Post Date
Mar 7 2021

Engineering of HIV-1 and HCV Entry Inhibitor Lectins and Understanding the Atomic Level Details of HIV Cellular Entry Proces

Year
2020
Supervisor:
Dr. Syed Shahzad ul Hussan
Students:
Munazza Shahid
MS/PhD
PhD
Reference / Filters
Chemistry & Chemical Engineering
Abstract:
Human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) infections occur in millions of people all over the world. Due to the rapidly evolving nature of these viruses, drug resistant variants emerge quickly. Therefore, a continuous effort is required to develop new drugs and understand the infection to explore new drug targets. Envelopes of both of these viruses exhibit a dense array of glycans that could be exploited as a therapeutic target. In recent years, several lectins have been reported to inhibit these infections by targeting these glycans. However, none of these lectins have been developed into a drug owing to their potential cytotoxic and immunogenic effects. This study focuses on two associated objectives: 1. Engineering of viral entry inhibitor lectins with an aim to minimize or eradicate potential cytotoxicity and immunogenicity of the lectin. 2- Characterizing binding of the V3 domain of HIV-1 surface glycoprotein to its cellular receptor CCR5 to better understand the viral cellular entry process.

Microvirin (MVN) is a known anti-HIV lectin that consists of two structural domains with only one domain involved in binding to glycan epitopes on the surface of the virus. Generally, size and chemical heterogeneity are the two major contributing factors in immunogenicity of a protein. Considering these factors, we designed two types of MVN variants, one consisting of only the carbohydrate-binding domain with size almost half of the parent protein and the second consisting of two domains with identical amino acid sequence. We produced several mutants of each type of variants to optimize folding. Finally, we were able to engineer a variant that potently inhibited HIV and HCV in viral infectivity assays and showed significantly less cytotoxic and immunogenic effects as compared to MVN. We named this protein as LUMS1 that represents a potential drug candidate against HIV and HCV particularly co-infections of these viruses. In the second project, we engineered a soluble form of the CCR5 receptor by incorporating artificial linkers of amino acid sequences connecting the extracellular regions of the receptor in place of hydrophobic trans-membrane regions and optimized its functional conformation by in vitro folding. We also produced 13C15N-labelled V3 domain of HIV-1 glycoprotein by expressing its gene along with a fusion protein. We determined through SPR and NMR chemical shift perturbation experiments that only the cyclic form of V3 could bind to CCR5 with KD values of around 200 μM. Overall, in this study we successfully engineered a lectin that potently inhibits HIV-1 and HCV cellular entry, and with its extremely low cytotoxicity and negligible potential immunogenicity it represents a promising candidate for anti-viral drug development. Moreover, we obtained several key information related to the binding of the V3 domain to its cellular receptor.

Publications:

1.     Shahid, M.; Qadir, A.; Yang, J.; Ahmad, I.; Zahid, H.; Mirza, S.; Windisch, M. P.; Shahzad-Ul-Hussan, S., An Engineered Microvirin Variant with Identical Structural Domains Potently Inhibits Human Immunodeficiency Virus and Hepatitis C Virus Cellular Entry. Viruses 2020, 12, 1999-2011.

2.     Chantadul, V.; Wright, G. S.; Amporndanai, K.; Shahid, M.; Antonyuk, S. V.; Washbourn, G.; Rogers, M.; Roberts, N.; Pye, M.; O’Neill, P. M., Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy. Communications biology 2020, 3, 1-10.