Date of Award

Spring 5-17-2025

Document Type

Honors Project

University Scholars Director

Joshua Tom

First Advisor/Committee Member

Tracie Delgado

Keywords

Methotrexate, Gammaherpesvirus, Murine Herpesvirus-68, Antiviral, Drug

Abstract

It is estimated that ~15% of all cancers are caused by oncogenic virus infections. Two of the top seven cancer-causing human viruses are members of the gammaherpesvirus family: Epstein Barr Virus (EBV) and Kaposi’s Sarcoma Herpesvirus (KSHV). Our lab uses Murine Herpesvirus 68 (MHV-68), a mouse gammaherpesvirus with shares significant genetic homology to KSHV and EBV, as a model system to understand how gammaherpesviruses alter the metabolism of their host during lytic infection to promote their replication. We recently metabolically profiled MHV-68 infected host cells at various time points during the lytic infectious cycle. Our data showed nucleotide metabolism is significantly induced in MHV-68 infected NIH/3T3 cells, revealing a potential antiviral target. This study investigates the antiviral efficacy of Methotrexate (MTX), an FDA-approved nucleotide biosynthesis inhibitor currently used to treat cancer, rheumatoid arthritis, and psoriasis. MTX inhibits dihydrofolate reductase (DHFR), an enzyme crucial for producing thymidylate and purine nucleotides, which are essential for de novo nucleotide synthesis. We hypothesized that MTX can block MHV-68 production and be repurposed as an antiviral drug. To test our hypothesis, we first determined a safe concentration of MTX in NIH/3T3 cells using both qualitative (microscopy) and quantitative (trypan blue exclusion) cell viability assays. Next, we infected NIH/3T3 cells with MHV-68 and treated them with a safe level of MTX or solvent control. After 48 hours, we assessed viral production in control vs MTX treated cellular supernatants via viral plaque assays. Our results revealed that MTX significantly suppressed MHV-68 virion production by ~98.5-fold. These findings suggest that targeting host metabolic pathways could be an effective antiviral strategy against gammaherpesviruses in humans. Further research is needed to explore the use of MTX as a broad viral therapy against other viruses.

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Copyright held by author.

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