Understanding viral polyproteins: insights from studies on human immunodeficiency virus type 1 and severe acute respiratory syndrome coronavirus 2
Creators: name: Yadav, Ruchi
Publisher: name: No Publisher Supplied
Year: 2024

description: In recent decades, there has been a significant increase in viral epidemics and pandemics. From the global acquired immunodeficiency syndrome (AIDS) epidemic in the 1980s to the recent coronavirus disease 2019 (COVID-19) pandemic, both have caused thousands of deaths worldwide. These deadly viruses are evolving continuously despite the availability of FDA-approved direct antivirals for both HIV-1 and SARS-CoV-2, as well as successful vaccines for SARS-CoV-2. This constant viral evolution poses a significant challenge to scientists as they must deal with obstacles such as drug-resistance mutations or mutations to evade (natural or vaccine-mediated) immunity. Therefore, a comprehensive understanding of every aspect of the viral replication cycle is important for developing more effective treatments and potential cures.Human immunodeficiency virus 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS), while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19). They are classified as retroviruses and RNA viruses, respectively, and both utilize a “polyprotein strategy,” where the viral genome is translated into long precursor polyprotein(s) that undergo cleavage to produce essential viral proteins. This dissertation is dedicated to improving the understanding of viral polyproteins, particularly those from HIV-1 and SARS-CoV-2.As of 2023, ~40 million people globally are living with HIV-1. While highly active antiretroviral therapy (HAART) has revolutionized HIV-1 treatment, challenges such as drug-resistance mutations, adherence to daily regimens, and reinfections exist. An in-depth knowledge of all the stages of the HIV-1 replication cycle is necessary to develop alternative strategies. HIV-1 polyproteins, specifically Gag and Gag-Pol, play an important role in the assembly and maturation stages of the replication cycle. Despite 40 years of extensive research on HIV-1, there is limited understanding of the Gag-Pol polyprotein, primarily due to the lack of suitable heterologous systems to produce this protein. This dissertation details protocols developed for producing soluble Gag-Pol in bacterial cells with sufficient yields to perform structural and biophysical studies. The single-particle cryo-EM structure of Gag-Pol showed a dimeric organization with a heterodimeric reverse transcriptase (RT) core. The structure analysis also showed low resolution density corresponding to PR which has provided insights into the role of RT in PR dimerization during maturation. HIV-1 assembly has been widely studied using Gag virus-like particles (VLPs) as a model system. Even though the Gag VLPs have significantly improved our current knowledge of the assembly process, they are incomplete mimics of the actual virus particles due to the absence of Gag-Pol in them. This dissertation has attempted to fill this gap by assembling VLPs in vitro containing both Gag and Gag-Pol polyproteins. These Gag:Gag-Pol VLPs may provide effective model systems for understanding the intricate mechanisms of the assembly and maturation process. During the COVID-19 pandemic, SARS-CoV-2 has infected hundreds of millions and caused millions of deaths throughout the world. Polyprotein processing is a crucial process for viral replication. However, it is a poorly understood stage of the virus replication cycle. This dissertation has provided structural and biochemical insights into the processing of SARS-CoV-2 nsp7-11 and nsp7-8 polyprotein intermediates, whose mature products are key components of the replication-transcription complex (RTC). The integrative structural models of nsp7-11 and nsp7-8 were developed using data from cross-linking mass spectroscopy (XL-MS), hydrogen-deuterium exchange (HDX) MS, and small-angle X-ray scattering (SAXS) techniques. These models helped us understand the role of cleavage junction conformation and accessibility in determining the processing order. Additionally, this dissertation has also provided insights into two possible binding pathways by revealing the footprint of the polyprotein binding on Mpro. Finally, a proteolysis assay using the nsp7-11 polyprotein as substrate—benchmarked with nirmatrelvir—was used to characterize the effect of small molecule binders on Mpro-mediated processing. The results have provided insights into the allosteric regulation of Mpro activity. In summary, these structural and biochemical insights into SARS-CoV-2 polyproteins may help in understanding their role in the viral life cycle and provide a basis for targeting them using structure-based drug design and discovery.SARS-CoV-2 nsp7 and nsp8 are essential cofactors of nsp12, which together form the viral RNA-dependent RNA polymerase or RdRp. This dissertation describes the expression and purification protocols of these two important viral proteins. XL-MS and HDX-MS techniques have been utilized to understand the dynamics of full-length nsp7:nsp8 heterodimer formation. These results have provided insights into the pathways of RTC formation which is required for the gene replication. Lastly, this dissertation summarizes the polyprotein processing stage in the two viruses – HIV-1 and SARS-CoV-2. It combines the results from the above chapters to emphasize the pathways involved in polyprotein processing and the implications for antiviral drug discovery.