*Article* **Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase**

**Preeti Hooda 1,† , Mohd Ishtikhar 1,†, Shweta Saraswat <sup>1</sup> , Pooja Bhatia <sup>1</sup> , Deepali Mishra <sup>1</sup> , Aditya Trivedi <sup>1</sup> , Rajkumar Kulandaisamy <sup>2</sup> , Soumya Aggarwal <sup>3</sup> , Manoj Munde <sup>3</sup> , Nemat Ali <sup>4</sup> , Abdullah F. AlAsmari <sup>4</sup> , Mohd A. Rauf <sup>5</sup> , Krishna K. Inampudi 2,\* and Deepak Sehgal 1,\***


**Abstract:** Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 50 -end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33–353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg2+) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg2+ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity.

**Keywords:** hepatitis E virus; methyltransferase; fluorescence quenching; protein–ligand interaction; protein stability; enzyme assay

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