Viruses modulate and utilize many host cellular processes for their replication. Virus infection can result in changes in cellular metabolism and signaling that facilitate viral replication. These processes involve different molecules, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs). NF-κB is a DNA-binding protein that is required in the transcription of various genes involved in controlling cellular processes, in particular, inflammatory and immune responses. The modulation of NF-κB pathway by BBR is one of the mechanisms that inhibits virus infection. BBR downregulates virus-induced NF-κB activation and blocks degradation of the endogenous NF-κB inhibitor IκBα. MAPKs are involved in the regulation of key cellular signaling pathways, such as apoptosis, differentiation, proliferation, and immune responses. MAPKs play an essential role in infection and cellular stress. Moreover, they are known to promote survival and generation of progeny virions. Four subgroups of MAPKs have been identified, namely, extracellular-signal-regulated kinase (ERK) 1 and 2, ERK5, isoforms of p38 protein (p38), and c-Jun N-terminal kinases (JNK) [
16]. The JNK and p38 pathways play a key role in inflammation and tissue homeostasis. MAPKs are responsible for the phosphorylation of serine and threonine in many proteins. Phosphorylation plays a significant role in protein interactions, protein folding, and activation and deactivation of enzymes. ERK kinases phosphorylate multiple substrates, such as c-Fos and Elk 1, which are involved in the regulation of cell cycle progression and survival. Protein phosphorylation also plays an essential role in the infection cycle of many viruses [
17]. A number of viruses induce or inhibit the phosphorylation of cellular proteins at all levels of signal transmission pathways from the plasma membrane to the nucleus. Phosphorylation can affect a viral protein’s stability, activity, interaction with other cellular and viral proteins, and infectivity. Viruses such as Epstein-Barr virus (EBV) [
18], hepatitis C virus (HCV) [
19], and coronavirus type 2 [
20] activate the phosphorylation of MAPK. Changes in the phosphorylation of proteins have been observed during viral replication. For example, the human immunodeficiency virus (HIV) protein p6 is phosphorylated at a specific site (Thr 23) by MAPK and ERK-2. Mutational analysis has demonstrated that Thr 23 is important for the infectivity, maturation, and budding of viral particles [
21]. Phosphorylation of the coat protein (CP) of RNA viruses can significantly affect CP-RNA interactions, the stability of viral particles, and the viral infection processes [
22,
23]. Moreover, many viruses use phosphorylation of proteins to modulate signaling in order to prevent apoptosis and promote cellular survival and proliferation [
24].
An example is chikungunya virus (CHIKV). It is transmitted to humans mainly by infected mosquitoes, specifically
Aedes aegypti and
Aedes albopictus. CHIKV causes fever, joint swelling, headache, and severe persistent muscle and joint pain in humans. CHIKV belongs to the family
Togaviridae and has a positive-sense RNA genome. Host macrophages are the major cellular reservoirs of CHIKV during infection, and the production of the cytokines TNF, IL-1β, and IL-6, IL-8 may be associated with viral pathogenesis. During CHIKV infection, the major mitogen-activated protein kinase signaling pathways, p38, JNK, and ERK are activated. Activation of ERK and JNK kinases are essential for generation of CHIKV progeny virions. The specific molecular mechanism of CHIKV-induced MAPKs is not known. It has been shown that CHIKV can modulate the phosphorylation of p38 and JNK. Recently, it was demonstrated that the CHIKV nsP2 protein interacts with p38 and JNK in host macrophages [
25]. In addition, JNK modulates the replication of certain viruses. For example, replication of HSV, HIV, and rotaviruses is suppressed by JNK inhibition, while the replication of influenza virus is increased. CHIKV induces MAPK activation as well as expression of viral nonstructural and structural proteins. Varghese et al. showed that BBR significantly reduces CHIKV-induced activation of MAPKs. The P38, ERK and JNK signaling pathways are strongly inhibited upon treatment with BBR, which especially targets the ERK signaling pathway, resulting in a marked reduction in particle formations. The reduction in viral protein expression after BBR treatment is probably a consequence of a decrease in virus-induced signaling. BBR treatment does not inhibit virus entry or the enzymatic activity of the viral replicase [
26]. Furthermore, CHIKV induces prosurvival signal cascades such as PI3K-AKT and autophagy [
27]. It has also been reported that BBR significantly reduces phosphorylation of p38 MAPK during respiratory syncytial virus (RSV) infection [
28]. p38 MAPK is induced in the early stage of RSV infection [
29]. Inhibition of the activity of p38 MAPK by BBR has also been observed during hepatitis B virus (HBV) infection. HBV is a member of the family
Hepadnaviridae. Its virion contains a partially double-stranded relaxed circular DNA (rcDNA) genome, which, in the nucleus of an infected cell, is converted to covalently closed circular DNA (cccDNA). p38 MAPK plays a central role in the maintenance of HBV cccDNA in infected cells [
30]. The cccDNA acts as a template for RNA synthesis, including mRNAs and pregenomic RNAs (pgRNAs). During the life cycle of HBV, pgRNA is converted to partially double-stranded rcDNA in the capsid by reverse transcriptase (RT). Inhibition of the activity of p38 MAPK is positively correlated with the suppression of HBV surface antigen (HBsAg) production, HBV e-antigen (HBeAg) secretion, and inhibition of HBV replication. It has been reported that BBR also intercalates into DNA, inhibiting DNA synthesis and reverse transcriptase activity [
31‐
34]. HBV infection is associated with a broad spectrum of liver diseases, including acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. About a million deaths globally are caused by HBV-related diseases each year, with more than a million people suffering from chronic hepatitis B worldwide [
35]. The ability of BBR to inhibit MAPK might make it a potential candidate for a new antiviral agent against HBV infection. p38 MAPK has been suggested as a possible target for anti-HBV therapy. Kim et al. showed that biphenyl amides act as p38 MAPK inhibitors and suppress HBsAg secretion [
36]. Curcumin, another well-known alkaloid, decreases the level of HBsAg and reduces the number of cccDNA copies, thus inhibiting HBV replication and expression. Moreover, this natural plant compound reduces the acetylation level of cccDNA-bound histone H3 and H4 [
37]. The current anti-HBV therapy options have disadvantages. These include high cost and cumulative toxicity, which results in bone disease and renal injury. Additionally, the therapy is limited to patients with viremia and elevated alanine aminotransferase (ALT) or fibrosis [
38], and the MAPK pathway appears to be activated by other viruses, such as HCV [
39], dengue virus [
40], coronavirus [
41], Venezuelan equine encephalitis virus (VEEV) [
42], and enterovirus 71 (EV71) [
43].
Another strategy that plays an important role in the replication of some viruses is autophagy. Autophagy is an intracellular degradation process that helps to maintain cellular homeostasis under both normal and stress conditions. It is a dynamic process starting with autophagosome formation, followed by fusion with lysosomes and degradation of the enclosed cargo. Autophagy is also an antiviral mechanism that selectively degrades viral components or viral particles inside lysosomes [
44]. One example of a virus that exploits autophagy is the enterovirus EV71, a member of the family
Picornaviridae. Picornaviruses are nonenveloped viruses with a positive-stranded RNA genome of 7.5 kb that contains a single open reading frame (ORF) flanked by 5’ and 3’ untranslated regions. This RNA serves as a template for translation of the viral polyprotein and for the amplification of the viral genome. Viral replication takes place on intracellular membranous structures and is dependent on autophagy. Enteroviruses induce autophagy to promote their own replication [
45]. The mechanism responsible for this pro-viral function of autophagy is unknown. EV disrupts autophagosome-lysosome fusion, which allows viral RNA and proteins to escape degradation and consequently facilitates viral replication by providing membranes for replication, leading to pathogenesis in the host. EVs are associated with neurological disorders, cardiovascular damage, and metabolic disease. Recently, it has been reported that BBR can inhibit virus-induced autophagy and reduce viral RNA and protein synthesis [
46]. BBR inhibits EV71-induced autophagy by affecting JNK, PI3KIII and AKT signaling. For example, BBR treatment increases AKT phosphorylation and reduces JNK and PI3KIII phosphorylation. JNK signaling is involved in autophagy, and its inhibition can inhibit this process. BBR also inhibits the MEK/ERK signaling pathway, which is important for mediating innate immunity to viral infections and plays a significant role in EV71 replication and pathogenesis [
43,
46]. This suggests that BBR acts by inhibiting MAPK signaling and is a potential agent for the treatment of enterovirus infection, especially since there are no other effective and licensed drugs available.