About

Paul Ahlquist is a professor in the Department of Plant Pathology at the University of Wisconsin-Madison. He holds a Ph.D. in Biophysics from the University of Wisconsin-Madison. His research focuses on studying the novel, RNA-based pathways and virus-host interactions underlying replication, gene expression, and evolution by positive-strand RNA viruses, which include many important human pathogens such as hepatitis C virus and the SARS coronavirus. His work integrates molecular genetics, genomics, biochemistry, and cell biology to address fundamental questions in virus replication and virus-cell interactions. Ahlquist's research has led to significant discoveries, including the identification of structural and functional parallels among the replication complexes of different virus classes, implying a functional and evolutionary unification within virology. He has also identified the first higher eukaryotic viruses capable of directing genome replication, gene expression, and virion assembly in the genetically tractable yeast Saccharomyces cerevisiae. Using yeast genetics and genomics, his studies aim to identify host genes required for viral RNA replication and to understand how these genes function with virus-encoded factors to facilitate viral processes.

Research topics

• Molecular biology
• Virology
• Biology
• Cell biology
• Biochemistry

Selected publications

• Nodavirus protein A’s interdomain elbow controls RNA replication organelle formation and function

  Nucleic Acids Research · 2026-02-14

  articleOpen accessSenior author

  Positive-strand [(+)RNA] viruses replicate their RNA genomes in poorly understood membrane-associated replication organelles (ROs). Cryo-electron microscopy of nodaviral ROs revealed that viral RNA replication protein A, with polymerase and RNA capping domains, forms a "crown" of two stacked 12-mer rings at the RO's opening to the cytosol, providing powerful foundations for analyzing RO formation and function. The lower proto-crown is a ring of 12 polymerases with RNA capping domains clustered to form a central floor. The upper crown mirrors the polymerase ring but has RNA capping domains projecting radially outward. Here, we identify a critical protein A "elbow" segment of only 17 amino acids that coordinates most interactions between crown floor subunits. Our extensive mutational and genetic complementation analyses reveal that distinct elbow subsegments cooperatively support proto-crown formation and separately activate the neighboring RNA capping and distal polymerase domains. These and other findings establish the elbow as a master regulator whose separable interactions drive proto-crown assembly, license RNA polymerase and capping and, with an adjacent flexible linker, control protein A's conformational changes. They also further localize RNA capping to the crown floor. This work illuminates (+)RNA viruses' elegant regulation of genome replication steps and future control strategies.

  PublisherDOI

• Genetic complementation reveals structure-function links in nodavirus RNA replication complex crowns

  PLoS Pathogens · 2025-12-11 · 1 citations

  preprintOpen accessSenior author

  Abstract Positive-strand RNA viruses replicate their RNA genomes in virus-induced, membrane-bounded organelles. As first found for nodaviruses, the necked cytosolic portals of these organelles bear ringed “crown” complexes of viral RNA replication proteins that drive synthesis, capping and release of new RNA genomes. Nodavirus crowns contain two 12-mer rings of viral protein A with C-proximal polymerase domains stacked. In the basal ring, protein A’s N-proximal RNA capping domains form a central, toroidal floor, while in the apical ring these domains extend radially outward. A third protein A conformation provides a putative central Pol domain interacting with the viral dsRNA replication intermediate in a vesicle beneath the crown. Protein A’s multiple conformations likely differentially contribute to crown assembly, RNA template recruitment, (−) and (+) strand synthesis, RNA capping, and progeny RNA release. Protein A’s high copy numbers may provide robustness to these processes. To test such concepts, we combined mutational, complementation and functional analyses. Strong complementation between null mutants in protein A’s polymerase and RNA capping active sites showed that they operate in independent protein A copies, likely at distinct sites. Thus, neither function is required in all protein A copies, nor are both required in any single copy. Lack of complementation between mutants in distinct RNA capping steps implied that major RNA capping steps must be performed in the same protein. Although RNA polymerase and capping activity were not required in all protein A subunits, none of a series of deletions across these domains were complementable, showing the importance of structural and other requirements for crown assembly, etc.. Surprisingly, RNA replication was more sensitive to depleting the fraction of subunits retaining protein A’s C-terminal intrinsically disordered region than polymerase or capping activity. These and other results reveal and illuminate the cooperative, interdependent nature of protein A’s diverse functions. Author summary Positive-strand RNA viruses represent the largest genetic class of viruses and include human, animal, and plant pathogens causing major agricultural, economic, and environmental consequences. Using no DNA intermediates to multiply their RNA genomes, these viruses modify cellular membranes into novel, infection-specific RNA replication organelles. Emerging results show that RNA replication proteins encoded by many or most of these viruses assemble into ringed, crown-like viral protein complexes gating portals to these compartments. We previously revealed that nodavirus crowns contain two stacked 12-mer rings of viral replicase protein A, which contains polymerase, RNA capping and other domains. The nodavirus experiments reported here are among the earliest explorations in cells to illuminate the functions and interactions of such multi-domain RNA replication proteins in the context of their highly multimeric crowns. Critical questions include whether all domains and interactions are required in all protein A conformations, whether protein A multiplicity might provide dose-responsive redundancy for any crown functions, or whether defects in individual protein copies might inhibit or even poison operation of the entire crown. The results have significant implications for positive-strand RNA virus biology and thus for efforts toward virus control and beneficial uses.

  PublisherDOI

• MPAC: a computational framework for inferring pathway activities from multi-omic data

  Bioinformatics · 2025-09-10

  articleOpen access

  MOTIVATION: Fully capturing cellular state requires examining genomic, epigenomic, transcriptomic, proteomic, and other assays for a biological sample and comprehensive computational modeling to reason with the complex and sometimes conflicting measurements. Modeling these so-called multi-omic data is especially beneficial in disease analysis, where observations across omic data types may reveal unexpected patient groupings and inform clinical outcomes and treatments. RESULTS: We present Multi-omic Pathway Analysis of Cells (MPAC), a computational framework that interprets multi-omic data through prior knowledge from biological pathways. MPAC leverages network relationships encoded in pathways through a factor graph to infer consensus activity levels for proteins and associated pathway entities from multi-omic data, runs permutation testing to eliminate spurious activity predictions, and groups biological samples by pathway activities to allow identifying and prioritizing proteins with potential clinical relevance, e.g. associated with patient prognosis. Using DNA copy number alteration and RNA-seq data from head and neck squamous cell carcinoma patients from The Cancer Genome Atlas as an example, we demonstrate that MPAC predicts a patient subgroup related to immune responses not identified by analysis with either input omic data type alone. Key proteins identified via this subgroup have pathway activities related to clinical outcome as well as immune cell composition. Our MPAC R package enables similar multi-omic analyses on new datasets. AVAILABILITY AND IMPLEMENTATION: The MPAC package is available at Bioconductor https://bioconductor.org/packages/MPAC.

  PublisherDOI

• MPAC: a computational framework for inferring pathway activities from multi-omic data

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-06-17 · 1 citations

  preprintOpen access

  Fully capturing cellular state requires examining genomic, epigenomic, transcriptomic, proteomic, and other assays for a biological sample and comprehensive computational modeling to reason with the complex and sometimes conflicting measurements. Modeling these so-called multi-omic data is especially beneficial in disease analysis, where observations across omic data types may reveal unexpected patient groupings and inform clinical outcomes and treatments. We present Multi-omic Pathway Analysis of Cells (MPAC), a computational framework that interprets multi-omic data through prior knowledge from biological pathways. MPAC leverages network relationships encoded in pathways through a factor graph to infer consensus activity levels for proteins and associated pathway entities from multi-omic data, runs permutation testing to eliminate spurious activity predictions, and groups biological samples by pathway activities to allow identifying and prioritizing proteins with potential clinical relevance, e.g., associated with patient prognosis. Using DNA copy number alteration and RNA-seq data from head and neck squamous cell carcinoma patients from The Cancer Genome Atlas as an example, we demonstrate that MPAC predicts a patient subgroup related to immune responses not identified by analysis with either input omic data type alone. Key proteins identified via this subgroup have pathway activities related to clinical outcome as well as immune cell compositions. Our MPAC R package, available at https://bioconductor.org/packages/MPAC, enables similar multi-omic analyses on new datasets.

  PublisherOA PDFDOI

• Key aspects of papillomavirus infection influence the host cervicovaginal microbiome in a preclinical murine papillomavirus (MmuPV1) infection model

  mBio · 2024-05-14 · 7 citations

  articleOpen access

  ABSTRACT Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States and are a major etiological agent of cancers in the anogenital tract and oral cavity. Growing evidence suggests changes in the host microbiome are associated with the natural history and ultimate outcome of HPV infection. We sought to define changes in the host cervicovaginal microbiome during papillomavirus infection, persistence, and pathogenesis using the murine papillomavirus (MmuPV1) cervicovaginal infection model. Cervicovaginal lavages were performed over a time course of MmuPV1 infection in immunocompetent female FVB/N mice and extracted DNA was analyzed by qPCR to track MmuPV1 viral copy number. 16S ribosomal RNA (rRNA) gene sequencing was used to determine the composition and diversity of microbial communities throughout this time course. We also sought to determine whether specific microbial communities exist across the spectrum of MmuPV1-induced neoplastic disease. We, therefore, performed laser-capture microdissection to isolate regions of disease representing all stages of neoplastic disease progression (normal, low- and high-grade dysplasia, and cancer) from female reproductive tract tissue sections from MmuPV1-infected mice and performed 16S rRNA sequencing. Consistent with other studies, we found that the natural murine cervicovaginal microbiome is highly variable across different experiments. Despite these differences in initial microbiome composition between experiments, we observed that MmuPV1 persistence, viral load, and severity of disease influenced the composition of the cervicovaginal microbiome. These studies demonstrate that papillomavirus infection can alter the cervicovaginal microbiome. IMPORTANCE Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. A subset of HPVs that infect the anogenital tract (cervix, vagina, anus) and oral cavity cause at least 5% of cancers worldwide. Recent evidence indicates that the community of microbial organisms present in the human cervix and vagina, known as the cervicovaginal microbiome, plays a role in HPV-induced cervical cancer. However, the mechanisms underlying this interplay are not well-defined. In this study, we infected the female reproductive tract of mice with a murine papillomavirus (MmuPV1) and found that key aspects of papillomavirus infection and disease influence the host cervicovaginal microbiome. This is the first study to define changes in the host microbiome associated with MmuPV1 infection in a preclinical animal model of HPV-induced cervical cancer. These results pave the way for using MmuPV1 infection models to further investigate the interactions between papillomaviruses and the host microbiome.

  PublisherDOI

• Positive-strand RNA virus genome replication organelles: structure, assembly, control

  Trends in Genetics · 2024-05-08 · 36 citations

  reviewOpen accessSenior authorCorresponding

  Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.

  PublisherOA PDFDOI

• Exploiting rodent cell blocks for intrinsic resistance to HIV-1 gene expression in human T cells

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-04-08

  preprintOpen access

  Abstract HIV-1 virion production is inefficient in cells derived from mice and other rodents reflecting cell-intrinsic defects to interactions between the HIV-1 auxiliary proteins Tat and Rev and host dependency factors CCNT1 (Cyclin T1) and XPO1 (Exportin-1, also known as CRM1), respectively. In human cells, Tat binds CCNT1 to enhance viral RNA transcription and Rev recruits XPO1 to mediate the nuclear export of intron-containing viral RNA. In mouse cells, Tat’s interactions with CCNT1 are inefficient, mapped to a single species-specific residue Y261 instead of C261 in human. Rev interacts poorly with murine XPO1, mapped to a trio of amino acids T411/V412/S414 instead of P411/M412/F414 in humans. To determine if these discrete species-specific regions of otherwise conserved housekeeping proteins represent viable targets for inhibiting Tat and Rev function in humans, herein we recoded (“mousified”) each in human CD4+ T cells using precision CRISPR/Cas9-facilitated gene editing. Both edits yielded cells refractory to Rev or Tat activity, respectively, with isolated, isogenic CCNT1.C261Y cell lines remarkable in their capacity to exhibit near total inactivation of viral gene expression for all X4 and R5-tropic HIV-1 strains tested, and even the more distantly related lentiviruses including HIV-2 and SIV agm . These studies validate minor and naturally-occurring, species-specific differences in otherwise conserved human host factors as compelling targets for achieving broad-acting cell-intrinsic resistance to HIV’s post-integration phases. Importance Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 editing to engineer “mousified” versions of two of these proteins, CCNT1 and XPO1, in human T cells. CCNT1 and XPO1 are essential for efficient HIV-1 transcription and viral RNA transport, respectively, making them intriguing targets for gene-based inactivation of virus replication. Targeting either gene yielded antiviral phenotypes, with isogenic CCNT1-modified cell lines confirmed to exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cells. These results provide proof of concept for targeting CCNT1 (and potentially XPO1) in the context of one or more functional HIV-1 cure strategies.

  PublisherOA PDFDOI

• Supplementary Methods from Genome-Wide Expression Profiling Reveals EBV-Associated Inhibition of MHC Class I Expression in Nasopharyngeal Carcinoma

  2023-03-30

  preprintOpen accessSenior author

  Supplementary Methods from Genome-Wide Expression Profiling Reveals EBV-Associated Inhibition of MHC Class I Expression in Nasopharyngeal Carcinoma

  PublisherDOI

• Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells

  mBio · 2023-09-07 · 5 citations

  articleOpen access

  ABSTRACT HIV-1 virion production is inefficient in cells derived from mice and other rodents reflecting cell-intrinsic defects to interactions between the HIV-1 auxiliary proteins Tat and Rev and host dependency factors CCNT1 (Cyclin T1) and XPO1 (exportin-1, also known as CRM1), respectively. In human cells, Tat binds CCNT1 to enhance viral RNA transcription and Rev recruits XPO1 to mediate the nuclear export of intron-containing viral RNA. In mouse cells, Tat’s interactions with CCNT1 are inefficient, mapped to a single species-specific residue Y261 instead of C261 in humans. Rev interacts poorly with murine XPO1, mapped to a trio of amino acids T411/V412/S414 instead of P411/M412/F414 in humans. To determine if these discrete species-specific regions of otherwise conserved housekeeping proteins represent viable targets for inhibiting HIV-1 replication in humans, herein, we employed CRISPR/Cas9 to recode the relevant regions of CCNT1 and XPO1 in human CD4+ T cells. While efforts to modify XPO1 were inconclusive, we generated isogenic CCNT1.C261Y cell lines exhibiting remarkable resistance to HIV-1 Tat, exhibiting near total inactivation of viral gene expression for all X4- and R5-tropic HIV-1 strains tested, as well as the more distantly related primate lentiviruses HIV-2 and SIV agm . Induction of viral reactivation using latency reversal agents (LRAs) was also restricted in CCNT1.C261Y cells. These studies validate a minor and naturally occurring, species-specific difference in a conserved human host factor as a compelling potential target for achieving broad-acting cell-intrinsic resistance to HIV’s post-integration phases. Importance Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a “mousified” version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting CCNT1 in the context of one or more functional HIV-1 cure strategies.

  PublisherDOI

• Data from Genome-Wide Expression Profiling Reveals EBV-Associated Inhibition of MHC Class I Expression in Nasopharyngeal Carcinoma

  2023-03-30

  preprintOpen accessSenior author

  <div>Abstract<p>To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (<i>EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1</i>, and <i>LMP2A</i>) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC. (Cancer Res 2006; 66(16): 7999-8006)</p></div>

  PublisherDOI

Recent grants

• Core C - Virus/Vector

  NIH · $113.8M · 1997–2029

• NIH Grant R01CA097944

  NIH · $1.4M · 2007

• NIH Grant R01GM035072

  NIH · $3.3M · 2011

• NIH Grant R01GM051301

  NIH · $1.1M · 1999

• Molecular Genetic Analysis of Host Specificity in Plant Viruses

  NSF · $260k · 1990–1994

Frequent coauthors

• Hong Zhan

  University of Wisconsin–Madison

  161 shared

• Nuruddin Unchwaniwala

  161 shared

• Janice Pennington

  University of Wisconsin–Madison

  160 shared

• Masaki Nishikiori

  Morgridge Institute for Research

  157 shared

• Johan A. den Boon

  Morgridge Institute for Research

  137 shared

• Irina Novikova

  Pacific Northwest National Laboratory

  129 shared

• Nancy Meyer

  Oregon Health & Science University

  89 shared

• Michael Janda

  68 shared