MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway

Abstract

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotidereleasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection. Herpes simplex virus 1 (HSV-1) is a member of the Alphaherpesvirinae subfamily and a human DNA virus that is known to cause a number of clinical manifestations, including cold sores, keratitis, meningitis and encephalitis 1,2. HSV-1 can establish latent infections in sensory neurons and periodically reactivate at the original site of infection, resulting in lesions 3. During latent infection, the HSV genome circularizes to form an episome in the nucleus, leading to expression of latency-associated transcripts (LATs) that are thought to be necessary for latency and reactivation. Upon reactivation, lytic-related genes are expressed in a temporal and sequential manner, which can be divided into three transcriptional stages: immediate early (IE/α), early (E/β), and late (L/γ). Some IE products function as triggers for transcriptional activation of E genes associated with viral DNA replication. L genes encode structural and functional proteins for producing viral progeny. Although acyclovir (ACV) and its analogues have been the standard therapy for HSV infection, their widespread and long-term use has recently led to the emergence of drug-resistant HSV strains 4-6. Thus, due to a lack of effective vaccines, side effects associated with ACV, such as nephrotoxicity, and appearance of ACV-resistant strains, new anti-HSV compounds with mechanisms of inhibition distinct from ACV are urgently needed for the treatment of HSV infection 7. HSV infection alters several signaling pathways, which can be triggered by viral molecules known as pathogen assoc...

Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro/3CLpro) and Human Cathepsin-L

“…MG-132 antiviral activity was also observed for other viruses, such as Herpes simplex virus 1 [15], hepatitis E [16], human cytomegalovirus [17], porcine circovirus type 2 [18], and coxsackievirus B3 [19]. In all these cases the control of viral cellentry was directly related to the interference of the ubiquitin-proteasome system rather than direct inhibition of protease-driven viral replication [15][16][17][18]. A different mechanism was observed for the Hendra virus, a representative of the paramyxoviruses, where inhibition of infection was dependent on proteolytic processing of paramyxovirus fusion proteins that are essential for virus entry.…”

“…The effect on viral infection was mainly linked with the inhibition of the host protease, calpain-m [14], and not due to proteasome or autophagy impairment. MG-132 antiviral activity was also observed for other viruses, such as Herpes simplex virus 1 [15], hepatitis E [16], human cytomegalovirus [17], porcine circovirus type 2 [18], and coxsackievirus B3 [19]. In all these cases the control of viral cellentry was directly related to the interference of the ubiquitin-proteasome system rather than direct inhibition of protease-driven viral replication [15][16][17][18].…”

Viral entry and the ubiquitin‐proteasome system

“…The Ras–Raf–MEK–ERK signalling pathway might play a role in proteasome‐dependent entry of HSV. HSV infection induces the ubiquitination of Ras‐GRF2 as well as its degradation (Ishimaru et al, 2020). A connection between this pathway and viral nucleocapsid transport and any role for ICP0‐mediated degradation remains to be determined.…”

Pax5 mediates the transcriptional activation of the CD81 gene

“…B-lymphoma cell lines (Ramos, BJAB, Raji, Akata, MutuIII, and BC3) were maintained in RPMI 1640 medium supplemented with 10% FBS. Ramos, BJAB, Raji, Akata, MutuIII, SW480, and BC3 were kindly provided by Dr. S. D. Hayward (Johns Hopkins University School of Medicine, Baltimore, Maryland, USA) 38 , 39 . HeLa and 293T were provided by the RIKEN BioResource Research Center.…”

“…The sample was boiled for 5 min and sonicated for 30 s with a probe type sonicator in order to shear the chromosomal DNA. The resulting lysate was subjected to SDS-PAGE on 8 or 12% polyacrylamide gel followed by Western blotting 38 , 39 . The proteins were transferred onto a ClearTrans® nitrocellulose membrane (Wako, Osaka, Japan), and the membrane was incubated with 3% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-T) for 1 h at room temperature.…”