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

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

Seth M Schneider et al. mBio.

Abstract

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.

Keywords: antiviral agents; bortezomib; herpes simplex virus; human herpesviruses; proteasome.

Figures

FIG 1
FIG 1
Chemical structure of the proteasome inhibitor bortezomib (C19H25BN4O4). Bortezomib [N-(2,3-pyrazine) carbonyl-l-phenylalanine-l-leucine boronic acid] inhibits the proteasome via binding of its boron atom (red) to the chymotrypsin-like active site of the proteasome. The figure was drawn with PubChem Sketcher (103).
FIG 2
FIG 2
Bortezomib inhibits HSV infection. The indicated strains of HSV were added to (A) Vero cells (MOI of 0.004) or (B) HFF cells (MOI of 0.004) in the presence of increasing concentrations of bortezomib. At 18 to 24 h p.i., cells were fixed and assayed for HSV plaque formation. Plaque reduction is indicated as inhibition represented as a percentage of PFU obtained in the absence of drug. EC50 values for each virus as shown were calculated using GraphPad Prism software and range from 3.7 to 50.6 nM. Data are presented as graphed representatives of results from at least three experiments for each strain. Error bars, standard deviations (SD). EC50 data are presented as means ± standard errors of the means (SEM).
FIG 3
FIG 3
Bortezomib exhibits low cytotoxicity at concentrations effective against HSV infection. Bortezomib was added to Vero cells. At 24 h, LDH activity in the supernatant was assayed as a measure of cytotoxicity. Values are shown as percentages of detergent-lysed control values. Data presented are representative of results from at least three experiments. Error bars, SD.
FIG 4
FIG 4
Bortezomib is effective when added prior to 3 h p.i. HSV-1 KOS was added to Vero cells (MOI of 0.001). At the indicated times p.i., 200 nM bortezomib was added. At 18 to 24 h p.i., plaques were enumerated. The values representing mock-treated samples were set to 100%. Data presented are representative of results from three experiments. Error bars, SEM.
FIG 5
FIG 5
Bortezomib does not exhibit virucidal activity. HSV-1 KOS virions were treated with 100 nM bortezomib at 37°C for 1 h. Bortezomib was diluted to reach noninhibitory concentrations, and titers were determined on Vero cells. Data presented are representative of results from three experiments. Error bars, SEM. ns, not significant (compared to no-drug treatment).
FIG 6
FIG 6
Bortezomib does not affect HSV attachment to cells but inhibits transport of the entering capsid to the nucleus. (A) HSV-1 KOS was added to Vero cells (40 genome copies/cell) in the presence of DMSO control (No drug), 500 nM bortezomib, or 2 µg/ml heparin control. Samples were subjected to spinoculation at 200 × g at 4°C for 1 h. After three washes, cell-associated HSV levels were determined by qPCR. Data presented represent means of results from three experiments. Error bars, SEM; ns, not significant; *, P value of <0.05 (compared to no drug). (B to D) HSV-1 K26GFP was added to Vero cells on coverslips in the presence of (B) DMSO control or (C) 100 nM bortezomib or (D) 500 nM bortezomib for 2.5 h. Cells were fixed and stained with DAPI nuclear stain and visualized. Data presented are representative of results from at least two experiments.
FIG 7
FIG 7
Bortezomib prevents virus-induced ND10 disruption. Vero cells were pretreated with (A to D) DMSO control or (E to H) 200 nM bortezomib or (I to L) 500 nM bortezomib for 15 to 18 min at 37°C. HSV-1 KOS was added to Vero cells (MOI of ∼0.8) for 6 h at 37°C in the continued presence of agent. Cells were fixed, permeabilized, and stained for PML and ICP4. Panels D, H, and L represent zoomed-out views to show more of the surrounding cells. The ICP4 staining results were consistent with ICP4 expressed by the infected cell (37). Data presented are representative of results from three experiments.
FIG 8
FIG 8
Bortezomib and acyclovir act synergistically to inhibit HSV infection. HSV-1 KOS was added to Vero cells (MOI of 0.1) in the presence of various combinations of acyclovir and bortezomib. At 24 h p.i., cells were fixed, and titers were determined on Vero cells. (A) 3D graph depicting viral titers at the various drug combinations. (B) Isobologram depicting synergistic profiles of bortezomib and acyclovir. “Fa” (fraction affected) refers to fraction inhibition. Each colored line depicts a certain level of fraction inhibition, with endpoints signifying the amount of each drug alone needed to achieve that amount of inhibition. Colored symbols signify how much of each of the two drugs working together is needed to achieve the same inhibition. Symbols below the respective colored lines indicate synergism, those on or near the respective lines indicate additivity, and those above the respective lines indicate antagonism. Data presented are representative of results from three experiments. (C) Software-determined CI values at the specified fractions affected (Fa). Here, “Fa” refers to inhibition of HSV-1 plaque formation (fraction of control). Data are presented as means of results from three experiments ± SEM.

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