**1. Introduction**

Hepatitis E virus (HEV) is inherently hepatotropic, causing acute hepatitis and chronic infection in immunocompromised patients, as well as leading to extrahepatic manifestations in some patients [1–4]. Globally, hepatitis E accounts for an estimated mortality rate of ~3.3% in the infected population and causes fulminant hepatitis failure in 25–30% of infected pregnant women [5]. HEV has a genome of ~7.2 kb plus-stranded RNA with a 5 0 -methylguanine (m7G) cap accorded by guanylyltransferase (GTase) and methyltransferase (MTase). The RNA capping is essential for the viruses to evade the host immune system and produce other viral proteins by protecting the viral mRNA from nucleases.

**Citation:** Hooda, P.; Ishtikhar, M.; Saraswat, S.; Bhatia, P.; Mishra, D.; Trivedi, A.; Kulandaisamy, R.; Aggarwal, S.; Munde, M.; Ali, N.; et al. Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase. *Molecules* **2022**, *27*, 1505. https:// doi.org/10.3390/molecules27051505

Academic Editor: Luigi A. Agrofoglio

Received: 28 December 2021 Accepted: 18 February 2022 Published: 23 February 2022

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In the case of HEV, the 5<sup>0</sup> m7G cap has been demonstrated to play an additional role, increasing infectivity in non-human primates and cultured hepatoma cells [6,7]. Despite being an important enzyme, few studies have examined the functional and structural aspects of the HEV MTase. Magden et al. 2001 expressed a 110 kDa protein from the 1–979 amino acid region of HEV cDNA and demonstrated both the activities of MTase and GTase [8]. Before that, using computational homology modelling, Koonin et al. derived that the MTase domain lay in the region of aa 56–240 of the HEV genome [9]. Through bioinformatic studies, Emerson et al. predicted that the MTase lay in the region of aa 33–353 of HEV genotype 1 [6]. This fragment was further preferred since it formed a section of the MTase domain of aa 1–979 expressed by Magden and overlapping the region of amino acids 56–240, computationally predicted to be MTase by Koonin. Howver, expressing the protein containing this predicted fragment and validating the MTase activity is an unexplored area.

Other viruses in which capping is essential for MTase activity include the Dengue virus [10], coronaviruses [11], and flaviviruses [12]. MTase is an integral enzyme required for capping, which is dependent on magnesium (Mg2+) as a cofactor for its activity [13]. The dependence of MTase on divalent cations for its activity has been observed in the case of many viruses, such as the Zika virus [14] and respiratory syncytial virus (RSV) [15]. Many viruses, such as the Chlorella virus and hepatitis C virus (HCV), coronaviruses, and flaviviruses, also need Mg2+ for the activity of viral enzymes [16–20]. Alteration of the activity may be due to conformational changes resulting from the binding of magnesium to the enzyme. Following this hypothesis, the role of Mg2+ in the MTase activity of coronaviruses has been attributed to the conformational changes in the nsp10/nsp16 enzyme complex for 20O-methylation [21]. The binding is postulated to induce the change in the structural, conformational, and interactional properties of MTase. As seen in the Dengue virus, Mg2+ stabilises the RNA cap by coordinating with the inverted triphosphate moiety from the solvent-exposed side of the RNA cap [22].

Briefly, in the present study, we have reported and expressed the HEV MTase from the predicted domain of the 33–353 amino acid fragment of HEV genotype 1. The molecular weight of the enzyme with MTase activity was found to be 37 kDa. The enzyme activity was shown to be associated with Mg2+ using biochemical and biophysical studies.

Our work suggested that HEV MTase requires Mg2+ for its activity, and future studies should help to establish the direct relationship between RNA capping and host cellular metal ions. Further, the identification of drug-like inhibitors that are structurally compatible with the domains responsible for the enzyme activity could be performed. Using this information, the co-crystallisation of MTase with magnesium can be performed, to elucidate the exact binding pocket of Mg2+ and hence design structurally compatible inhibitors.
