scholarly journals Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Judy J. Wan ◽  
Rebecca S. Brown ◽  
Margaret Kielian
Keyword(s):  
Small Molecule ◽  
Particle Production ◽  
Rna Virus ◽  
Virus Production ◽  
Free Virus ◽  
Genome Packaging ◽  
Berberine Chloride ◽  
Late Step ◽  
Infected Cells

ABSTRACT Alphaviruses are enveloped positive-sense RNA viruses that can cause serious human illnesses such as polyarthritis and encephalitis. Despite their widespread distribution and medical importance, there are no licensed vaccines or antivirals to combat alphavirus infections. Berberine chloride (BBC) is a pan-alphavirus inhibitor that was previously identified in a replicon-based small-molecule screen. This work showed that BBC inhibits alphavirus replication but also suggested that BBC might have additional effects later in the viral life cycle. Here, we show that BBC has late effects that target the virus nucleocapsid (NC) core. Infected cells treated with BBC late in infection were unable to form stable cytoplasmic NCs or assembly intermediates, as assayed by gradient sedimentation. In vitro studies with recombinant capsid protein (Cp) and purified genomic RNA (gRNA) showed that BBC perturbs core-like particle formation and potentially traps the assembly process in intermediate states. Particles produced from BBC-treated cells were less infectious, despite efficient particle production and only minor decreases in genome packaging. In addition, BBC treatment of free virus particles strongly decreased alphavirus infectivity. In contrast, the infectivity of the negative-sense RNA virus vesicular stomatitis virus was resistant to BBC treatment of infected cells or free virus. Together, our data indicate that BBC alters alphavirus Cp-gRNA interactions and oligomerization and suggest that this may cause defects in NC assembly and in disassembly during subsequent virus entry. Thus, BBC may be considered a novel alphavirus NC assembly inhibitor. IMPORTANCE The alphavirus chikungunya virus (CHIKV) is an example of an emerging human pathogen with increased and rapid global spread. Although an acute CHIKV infection is rarely fatal, many patients suffer from debilitating chronic arthralgia for years. Antivirals against chikungunya and other alphaviruses have been identified in vitro, but to date none have been shown to be efficacious and have been licensed for human use. Here, we investigated a small molecule, berberine chloride (BBC), and showed that it inhibited infectious virus production by several alphaviruses including CHIKV. BBC acted on a late step in the alphavirus exit pathway, namely the formation of the nucleocapsid containing the infectious viral RNA. Better understanding of nucleocapsid formation and its inhibition by BBC will provide important information on the mechanisms of infectious alphavirus production and may enable their future targeting in antiviral strategies.

scholarly journals Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rebecca S. Brown ◽  
Dimitrios G. Anastasakis ◽  
Markus Hafner ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Binding Sites ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Genome Packaging ◽  
Genome Recognition ◽  
Infected Cells

Abstract The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.

scholarly journals Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production

2008 ◽  
Vol 89 (5) ◽  
pp. 1212-1219 ◽  
Author(s):  
Hai-Jun Xu ◽  
Zhang-Nv Yang ◽  
Jin-Fang Zhao ◽  
Cai-Hong Tian ◽  
Jun-Qing Ge ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cultured Cells ◽  
Virus Production ◽  
Core Gene ◽  
Virus Protein ◽  
Infected Cells ◽  
Budded Virus ◽  
Larval Bioassay

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase–Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16–18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

scholarly journals Trypsin increases pseudorabies virus production through activation of the ERK signalling pathway

2006 ◽  
Vol 87 (5) ◽  
pp. 1109-1112 ◽  
Author(s):  
Béatrice Riteau ◽  
Christiane de Vaureix ◽  
François Lefèvre
Keyword(s):  
Pseudorabies Virus ◽  
Virus Production ◽  
Signalling Pathway ◽  
Herpesvirus Infection ◽  
Extracellular Proteases ◽  
Extracellular Signal ◽  
Inflammatory Processes ◽  
Infected Cells

Extracellular proteases that are expressed in primary and secondary foci of viral infection are potentially important mediators of infectious inflammatory processes. For some viruses, such as influenza virus and rotaviruses, proteases such as trypsin enhance infectivity by a direct proteolytic effect on some virion proteins. By using an in vitro model of herpesvirus infection, the possibility that proteases modulate the viral cycle through signalling delivered to the infected cell was investigated. It is reported that exposure of pseudorabies virus-infected cells to trypsin increased virus production. Moreover, this treatment induced synergistic and sustained activation of the extracellular signal-regulated kinase (ERK) 1/2 signalling pathway, which appeared to be necessary for this increased viral production. These results suggest that herpesviruses could take advantage of the inflammatory context and particularly of the presence of proteases to increase their replication. Thus, these data point to a potentially important role of extracellular proteases in herpesvirus infection.

scholarly journals Bepridil is potent against SARS-CoV-2 In Vitro

2020 ◽  
Author(s):  
Erol C. Vatansever ◽  
Kai Yang ◽  
Kaci C. Kratch ◽  
Aleksandra Drelich ◽  
Chia-Chuan Cho ◽  
...  
Keyword(s):  
Small Molecule ◽  
A549 Cells ◽  
Live Virus ◽  
Docking Analysis ◽  
Computational Docking ◽  
Main Protease ◽  
Infected Cells ◽  
Endosomal Ph ◽  
Cytopathogenic Effect

ABSTRACTGuided by a computational docking analysis, about 30 FDA/EMA-approved small molecule medicines were characterized on their inhibition of the SARS-CoV-2 main protease (MPro). Of these tested small molecule medicines, six displayed an IC50 value in inhibiting MPro below 100 μM. Three medicines pimozide, ebastine, and bepridil are basic small molecules. Their uses in COVID-19 patients potentiate dual functions by both raising endosomal pH to slow SARS-CoV-2 entry into the human cell host and inhibiting MPro in infected cells. A live virus-based microneutralization assay showed that bepridil inhibited cytopathogenic effect induced by SARS-CoV-2 in Vero E6 cells completely at and dose-dependently below 5 μM and in A549 cells completely at and dose-dependently below 6.25 μM. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.

scholarly journals Multiple Capsid Protein Binding Sites Mediate Selective Packaging of the Alphavirus Genomic RNA

2020 ◽  
Author(s):  
Rebecca S. Brown ◽  
Dimitrios G. Anastasakis ◽  
Markus Hafner ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Binding Sites ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Genome Packaging ◽  
Genome Recognition ◽  
Infected Cells

ABSTRACTThe alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, we comprehensively defined binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrated Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments showed that PS was not required for production of infectious SFV or Chikungunya virus. Instead, we identified multiple novel Cp binding sites that were enriched on gRNA-specific regions and promoted infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrated that budding caused discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site was maintained throughout virus assembly, and specifically bound and assembled with Cp into core-like particles in vitro. Together our data suggest a new model for selective alphavirus genome recognition and assembly.

scholarly journals Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

2014 ◽  
Vol 89 (1) ◽  
pp. 523-534 ◽  
Author(s):  
Mayuri Sharma ◽  
Brian J. Bender ◽  
Jeremy P. Kamil ◽  
Ming F. Lye ◽  
Jean M. Pesola ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Continuous Distribution ◽  
Virus Production ◽  
Nuclear Lamina ◽  
Dominant Negative ◽  
Lamin A ◽  
Nuclear Egress ◽  
Dividing Cells ◽  
Infected Cells

ABSTRACTHerpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with aUL97mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses.

scholarly journals The effect of in vitro human immunodeficiency virus infection on dendritic-cell differentiation and function

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4215-4228 ◽  
Author(s):  
B Canque ◽  
M Rosenzwajg ◽  
S Camus ◽  
M Yagello ◽  
ML Bonnet ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Virus Production ◽  
Polymerase Chain Reaction Analysis ◽  
Viral Dna ◽  
Human Cord Blood ◽  
Necrosis Factor Alpha ◽  
Dendritic Cell Differentiation ◽  
Immunodeficiency Virus ◽  
Infected Cells

Abstract CD1a+ dendritic cells (DC) differentiate from a major population of nonadherent CD13(hi)lin-cells that appear when human cord blood CD34+ hematopoietic progenitor cells are cultured with stem-cell factor, granulocyte/macrophage (MA) colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha) for 5 days. CD13hilin- cells, which also comprise MA and granulocyte precursors, are CD4+ and can thus be targets of human immunodeficiency virus (HIV). Low replication was noted when these day 5 cells were infected with lymphotropic HIV-1LA1 (p24: < or = 4 ng/mL on day 8 postinfection [PI]), while high virus production occurred with MA-tropic HIV-1Ba-L, HIV-1Ada, or HIV-1-m-n. (p24: 50 to > or = 1,000 ng/mL). Strong cytopathicity (CPE) was then observed in nonadherent cells as in adherent MA. However, FACS analysis on day 7 PI showed that HIV did not affect differentiation of DC that survived CPE: apart from CD4 downmodulation related to HIV production, overall expression of CD40, CD80, and CD86 costimulatory molecules, and of HLA-DR, was unchanged relative to controls. At that time, the capacity of DC from HIV-infected cultures to stimulate the mixed leukocyte reaction was only altered less than 10-fold. Immunocytochemistry on day 7 PI showed that most HIV-infected cells were included in syncytia that were stained by anti-CD1a, anti-S100, and anti-CD14 antibodies, indicating that syncytia consisted of DC and cells of the MA lineage. Polymerase chain reaction analysis of FACS- sorted CD1a+ cells confirmed that they harbored then HIV DNA. Viral DNA was also detected in CD1a+ DC from noninfected cultures that had been exposed to HIV only after sorting. Therefore, we examined whether in infected cultures DC precursors were infected at the onset or if virus spread later from other infected cells to differentiated DC. This was answered by showing that, 24 hours postexposure to HIV, viral DNA was preferentially detected in day 5 sorted CD13hilin-versus CD13hilin- cells, and that it was found in the CD1a+ progeny of CD13(hi)lin-cells 48 hours later. In addition, HIV replication did not affect myeloid clonogenic progenitors in day 0 to day 7 PI cultures, although viral DNA was detected in colony-forming unit-granulocyte/macrophage (CFU-GM)/CFU-M colonies derived from day 3 and 7 PI cultures. Thus, precursors of DC and their progeny are susceptible to HIV in vitro, but, apart from CPE, the effect of virus production on DC differentiation or function is limited.

scholarly journals Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

2012 ◽  
Vol 87 (3) ◽  
pp. 1385-1399 ◽  
Author(s):  
Thomas H. R. Carlsen ◽  
Troels K. H. Scheel ◽  
Santseharay Ramirez ◽  
Steven K. H. Foung ◽  
Jens Bukh
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Core Protein ◽  
Particle Density ◽  
Virus Production ◽  
Envelope Proteins ◽  
Late Step

ABSTRACTThe hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexesin vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ∼100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.

scholarly journals Cytomegalovirus Assemblin (pUL80a): Cleavage at Internal Site Not Essential for Virus Growth; Proteinase Absent from Virions

2002 ◽  
Vol 76 (17) ◽  
pp. 8667-8674 ◽  
Author(s):  
Chee-Kai Chan ◽  
Edward J. Brignole ◽  
Wade Gibson
Keyword(s):  
Virus Production ◽  
Artificial Chromosome ◽  
Protein Product ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Apparent Difference ◽  
Virus Growth ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT The human cytomegalovirus (HCMV) maturational proteinase is synthesized as an enzymatically active 74-kDa precursor that cleaves itself at four sites. Two of these, called the maturational (M) and release (R) sites, are conserved in the homologs of all herpesviruses. The other two, called the internal (I) and cryptic (C) sites, have recognized consensus sequences only among cytomegalovirus (CMV) homologs and are located in the 28-kDa proteolytic portion of the precursor, called assemblin. I-site cleavage cuts assemblin in half without detected effect on its enzymatic behavior in vitro. To investigate the requirement for this cleavage during virus infection, we used the CMV-bacterial artificial chromosome system (E. M. Borst, G. Hahn, U. H. Koszinowski, and M. Messerle, J. Virol. 73:8320-8329, 1999) to construct a virus encoding a mutant I site (Ala143 to Val) intended to be blocked for cleavage. Characterizations of the resulting mutant (i) confirmed the presence of the mutation in the viral genome and the inability of the mutant virus to effect I-site cleavage in infected cells; (ii) determined that the mutation has no gross effect on the rate of virus production or on the amounts of extracellular virions, noninfectious enveloped particles (NIEPs), and dense bodies; (iii) established that assemblin and its cleavage products are present in NIEPs but are absent from CMV virions, an apparent difference from what is found for virions of herpes simplex virus; and (iv) showed that the 23-kDa protein product of C-site cleavage is more abundant in mutant virus-than in wild-type virus-infected cells and NIEPs. We conclude that the production of infectious CMV requires neither I-site cleavage of assemblin nor the presence of assemblin in the mature virion.

scholarly journals Chloroquine inhibits Zika Virus infection in different cellular models

2016 ◽  
Author(s):  
Rodrigo Delvecchio ◽  
Luiza M Higa ◽  
Paula Pezzuto ◽  
Ana Luiza Valadão ◽  
Patrícia P Garcez ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Zika Virus ◽  
Antiviral Agents ◽  
Virus Production ◽  
Congenital Defects ◽  
Zikv Infection ◽  
Cytotoxic Effects ◽  
Cellular Models ◽  
Infected Cells

SummaryZika virus (ZIKV) infectionin uteromight lead to microcephaly and other congenital defects. In adults, cases of Guillain-Barré syndrome and meningoencephalitis associated with ZIKV infection have been reported, and no specific therapy is available so far. There is urgency for the discovery of antiviral agents capable of inhibiting viral replication and its deleterious effects. Chloroquine is widely administered as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in VERO, human brain microvascular endothelial, and neural stem cells. We demonstratedin vitrothat chloroquine reduces the number of ZIKV-infected cells, virus production and cell death promoted by ZIKV infection without cytotoxic effects. Our results suggest that chloroquine is a promising candidate for ZIKV clinical trials, since it is already approved for clinical use and can be safely administered to pregnant woman.

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