Controversies’ clarification regarding ribavirin efficacy in measles and coronaviruses: Comprehensive therapeutic approach strictly tailored to COVID-19 disease stages

Table 2 Ribavirin multimodal mechanisms of action with distinct examples

Type	Mechanism
Direct
1	Inhibition of RdRp-RNA synthesis
	Direct interaction with RTP. RTP has been reported to competitively inhibit the influenza virus RNA polymerase, with respect to ATP and GTP, whereas RMP has no such observable effect[32]; however, for HCV, there are conflicting data regarding the impact of RBV on the RdRp, with reports of both no direct inhibition[33] as well as observations that RBV-containing RNA templates can cause a significant blockage of RNA elongation[34]
	Shown for Influenza A, La Crosse virus and for the key HIV polymerase, reverse transcriptase[24,32,35]
	Influenza virus: For cells treated with either RBV or methotrexate (a purine synthesis inhibitor that decreases intracellular concentrations of purines), the loss of polymerase activity at low concentrations of nucleotide is the culprit[36]
	Hantaan virus: The observed increase in RBV-5’-triphosphate supports the direct interaction and inhibition of the virus RdRp[37,38]
2	Increasing viral mutation rates through the misincorporation of RBV into the genome, leading to population extinction[39]
	RBV triphosphate is incorporated into the viral RNA by poliovirus polymerase, where it templates cytidine and uridine equally efficiently[40]. It has been suggested that RBV enhances viral mutagenesis, leading to error catastrophe by incorrect substitution of RTP for GTP[34,40,41] into viral RNA as most viral RdRps lack proofreading capability; although, this mechanism has been disputed for some viruses[42]. For example, for poliovirus, a 9.7-fold increase in mutagenesis following RBV treatment resulted in 99.3% loss in infectivity[40]. RTP is incorporated into the viral RNA by poliovirus by poliovirus polymerase, where it templates cytidine and uridine equally efficiently[40]. It has been suggested that RBV enhances viral mutagenesis, leading to error catastrophe by incorrect substitution of RTP for GTP[34,40,41] into viral RNA as most viral RdRps lack proofreading capability; although, this mechanism has been disputed for some viruses[42]. For example, for poliovirus, a 9.7-fold increase in mutagenesis following RBV treatment resulted in 99.3% loss in infectivity[40]. RTP is incorporated into the viral RNA by poliovirus polymerase, where it is mutagenic, since it templates cytidine and uridine equally efficiently[9]. Unlike GTP, RTP has ambiguous base-pairing capacity and can form two hydrogen bonds with uridine triphosphate or cytidine triphosphate with equal efficiency[40], leading to a subsequent increase in G-to-A and C-to-U single nucleotide variations throughout the entire HCV open reading frame[43]. Recent in vivo studies indicate that similar RBV-induced mutagenesis occurs in HEV[44]
	HCV: Initial studies showed no mutagenic effects, while in later results a mutagenic activity was indeed observed[45-47]
	LCMV: Along with the inhibition of IMPDH enzyme, a mutagenic activity also occurs[27]
3	Interference with formation of the 5’ cap structure of viral mRNA (capping activity)
	This is probably due to competitive inhibition of both guanyltransferase and methyltransferase capping enzymes
	mRNAs contain extensive modification on the 5´ end (known as the “five prime cap”), often utilizing guanine which is methylated in the 7-position, as this is essential for the stability and efficient translation of mRNA[39]. Thus, RNA capping has major secondary impact on the translation of both viral and host cell mRNAs. Interestingly, RTP reportedly acts as a competitive inhibitor for the capping of mRNAs, subsequently leading to impaired translation[48] by forming a covalent RMP-capping enzyme intermediate in place of the normally observed GMP-enzyme intermediate[49]
	Thus, virus which do not form capped mRNA are relative insensitive to RBV
	Mutants of Sindbis virus with an altered guanyltransferase demonstrate acquired resistance to RBV[50]
Indirect
1	Inhibition of IMPDH by RBV-5’-monophosphate
	RBV is a structural analogue of guanosine and acts as a potent competitive inhibitor of the enzyme IMPDH by RMP, leading to reduced GMP biosynthesis by diminution of the conversion of inosine monophosphate to XMP and resulting in the depletion of intracellular GTP[51]; this process is reversible in vitro by the addition of exogenous guanosine[52]. XMP can then be aminated to GMP by the GMP synthase enzyme. GMP is further converted to guanine metabolites, such as GTP and dGTP, essential precursors for RNA and DNA synthesis, respectively. This inhibition of IMPDH may occur even at relatively low RBV concentrations (10 μmol/L)[53] and leads to marked changes in the balance of the GTP pool in cells, with subsequent major impact on the host cell and viral gene expression as well as on viral replication[54-58]. This effect may be reversible in vitro by the addition of exogenous guanosine[59]
	HEV replication in vitro: MPA (an IMPDH inhibitor) has the same antiviral effect as RBV, which can be neutralized by the addition of guanosine[60]
	HCV: RBV acts through the inhibition of IMPDH, since the addition of guanosine negates this effect[45]
	LCMV: Inhibition is also mediated through a decrease in GTP levels[27]
2	Immunomodulatory effects of RBV
	Initially, a possible effect on T-cell subset balance was suggested[40,41]. RBV was also shown to inhibit lymphocyte proliferation, possibly due to the depletion of GTP which is essential for proliferating T-cells[61,62]. In vitro, IL-2 and IL-4 production was affected at lower RBV concentrations than IFN-γ production, suggesting a differential effect on Th1 and Th2 lymphocytes[63]. RBV administration in mice infected with influenza virus significantly attenuated respiratory immune responses as well as secretory and total IgA mucosal responses[64]. Affects T-cell subset balance[61,62]. Ameliorates spontaneous autoimmune disease in mice[65]. Inhibits lymphocyte proliferation due to depletion of GTP, which is essential for proliferating T-cells[61,66]. Enhances Th-1 over Th-2 responses or up-regulates the IFN-stimulated response element[63,67]. Clinical HCV studies have demonstrated that RBV monotherapy down-regulates the expression of IFN-stimulated genes in addition to reducing systemic concentrations of liver enzymes[68], and similarly lowered systemic concentrations of IP-10 (also known as CXCL10) associated with successful therapeutic outcome[69] in spite of only modest impact on viral levels
	In an animal model[67] for acute and chronic liver disease induced by the first coronavirus, many years before SARS emerged, MHV-3 was examined. MHV-3 has been the main model in studies on coronavirus replication and pathogenesis. Resistance to MHV-3 is associated with predominant Th1 response, the production of IFNs, neutralizing antibodies, and cytotoxic T cells. Viral infection of macrophages leads to a marked inflammatory response, including sustained production of TNF, IL-1, and procoagulant Fg12 prothrombinase and is associated with a Th2 cellular immune response and production of non-neutralizing antibodies. In hepatocellular necrosis (viral, toxins, etc.) resident macrophages (Kupffer cells) are activated and release a number of inflammatory mediators, including TNF, IL-1, proteolytic and enzymes, and inactivation of these macrophages prevents hepatic necrosis. RBV has minimal inhibitory effects on replication of MHV-3 in vitro, even at high concentrations. However, at concentrations achievable in vivo, it almost totally inhibits the production of the proinflammatory mediators TNF, IL-1 and procoagulant activity in macrophages in vitro[67]. RBV diminishes IL-4 production both by the Th1/Th2 lines as well as by the MHV-3 specific Th2 cell line, while it has no effects on IFN-γ production by Th1 cells, thereby preventing the shift to a Th2 response. The beneficial effect of RBV may be related to its ability to markedly reduce macrophage activation, thereby inhibiting the production of proinflammatory mediators from virally-activated macrophages; in addition, it diminishes Th2 cytokine production, while preserving Th1 cytokine production. RBV activates p53 by stimulating the mTOR protein and promoting the interaction between mTOR and p53. Activated p53 stimulates the transcription of IFN regulatory factor 9 and subsequently enhances IFN signaling (plasmids of lentiviruses that express either scrambled sequence or short-hairpin RNA against mTOR[70]. Furthermore, RBV-induced activation of mTOR and p53 enhances IFN-dependent signaling for the IFN-α/RBV combination treatment[71]. RBV stimulates the ERK1/2 pathway and subsequently promotes p53 activity, which at least partly contributes to the enhanced antiviral response of IFN-α plus RBV against HCV[71]. Regarding which is the predominant RBV antiviral mechanism, it is likely that for most viruses, RBV does indeed exert pleiotropic effects. However, a trend in the last decade has been to explore mutagenesis as the primary antiviral effect against RNA viruses, while clinical studies could determine whether mutagenesis occurs in vivo and how to optimize this activity in a therapeutic context[26]

ATP: Adenosine triphosphate; dGTP: Deoxyguanosine triphosphate; GMP: Guanosine monophosphate; GTP: Guanosine triphosphate; HCV: Hepatitis C virus; HEV: Hepatitis E virus; HIV: Human immunodeficiency virus; IFN: Interferon; IL: Interleukin; IMPDH: Inosine monophosphate dehydrogenase; IP-10: IFN-γ inducible protein 10; MHV-3: Mouse hepatitis virus strain-3; MPA: Mycophenolic acid; RBV: Ribavirin; RdRp: RNA-dependent RNA polymerase.