NMR FACILITY AT CNPEM: A CASE STUDY USING EXOMETABOLOMICS TO INVESTIGATE VIRAL INFECTION

The Nuclear Magnetic Resonance (NMR) facility at CNPEM, operational since 2001, houses two spectrometers (500 MHz and 600 MHz equipped with a cryogenic probe) primarily dedicated to metabolomics and the structural and functional analysis of proteins and their interactions. In accordance with the institution’s policy to provide open access to instrumentation for the wider scientific community, the facility also offers expert support in sample preparation, experimental design, and data interpretation. Within this context, we present a case study employing NMR-based metabolomics to investigate metabolic alterations induced by Tupanvirus altamarinense (TPV) infection in Acanthamoeba castellanii. TPV is a giant virus infecting A. castellanii, characterized by a large genome (~1.5 megabases) encoding nearly 1300 proteins. Uncommonly for viruses, its genome includes genes involved in translation, DNA repair, and lipid metabolism—functions generally absent in most viral lineages. Despite its genetic complexity, the mechanisms by which TPV modulates host metabolism remain poorly understood. Thus, studying TPV offers critical insights into viral diversity and evolution, challenges conventional virus definitions, and provides novel perspectives on virus–host interactions, particularly among giant viruses.To investigate these metabolic impacts, a standardized protocol was developed for culturing A. castellanii, followed by TPV infection and collection of culture media for NMR analysis. Initially, A. castellanii were seeded at a density of 1 × 10⁷ cells per 10 cm Petri dish in PYG medium. Subsequently, cells were infected with TPV and incubated for 24 hours. Three independent experiments were performed, each comprising two biological replicates (n = 6). Culture media from both infected and non-infected samples were collected and filtered using 3 kDa centrifugal filters (Amicon Ultra – 0.5 mL). A volume of 300 µL filtrate was then mixed with phosphate buffer containing 0.5 mM TMSP and transferred into 5 mm NMR tubes. One-dimensional ¹H NMR spectra were acquired on a Bruker NeoAdvance 600 MHz spectrometer equipped with a cryoprobe. Spectral processing and metabolite identification were conducted using Chenomx software (v12.0; available to facility users). A total of 42 metabolites were identified. Baseline metabolic profiling revealed uptake of eight metabolites from the culture medium (adenosine, inosine, lactate, methionine, sucrose, glucose, acetate, acetoin, and citrate) and excretion of 22 metabolites. Several excreted compounds showed increases exceeding 800% relative to control, including guanosine, UMP, adenine, GTP, AMP, urocanate, and cytosine. TPV infection induced increased consumption of lactate and citrate, alongside marked elevations in excretion of uridine, guanosine, adenine, urocanate, and cytosine. These findings demonstrate that a rapid exometabolomics approach can reveal significant alterations in metabolites associated with nucleic acid synthesis, likely reflecting viral strategies to support replication. This case study underscores the critical role of the CNPEM NMR facility in facilitating metabolomics research essential for elucidating complex biochemical responses, such as those elicited by TPV infection, thereby contributing to our understanding of giant virus biology.