Date of Award

Spring 2026

Document Type

Thesis

Publisher

Santa Clara : Santa Clara University, 2026

Degree Name

Master of Science (MS)

Department

Bioengineering

First Advisor

Prashanth Asuri

Second Advisor

Ian Carter-O’Connell

Abstract

Herpes simplex virus (HSV) is a highly prevalent human pathogen whose symptoms are characterized by the formation of painful blisters and ulcers. There are two major families of HSV: HSV-1, primarily transmitted through oral contact, and HSV-2, predominantly spread through sexual contact. Periodic reactivation of the virus enables continued transmission and recurrent disease. Antiviral drugs that suppress viral replication are available to the public; however, no curative therapy exists.

Recent studies suggest that HSV reprograms host cellular metabolism to support its replication. Notably, this includes the manipulation of nicotinamide adenine dinucleotide (NAD⁺) homeostasis. Under standard physiological conditions, NAD⁺ is consumed primarily by poly(ADP-ribose) polymerases (PARPs), sirtuin deacetylases (SIRTs), and various glycohydrolases. PARPs, in particular, transfer ADP-ribose moieties from NAD⁺ onto target proteins, which is a post-translational modification known as ADP-ribosylation. These enzymes signal and regulate cellular functions, including DNA damage repair, transcriptional regulation, viral recognition, and stress responses. The rate-limiting enzyme within the NAD⁺ salvage pathway is nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the conversion of nicotinamide (NAM) to nicotinamide mononucleotide (NMN). Interestingly, emerging data indicate that NAMPT exhibits antiviral activity against HSV-1.

Recent studies have shown that NAMPT and HSV-1 appear to be in direct competition, as HSV-1 depletes cellular NAD⁺ pools, whereas NAMPT replenishes NAD⁺ levels. Moreover, NAMPT appears to act outside of its canonical role in NAD⁺ biosynthesis by directly targeting viral particles and removing phosphoribose units from the virion surface. This represents a non-metabolic antiviral function distinct from its enzymatic activity on free NAD⁺, though it remains unclear how the ADP-ribose is processed into phosphoribose. A major obstacle to understanding this antiviral mechanism is the limited availability of well-defined phosphoribosylated substrates to investigate each of the processing steps. Traditional approaches that use snake venom phosphodiesterase (SVP) to generate phosphoribose-modified peptides are hindered by low enzyme purity, variable reaction efficiency, and poor reproducibility. In this thesis, I propose the use of the human Nudix hydrolase NUDT16 as a strategy to generate phosphoribosylated peptides through controlled cleavage of ADP-ribose modifications by PARP14. PARP14, a mono-ADP-ribosyltransferase, serves as the upstream enzyme in this pipeline, writing ADP-ribose marks onto substrate peptides that can then be processed in a defined and reproducible manner. NUDT16 is a well-defined enzymatic tool used to precisely remove adenosine phosphate from ADP-ribose, enabling the controlled generation of phosphoribosylated peptides, which I validate by MALDI-TOF mass spectrometry.

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