Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

Piret, Jocelyne; Goyette, Nathalie; Boivin, Guy

doi:10.1128/jcm.03274-15

Cited by 30 publications

(17 citation statements)

References 40 publications

(43 reference statements)

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“…In the case of LMV, the EC 50 value of the first‐step luciferase activity could not be determined. The EC 50 value of second‐step luciferase activity was similar to those of the virus yield and previously reported values . These results indicate that the two‐step culture protocol enhances detection of anti‐HCMV compounds.…”

Section: Resultssupporting

confidence: 87%

A two‐step culture method utilizing secreted luciferase recombinant virus for detection of anti‐cytomegalovirus compounds

Koshizuka

Sato

Kobiyama

et al. 2018

Microbiology and Immunology

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Quantification of human cytomegalovirus (HCMV) replication by plaque assay reflects viral infectivity but has several drawbacks. Recombinant virus expressing reporter genes can facilitate quantification of HCMV replication. In this study, a recombinant virus, Towne-BAC(dTT)-MetLuc, was constructed and the secretable Metridia luciferase (MetLuc) gene inserted into it under UL146 promoter. In addition, the UL130 gene was repaired to allow growth of the recombinant virus in both fibroblasts and epithelial cells. As predicted, luciferase activity was secreted into the culture medium and correlated with virus replication in infected fibroblasts and epithelial cells. Furthermore, secreted luciferase activity was correlated with the size of the recombinant virus inoculum with a dynamic range of 3 logs. This recombinant virus was used in a two-step culture protocol for detection of the anti-HCMV activity of compounds; that is, the supernatant of a first-step culture with anti-viral compounds was collected and inoculated into uninfected cells to create a second-step culture. Although secreted luciferase activity leaked in the first-step culture supernatant in the presence of some antiviral compounds, luciferase activity in the second-step culture supernatant reflected the virus yield in the first-step culture. Therefore, comparison of luciferase activity in the first- and second-step cultures indicated the anti-viral activity of the compounds. This two-step culture protocol facilitates screening of anti-viral compounds.

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Section: Resultssupporting

confidence: 87%

A two‐step culture method utilizing secreted luciferase recombinant virus for detection of anti‐cytomegalovirus compounds

Koshizuka

Sato

Kobiyama

et al. 2018

Microbiology and Immunology

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“…We were unable to reach 50% Vero cell cytotoxicity with concentrations as high as 1 mM. The foscarnet and cidofovir EC 50 values for HSV-1 on Vero cells are 32.6 µM and 6.4 µM, respectively (69, 70). Bortezomib yields ∼1,000-times-lower EC 50 values.…”

Section: Discussionmentioning

confidence: 77%

Early Steps in Herpes Simplex Virus Infection Blocked by a Proteasome Inhibitor

Schneider

Pritchard

Wudiri

et al. 2019

mBio

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Viruses commandeer host cell 26S proteasome activity to promote viral entry, gene expression, replication, assembly, and egress. Proteasomal degradation activity is critical for herpes simplex virus (HSV) infection. The proteasome inhibitor bortezomib (also known as Velcade and PS-341) is a clinically effective antineoplastic drug that is FDA approved for treatment of hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Low nanomolar concentrations of bortezomib inhibited infection by HSV-1, HSV-2, and acyclovir-resistant strains. Inhibition coincided with minimal cytotoxicity. Bortezomib did not affect attachment of HSV to cells or inactivate the virus directly. Bortezomib acted early in HSV infection by perturbing two distinct proteasome-dependent steps that occur within the initial hours of infection: the transport of incoming viral nucleocapsids to the nucleus and the virus-induced disruption of host nuclear domain 10 (ND10) structures. The combination of bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV infection. Thus, bortezomib is a novel potential therapeutic for HSV with a defined mechanism of action. IMPORTANCE Viruses usurp host cell functions to advance their replicative agenda. HSV relies on cellular proteasome activity for successful infection. Proteasome inhibitors, such as MG132, block HSV infection at multiple stages of the infectious cycle. Targeting host cell processes for antiviral intervention is an unconventional approach that might limit antiviral resistance. Here we demonstrated that the proteasome inhibitor bortezomib, which is a clinically effective cancer drug, has the in vitro features of a promising anti-HSV therapeutic. Bortezomib inhibited HSV infection during the first hours of infection at nanomolar concentrations that were minimally cytotoxic. The mechanism of bortezomib’s inhibition of early HSV infection was to halt nucleocapsid transport to the nucleus and to stabilize the ND10 cellular defense complex. Bortezomib and acyclovir acted synergistically to inhibit HSV infection. Overall, we present evidence for the repurposing of bortezomib as a novel antiherpesviral agent and describe specific mechanisms of action.

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“…Both compounds were used at low/non cytotoxic concentration (i.e. 100 nM c-exNDI and 3 μM ACV) corresponding to 5 times their IC 50 values 34 , 35 . Cells infected with HSV-1 [V41] and treated with c-exNDI or ACV at 24 h.p.i were monitored for their GFP fluorescence.…”

Section: Resultsmentioning

confidence: 99%

A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication

Callegaro

Perrone

Scalabrin

et al. 2017

Sci Rep

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G-quadruplexes (G4s) are nucleic acids secondary structures, epigenetic regulators in cells and viruses. In herpes simplex virus 1 (HSV-1)-infected cells, G4s are massively present during viral replication. We here aimed at investigating the possibility to target the HSV-1 G4s by a core extended naphtalene diimide (c-exNDI) G4 ligand. Biophysical and biomolecular analysis proved that c-exNDI stabilized the HSV-1 G4s in a concentration dependent manner. In MS competition assays, c-exNDI preferentially recognized HSV-1 G4s over cellular telomeric G4s, the most represented G4s within cells; other less abundant cellular G4s were also recognized. Treatment of HSV-1 infected cells with c-exNDI at low nanomolar concentrations induced significant virus inhibition with no cytotoxicity. The mechanism of action was ascribed to G4-mediated inhibition of viral DNA replication, with consequent impairment of viral genes transcription. Our data suggest that the observed potent antiviral activity and low cytotoxicity mainly depend on a combination of c-exNDI affinity for HSV-1 G4s and their massive presence during infection. HSV-1 G4s may thus represent new effective antiviral targets: the fact that no current antiherpetic drug exploits them and their presence at the viral genome, responsible for both active and latent HSV infections, makes them particularly attracting.

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Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

Cited by 30 publications

References 40 publications

A two‐step culture method utilizing secreted luciferase recombinant virus for detection of anti‐cytomegalovirus compounds

A two‐step culture method utilizing secreted luciferase recombinant virus for detection of anti‐cytomegalovirus compounds

Early Steps in Herpes Simplex Virus Infection Blocked by a Proteasome Inhibitor

A core extended naphtalene diimide G-quadruplex ligand potently inhibits herpes simplex virus 1 replication

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