rosamund A. Dejardin
Keyword(s):  
Virus Infection ◽  
Penicillium Chrysogenum ◽  
Type Virus ◽  
Penicillium Notatum ◽  
Virus Like Particles ◽  
Virus Particles ◽  
Infected Cells

Virus-like particles have been shown to be present in all strains of Penicillium chrysogenum examined but not in penicillin producing strains of P. notatum. Attempts to "cure" P. chrysogenum of the virus infection were not successful, nor was it possible to infect P. notatum with the purified virus from P. chrysogenum. Examination of longitudinal sections of the mycelia of P. chrysogenum showed that the virus particles are enclosed in vesicles in the infected cells.

scholarly journals Identification of Diphenoxylate as an Antiviral Agent Against Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Author(s):  
Jin Soo Shin ◽  
Eunhye Jung ◽  
Yejin Jang ◽  
Soo Bong Han ◽  
Meehyein Kim
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Antiviral Treatment ◽  
Antiviral Agent ◽  
Selectivity Index ◽  
Dependent Manner ◽  
S Protein ◽  
Chemical Derivatives ◽  
Highly Active ◽  
Infected Cells ◽  
Dose Dependent

This is the first report to show that diphenoxylate is highly active against SARS-CoV-2 with EC<sub>50</sub> of 1.4 μM, CC<sub>50</sub> over 100 μM and selectivity index over 71.4 after screening of 14 diphenyl derivatives. Our results with highly purified solid diphenoxylate confirmatively demonstrate that the viral S protein is reduced in the virus-infected cells in a dose-dependent manner. It could provide insights for antiviral treatment of COVID-19 with diphenoxylate or its chemical derivatives

scholarly journals Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is Released in Membranous Structures from 3a Protein-Expressing Cells and Infected Cells

2006 ◽  
Vol 80 (1) ◽  
pp. 210-217 ◽  
Author(s):  
Cheng Huang ◽  
Krishna Narayanan ◽  
Naoto Ito ◽  
C. J. Peters ◽  
Shinji Makino
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Buoyant Density ◽  
Protein A ◽  
Biological Properties ◽  
Structural Protein ◽  
Membrane Structures ◽  
Capture Assay ◽  
Release Analysis ◽  
Infected Cells ◽  
Detergent Resistant Membranes

ABSTRACT Severe acute respiratory syndrome coronavirus (SCoV) accessory protein 3a is a virus structural protein. We demonstrate here that 3a protein was released efficiently in membranous structures from various cell lines expressing 3a protein. A subpopulation of the released 3a protein is associated with detergent-resistant membranes. The presence of the YxxΦ and diacidic motifs, located within the cytoplasmic tail of the 3a protein, was not required for its efficient release. Analysis of supernatant from SCoV-infected cells with sucrose gradient sedimentation and virus capture assay indicated that the 3a protein was released from infected cells in two distinct populations, as a component of SCoV particles, and in membrane structures with a lower buoyant density. These data provide new insights into the biological properties of SCoV 3a protein.

scholarly journals Two palmitylated cysteine residues of the severe acute respiratory syndrome coronavirus spike (S) protein are critical for S incorporation into virus-like particles, but not for M–S co-localization

2012 ◽  
Vol 93 (4) ◽  
pp. 823-828 ◽  
Author(s):  
Makoto Ujike ◽  
Cheng Huang ◽  
Kazuya Shirato ◽  
Shutoku Matsuyama ◽  
Shinji Makino ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Immunofluorescence Assay ◽  
M Protein ◽  
Cysteine Residues ◽  
S Protein ◽  
Virion Assembly ◽  
Virus Like Particles ◽  
S Proteins

The endodomain of several coronavirus (CoV) spike (S) proteins contains palmitylated cysteine residues and enables co-localization and interaction with the CoV membrane (M) protein. Depalmitylation of mouse hepatitis virus S proteins abolished this interaction, resulting in the failure of S incorporation into virions. In contrast, an immunofluorescence assay (IFA) showed that depalmitylated severe acute respiratory syndrome coronavirus (SCoV) S proteins still co-localized with the M protein in the budding site. Here, we determined the ability of depalmitylated SCoV S mutants to incorporate S into virus-like particles (VLPs). IFA confirmed that all SCoV S mutants co-localized with the M protein intracellularly. However, the mutants lacking two cysteine residues (C1234/1235) failed to incorporate S into VLPs. This indicated that these palmitylated cysteines are essential for S incorporation, but are not involved in S co-localization mediated by the M protein. Our findings suggest that M–S co-localization and S incorporation occur independently of one another in SCoV virion assembly.

scholarly journals Identification of Diphenoxylate as an Antiviral Agent Against Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Author(s):  
Jin Soo Shin ◽  
Eunhye Jung ◽  
Yejin Jang ◽  
Soo Bong Han ◽  
Meehyein Kim
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Antiviral Treatment ◽  
Antiviral Agent ◽  
Selectivity Index ◽  
Dependent Manner ◽  
S Protein ◽  
Chemical Derivatives ◽  
Highly Active ◽  
Infected Cells ◽  
Dose Dependent

This is the first report to show that diphenoxylate is highly active against SARS-CoV-2 with EC<sub>50</sub> of 1.4 μM, CC<sub>50</sub> over 100 μM and selectivity index over 71.4 after screening of 14 diphenyl derivatives. Our results with highly purified solid diphenoxylate confirmatively demonstrate that the viral S protein is reduced in the virus-infected cells in a dose-dependent manner. It could provide insights for antiviral treatment of COVID-19 with diphenoxylate or its chemical derivatives

scholarly journals Identifying Epitopes Responsible for Neutralizing Antibody and DC-SIGN Binding on the Spike Glycoprotein of the Severe Acute Respiratory Syndrome Coronavirus

2006 ◽  
Vol 80 (21) ◽  
pp. 10315-10324 ◽  
Author(s):  
Yi-Ping Shih ◽  
Chia-Yen Chen ◽  
Shih-Jen Liu ◽  
Kuan-Hsuan Chen ◽  
Yuan-Ming Lee ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Binding Capacity ◽  
Neutralizing Antibody ◽  
Observation Data ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Capture Assay ◽  
Immature Dendritic Cells ◽  
Glycosylation Sites ◽  
Peptide Display

ABSTRACT The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) uses dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) to facilitate cell entry via cellular receptor-angiotensin-converting enzyme 2. For this project, we used recombinant baculoviruses expressing different lengths of SARS-CoV spike (S) protein in a capture assay to deduce the minimal DC-SIGN binding region. Our results identified the region location between amino acid (aa) residues 324 to 386 of the S protein. We then generated nine monoclonal antibodies (MAbs) against the S protein to map the DC-SIGN-binding domain using capture assays with pseudotyped viruses and observed that MAb SIa5 significantly blocked S protein-DC-SIGN interaction. An enhancement assay using the HKU39849 SARS-CoV strain and human immature dendritic cells confirmed our observation. Data from a pepscan analysis and M13 phage peptide display library system mapped the reactive MAb SIa5 epitope to aa residues 363 to 368 of the S protein. Results from a capture assay testing three pseudotyped viruses with mutated N-linked glycosylation sites of the S protein indicate that only two pseudotyped viruses (N330Q and N357Q, both of which lost glycosylation sites near the SIa5 epitope) had diminished DC-SIGN-binding capacity. We also noted that MAb SIb4 exerted a neutralizing effect against HKU39849; its reactive epitope was mapped to aa residues 435 to 439 of the S protein. We offer the data to facilitate the development of therapeutic agents and preventive vaccines against SARS-CoV infection.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus 3a Protein Is a Viral Structural Protein

2005 ◽  
Vol 79 (5) ◽  
pp. 3182-3186 ◽  
Author(s):  
Naoto Ito ◽  
Eric C. Mossel ◽  
Krishna Narayanan ◽  
Vsevolod L. Popov ◽  
Cheng Huang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Severe Acute Respiratory Syndrome ◽  
Posttranslational Modifications ◽  
Accessory Protein ◽  
Structural Protein ◽  
Viral Structural Protein ◽  
Infected Cells

ABSTRACT The present study showed the association of a severe acute respiratory syndrome coronavirus (SCoV) accessory protein, 3a, with plasma membrane and intracellular SCoV particles in infected cells. 3a protein appeared to undergo posttranslational modifications in infected cells and was incorporated into SCoV particles, establishing that 3a protein was a SCoV structural protein.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus 7a Accessory Protein Is a Viral Structural Protein

2006 ◽  
Vol 80 (15) ◽  
pp. 7287-7294 ◽  
Author(s):  
Cheng Huang ◽  
Naoto Ito ◽  
Chien-Te K. Tseng ◽  
Shinji Makino
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Biological Activities ◽  
Protein Translation ◽  
Host Protein ◽  
Mitogen Activated Protein Kinase ◽  
Accessory Protein ◽  
Structural Protein ◽  
S Protein ◽  
Mitogen Activated Protein ◽  
Protein Incorporation

ABSTRACT Severe acute respiratory syndrome coronavirus (SCoV) 7a protein is one of the viral accessory proteins. In expressing cells, 7a protein exhibits a variety of biological activities, including induction of apoptosis, activation of the mitogen-activated protein kinase signaling pathway, inhibition of host protein translation, and suppression of cell growth progression. Analysis of SCoV particles that were purified by either sucrose gradient equilibrium centrifugation or a virus capture assay, in which intact SCoV particles were specifically immunoprecipitated by anti-S protein monoclonal antibody, demonstrated that 7a protein was associated with purified SCoV particles. Coexpression of 7a protein with SCoV S, M, N, and E proteins resulted in production of virus-like particles (VLPs) carrying 7a protein, while 7a protein was not released from cells expressing 7a protein alone. Although interaction between 7a protein and another SCoV accessory protein, 3a, has been reported, 3a protein was dispensable for assembly of 7a protein into VLPs. S protein was not required for the 7a protein incorporation into VLPs, and yet 7a protein interacted with S protein in coexpressing cells. These data established that, in addition to 3a protein, 7a protein was a SCoV accessory protein identified as a SCoV structural protein.

scholarly journals Vpr Co-assembles with Gag during HIV-1 Assembly

2020 ◽  
Author(s):  
Kate Bredbenner ◽  
Sanford M Simon
Keyword(s):  
Genetic Analysis ◽  
Accessory Protein ◽  
Accessory Proteins ◽  
Structural Protein ◽  
Virus Like Particles ◽  
Future Treatments ◽  
Gag Assembly ◽  
Kinetics Of ◽  
Hiv 1

AbstractThe HIV-1 accessory protein Vpr is packaged into new virions at a 7:1 ratio of Gag/Vpr. Previous biochemical and genetic analysis has shown that Vpr gets packaged into virions via an LXXLF motif on the p6 domain of the Gag structural polyprotein. The kinetics of Vpr packaging compared to Gag assembly was previously unknown. Here, we confirm via biochemistry and imaging that fluorescently tagged Vpr gets packaged into virus-like particles only when the LXXLF motif is intact. When the LXXLF motif is mutated, Vpr is no longer recruited to Gag assemblies. When Vpr and Gag assembly are imaged together, we see that Vpr co-assembles with a slight delay compared to Gag suggesting that Vpr is not being recruited to the membrane with Gag but is instead being recruited to actively assembling Gag.ImportanceHIV-1 affects over 30 million people around the globe, and although we have good treatments, there is still no cure. The virus encodes 15 distinct proteins, and four of those proteins are known as accessory proteins. Vpr is one of the accessory proteins that is packaged into HIV-1 by interacting with the Gag structural protein. Without Vpr, HIV-1 is not as infectious. Our research shows that Vpr is packaged into new viruses as the virus is being formed rather than being put in towards the end of the assembly of a virus. Getting a clearer view of each step in the process of assembling each virion will help inform future treatments and help with overall comparisons between the assembly of different viruses.

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