scholarly journals A Single Deletion in the Membrane-Proximal Region of the Sindbis Virus Glycoprotein E2 Endodomain Blocks Virus Assembly

2000 ◽  
Vol 74 (9) ◽  
pp. 4220-4228 ◽  
Author(s):  
Raquel Hernandez ◽  
Heuiran Lee ◽  
Christine Nelson ◽  
Dennis T. Brown
Keyword(s):  
Sindbis Virus ◽  
Virus Assembly ◽  
Specific Interaction ◽  
Proximal Region ◽  
Host Cells ◽  
C Protein ◽  
Mosquito Cells ◽  
Single Deletion ◽  
Binding Cleft ◽  
Virus C

ABSTRACT The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390–400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531–541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, ΔK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, ΔK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.

scholarly journals SARS-CoV-2 Nucleocapsid Assembly Inhibitors: Repurposing Antiviral and Antimicrobial Drugs Targeting Nucleocapsid-RNA Interaction

2020 ◽  
Author(s):  
Debica Mukherjee ◽  
UPASANA RAY
Keyword(s):  
Rna Binding ◽  
Virus Assembly ◽  
Drug Repurposing ◽  
Docking Studies ◽  
Host Cells ◽  
Antimicrobial Drugs ◽  
Binding Cleft ◽  
Novel Coronavirus ◽  
Approved Drugs ◽  
Rna Interaction

<p>SARS-CoV-2 pandemic has become a serious concern due to high transmission of this virus and unavailability of any definitive drugs yet in clinics. While novel antivirals are under investigation stage, scientists are also rigorously trying to use drug repurposing as an option to fight against this highly infectious novel coronavirus. Several drugs are under regular use for other diseases that are getting screened for their usability against SARS-CoV2. In this study we have targeted SARS-CoV-2 nucleocapsid assembly to shortlist FDA approved drugs that could be tested for inhibition of SARS-CoV-2 virus particles inside the host cell. We could shortlist seven antiviral and anti-microbial drugs. These showed good fit in docking studies inside the RNA binding cleft of the nucleocapsid protein. Also, these drugs have good lipophilic properties suggesting that they would enter the host cells. We propose that these shortlisted drugs could potentially compete out binding of viral RNA to nucleocapsid and thus inhibit successful virus assembly leading to poor virus progeny levels. </p>

scholarly journals SARS-CoV-2 Nucleocapsid Assembly Inhibitors: Repurposing Antiviral and Antimicrobial Drugs Targeting Nucleocapsid-RNA Interaction

2020 ◽  
Author(s):  
Debica Mukherjee ◽  
UPASANA RAY
Keyword(s):  
Rna Binding ◽  
Virus Assembly ◽  
Drug Repurposing ◽  
Docking Studies ◽  
Host Cells ◽  
Antimicrobial Drugs ◽  
Binding Cleft ◽  
Novel Coronavirus ◽  
Approved Drugs ◽  
Rna Interaction

<p></p><p>SARS-CoV-2 pandemic has become a serious concern due to high transmission of this virus and unavailability of any definitive drugs yet in clinics. While novel antivirals are under investigation stage, scientists are also rigorously trying to use drug repurposing as an option to fight against this highly infectious novel coronavirus. Several drugs are under regular use for other diseases that are getting screened for their usability against SARS-CoV2. In this study we have targeted SARS-CoV-2 nucleocapsid assembly to shortlist FDA approved drugs that could be tested for inhibition of SARS-CoV-2 virus particles inside the host cell. We could shortlist five antiviral and anti-microbial drugs. These showed good fit in docking studies inside the RNA binding cleft of the nucleocapsid protein. Also, these drugs have lipophilic properties suggesting that they have the potential to enter the host cells. We propose that these shortlisted drugs could potentially compete out binding of viral RNA to nucleocapsid and thus inhibit successful virus assembly leading to poor virus progeny levels. </p><br><p></p>

scholarly journals The Amino-Terminal Half of Sendai Virus C Protein Is Not Responsible for either Counteracting the Antiviral Action of Interferons or Down-Regulating Viral RNA Synthesis

2002 ◽  
Vol 76 (14) ◽  
pp. 7114-7124 ◽  
Author(s):  
Atsushi Kato ◽  
Yukano Ohnishi ◽  
Michiko Hishiyama ◽  
Masayoshi Kohase ◽  
Sakura Saito ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Sendai Virus ◽  
Virus Assembly ◽  
Viral Rna ◽  
Antiviral Action ◽  
C Protein ◽  
Amino Terminal ◽  
Inhibitory Activities ◽  
C Proteins ◽  
Virus C

ABSTRACT The Sendai virus C proteins, C′, C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.

scholarly journals SARS-CoV-2 Nucleocapsid Assembly Inhibitors: Repurposing Antiviral and Antimicrobial Drugs Targeting Nucleocapsid-RNA Interaction

2020 ◽  
Author(s):  
Debica Mukherjee ◽  
UPASANA RAY
Keyword(s):  
Rna Binding ◽  
Virus Assembly ◽  
Drug Repurposing ◽  
Docking Studies ◽  
Host Cells ◽  
Antimicrobial Drugs ◽  
Binding Cleft ◽  
Novel Coronavirus ◽  
Approved Drugs ◽  
Rna Interaction

<p></p><p>SARS-CoV-2 pandemic has become a serious concern due to high transmission of this virus and unavailability of any definitive drugs yet in clinics. While novel antivirals are under investigation stage, scientists are also rigorously trying to use drug repurposing as an option to fight against this highly infectious novel coronavirus. Several drugs are under regular use for other diseases that are getting screened for their usability against SARS-CoV2. In this study we have targeted SARS-CoV-2 nucleocapsid assembly to shortlist FDA approved drugs that could be tested for inhibition of SARS-CoV-2 virus particles inside the host cell. We could shortlist five antiviral and anti-microbial drugs. These showed good fit in docking studies inside the RNA binding cleft of the nucleocapsid protein. Also, these drugs have lipophilic properties suggesting that they have the potential to enter the host cells. We propose that these shortlisted drugs could potentially compete out binding of viral RNA to nucleocapsid and thus inhibit successful virus assembly leading to poor virus progeny levels. </p><br><p></p>

Sindbis virus maturation in cultured mosquito cells is sensitive to actinomycin D

Virology ◽  
1981 ◽  
Vol 110 (2) ◽  
pp. 292-301 ◽  
Author(s):  
Ursula Scheefers-Borchel ◽  
Hans Scheefers ◽  
Judy Edwards ◽  
Dennis T. Brown
Keyword(s):  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Maturation ◽  
Mosquito Cells

scholarly journals Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells

2008 ◽  
Vol 89 (11) ◽  
pp. 2651-2661 ◽  
Author(s):  
Hua Wang ◽  
Carol D. Blair ◽  
Ken E. Olson ◽  
Rollie J. Clem
Keyword(s):  
Virus Replication ◽  
Persistent Infection ◽  
Sindbis Virus ◽  
Actinomycin D ◽  
Virus Production ◽  
Cell Types ◽  
Lytic Infection ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Apoptotic Genes

Sindbis virus (SINV) is a mosquito-borne virus in the genus Alphavirus, family Togaviridae. Like most alphaviruses, SINVs exhibit lytic infection (apoptosis) in many mammalian cell types, but are generally thought to cause persistent infection with only moderate cytopathic effects in mosquito cells. However, there have been several reports of apoptotic-like cell death in mosquitoes infected with alphaviruses or flaviviruses. Given that apoptosis has been shown to be an antiviral response in other systems, we have constructed recombinant SINVs that express either pro-apoptotic or anti-apoptotic genes in order to test the effects of inducing or inhibiting apoptosis on SINV replication in mosquito cells. Recombinant SINVs expressing the pro-apoptotic genes reaper (rpr) from Drosophila or michelob_x (mx) from Aedes aegypti caused extensive apoptosis in cells from the mosquito cell line C6/36, thus changing the normal persistent infection observed with SINV to a lytic infection. Although the infected cells underwent apoptosis, high levels of virus replication were still observed during the initial infection. However, virus production subsequently decreased compared with persistently infected cells, which continued to produce high levels of virus over the next several days. Infection of C6/36 cells with SINV expressing the baculovirus caspase inhibitor P35 inhibited actinomycin D-induced caspase activity and protected infected cells from actinomycin D-induced apoptosis, but had no observable effect on virus replication. This study is the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells.

scholarly journals Sendai virus C protein limits NO production in infected RAW264.7 macrophages

2018 ◽  
Vol 24 (7) ◽  
pp. 430-438 ◽  
Author(s):  
Erdenezaya Odkhuu ◽  
Takayuki Komatsu ◽  
Naoki Koide ◽  
Yoshikazu Naiki ◽  
Kenji Takeuchi ◽  
...  
Keyword(s):  
Sendai Virus ◽  
Virus Multiplication ◽  
Inos Expression ◽  
No Production ◽  
Ns1 Protein ◽  
Wild Type ◽  
C Protein ◽  
Raw264.7 Macrophages ◽  
Virus C ◽  
Stat Pathway

To suppress virus multiplication, infected macrophages produce NO. However, it remains unclear how infecting viruses then overcome NO challenge. In the present study, we report the effects of accessory protein C from Sendai virus (SeV), a prototypical paramyxovirus, on NO output. We found that in RAW264.7 murine macrophages, a mutant SeV without C protein (4C(–)) significantly enhanced inducible NO synthase (iNOS) expression and subsequent NO production compared to wild type SeV (wtSeV). SeV 4C(-) infection caused marked production of IFN-β, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-β Ab, however, resulted in only marginal suppression of NO production. In contrast, NF-κB, a primarily important factor for transcription of the iNOS gene, was also activated by 4C(–) infection but not wtSeV infection. Induction of NO production and iNOS expression by 4C(–) was significantly suppressed in cells constitutively expressing influenza virus NS1 protein that can sequester double-stranded (ds)RNA, which triggers activation of signaling pathways leading to activation of NF-κB and IRF3. Therefore, C protein appears to suppress NF-κB activation to inhibit iNOS expression and subsequent NO production, possibly by limiting dsRNA generation in the context of viral infection.

scholarly journals The Host Range Phenotype Displayed by a Sindbis Virus Glycoprotein Variant Results from Virion Aggregation and Retention on the Surface of Mosquito Cells

2000 ◽  
Vol 74 (23) ◽  
pp. 11398-11406 ◽  
Author(s):  
Karl W. Boehme ◽  
Vsevolod L. Popov ◽  
Hans W. Heidner
Keyword(s):  
Cell Surface ◽  
Host Range ◽  
Sindbis Virus ◽  
Cell Type ◽  
Virus Variant ◽  
Cytoplasmic Vacuoles ◽  
Mosquito Cells ◽  
Transmission Electron ◽  
Maturation Defect ◽  
Processing Properties

ABSTRACT The Sindbis virus variant NE2G216 is a PE2-containing host range mutant that is growth restricted in cultured mosquito cells (C6/36) due to inefficient release of virions from this cell type. The maturation defect of NE2G216 has been linked to the structures of N-linked oligosaccharides synthesized by arthropod cells. Analysis of C6/36 cells infected with NE2G216 by transmission electron microscopy revealed the presence of dense virus aggregates within cytoplasmic vacuoles and virus aggregates adhered to the cell surface. The virus aggregation phenotype of NE2G216 was reproduced in vertebrate cells (Pro-5) by the addition of 1-deoxymannojirimycin, an inhibitor of carbohydrate processing which limits the processing of N-linked oligosaccharides to structures that are structurally similar, albeit not identical, to those synthesized in C6/36 cells. We conclude that defective maturation of NE2G216 in mosquito cells is due to virion aggregation and retention on the cell surface and that this phenotype is directly linked to the carbohydrate-processing properties of these cells.

scholarly journals Novel Intersubunit Interaction Critical for HIV-1 Core Assembly Defines a Potentially Targetable Inhibitor Binding Pocket

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Pierrick Craveur ◽  
Anna T. Gres ◽  
Karen A. Kirby ◽  
Dandan Liu ◽  
John A. Hammond ◽  
...  
Keyword(s):  
Viral Replication ◽  
Specific Interaction ◽  
Binding Pocket ◽  
Core Stability ◽  
Host Cells ◽  
Capsid Assembly ◽  
Dynamic Simulations ◽  
Virus Like Particle ◽  
Hiv 1

ABSTRACTHIV-1 capsid protein (CA) plays critical roles in both early and late stages of the viral replication cycle. Mutagenesis and structural experiments have revealed that capsid core stability significantly affects uncoating and initiation of reverse transcription in host cells. This has led to efforts in developing antivirals targeting CA and its assembly, although none of the currently identified compounds are used in the clinic for treatment of HIV infection. A specific interaction that is primarily present in pentameric interfaces in the HIV-1 capsid core was identified and is reported to be important for CA assembly. This is shown by multidisciplinary characterization of CA site-directed mutants using biochemical analysis of virus-like particle formation, transmission electron microscopy ofin vitroassembly, crystallographic studies, and molecular dynamic simulations. The data are consistent with a model where a hydrogen bond between CA residues E28 and K30′ from neighboring N-terminal domains (CANTDs) is important for CA pentamer interactions during core assembly. This pentamer-preferred interaction forms part of anN-terminaldomaininterface (NDI) pocket that is amenable to antiviral targeting.IMPORTANCEPrecise assembly and disassembly of the HIV-1 capsid core are key to the success of viral replication. The forces that govern capsid core formation and dissociation involve intricate interactions between pentamers and hexamers formed by HIV-1 CA. We identified one particular interaction between E28 of one CA and K30′ of the adjacent CA that appears more frequently in pentamers than in hexamers and that is important for capsid assembly. Targeting the corresponding site could lead to the development of antivirals which disrupt this interaction and affect capsid assembly.

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