**1. Introduction**

Human immunodeficiency virus (HIV-1) and hepatitis C virus (HCV) infections continue to be a healthcare challenge globally, accounting for an enormous disease burden [1–3]. An effective vaccine against these viruses remains to be developed. Within the past decade, advancement in the development of anti-viral regimens has improved the situation, particularly in controlling HCV infections [4,5]. However, the outcome of antiviral therapies could be limited by several factors, including the possible emergence of drug-resistant viral variants. This scenario, therefore, signifies the continuous efforts towards the development of new anti-viral therapies or preventive measures.

The common feature between HIV-1 and HCV is the presence of highly glycosylated outer envelope—the envelope glycoprotein 120 (gp120) of HIV-1 and E2 of HCV exhibits over 20 and 11

*N*-glycosylation sites, respectively [6–8]. This glycan shield decorating the surface of viruses has been exploited as a potential target for therapeutic or preventive interventions. In recent years, carbohydrate-binding agents, in particular, lectins have been identified which can inhibit cellular entry of the viruses by specifically binding to these viral surface glycans [9–12]. Given their potent nature, use of these lectins as topical microbicides has been suggested for the prevention of sexual transmission of HIV-1 [13–15]. As an example, griffithsin, a lectin from red algae, is currently in phase-1 clinical trials [16]. Two of the major challenges hampering the clinical application of these lectins are their potential cytotoxicity and immunogenicity. In general, chemical and structural heterogeneity of proteins is one of the primary factors responsible for their immunogenicity.

Microvirin (MVN) is one of the potent anti-HIV lectins, which was initially isolated from *Microcystis aeruginosa* and has been shown to have only minor cytotoxicity and mitogenic effects as compared to other antiviral lectins [17,18]. MVN has been reported to specifically recognize α(1-2)mannobiose present at the termini of branched high mannose type glycans on the viral surface. This 12 kDa lectin consists of two structural domains, which share 35% sequence identity, and unlike other anti-viral lectins, it exists as a monomer (Figure 1a). Moreover, there is a four residues long insertion in domain-A as compared to domain-B of MVN [19]. In this study, we engineered an MVN variant, LUMS1 (the name derived from Lahore University of Management Sciences), exhibiting 100% sequence identity between its two structural domains, thereby markedly decreasing the chemical heterogeneity. We investigated this protein for its potential to inhibit cellular entry of HIV and HCV, and studied its cytotoxicity, carbohydrate specificity, and preliminary effects on the activation of immune cell surface markers.

**Figure 1.** Description of the protein design: (**a**) microvirin (MVN) structure (PDB ID 2YHH) shown in cartoon presentation with two structural domains colored blue and green while bound glycan is colored yellow. Insertion of four amino acids in domain-A as compared to domain-B is indicated in magenta. The second putative carbohydrate binding site is indicated by a dotted circle. (**b**) The homology-modeled structure of LUMS1 was created through SWISS-MODEL online tools using MVN as a template. Qualitative model energy analysis (QMEAN) scoring function was used to access the quality of the model. Side chains of all cysteine residues in both proteins are shown in gold sticks. Alignment of amino acid sequence of two domains of MVN and LUMS1 is shown at the bottom of the respective protein structure. N, C indicates N- and C-termini of the protein sequences.
