Biology subjects

Sullivan, C. S.

Publications and source records attributed to Sullivan, C. S..

9 recordsLinked to original sources

bioRxiv2026

Divergent Fates of Kidney-Resident Polyomaviruses: Stable Shedding Versus Near-Silent Persistence

Polyomaviruses establish long-term infection in the kidney and are intermittently shed in urine. However, the relationship between kidney-resident viral genomes and urinary shedding during persistent infection remains poorly defined. Using a genetically barcoded murine polyomavirus library, we tracked thousands of viral lineages in vivo by pairing longitudinal urine sampling with endpoint barcode sequencing of kidney tissue in four mice. Across all animals, kidney infection consistently resolved into two stable viral populations, with near-silent persistence as the dominant fate. Most kidney-resident barcodes were never detected in late urine at late stages of infection, even though many reached substantial abundance within the kidney, demonstrating that kidney viral genome levels alone do not predict urinary shedding. In contrast, only a small minority of kidney barcodes contributed disproportionately to urine virus output at late timepoints, and these barcodes exhibited stable longitudinal behavior, with repeated detection in urine over time and markedly higher peak urine abundance than late non-shed or random barcode controls. Shedding behavior was not explained by input virus stock abundance, barcode sequence features, predicted miRNA targeting, or ongoing reseeding from blood or other tissues. Instead, barcodes that ultimately dominated late urine already showed elevated urine detection early after infection, indicating that shedding fate is established early and maintained throughout persistent infection. Together, these findings reveal that persistent kidney infection is a structured reservoir composed of a large population of deeply restricted viral genomes and a smaller, stable subset that repeatedly produces urine-detectable viruses, with concurrent smoldering infections and latency-like restriction representing one possible model to explain the sharply different probabilities of shedding among kidney-resident genomes.

microbiology↗

bioRxiv2025

Shedding dynamics of a DNA virus population during acute and long-term persistent infection

Although much is known of the molecular mechanisms of virus infection within cells, substantially less is understood about within-host infection. Such knowledge is key to understanding how viruses take up residence and transmit infectious virus, in some cases throughout the life of the host. Here, using murine polyomavirus (muPyV) as a tractable model, we monitor parallel infections of thousands of differentially barcoded viruses within a single host. In individual mice, we show that numerous viruses (>2600) establish infection and are maintained for long periods post-infection. Strikingly, a low level of many different barcodes is shed in urine at all times post-infection, with a minimum of at least 80 different barcodes present in every sample throughout months of infection. During the early acute phase, bulk shed virus genomes derive from numerous different barcodes. This is followed by long term persistent infection detectable in diverse organs. Consistent with limited productive exchange of virus genomes between organs, each displays a unique pattern of relative barcode abundance. During the persistent phase, constant low-level shedding of typically hundreds of barcodes is maintained but is overlapped with rare, punctuated shedding of high amounts of one or a few individual barcodes. In contrast to the early acute phase, these few infrequent highly shed barcodes comprise the majority of bulk shed genomes observed during late times of persistent infection, contributing to a stark decrease in bulk barcode diversity that is shed over time. These temporally shifting patterns, which are conserved across hosts, suggest that polyomaviruses balance continuous transmission potential with reservoir-driven high-level reactivation. This offers a mechanistic basis for polyomavirus ubiquity and long-term persistence, which are typical of many DNA viruses. Author Summary / ImportancePolyomavirus infections, mostly benign but potentially fatal for immunocompromised individuals, undergo acute and long-term persistent infections. Typically, polyomavirus-associated diseases arise due to virus infection occurring in the context of a persistently infected individual. However, little is understood regarding the mechanisms of how polyomaviruses establish, maintain, and reactivate from persistent infection. We developed a non-invasive virus shedding assay combining barcoded murine polyomavirus, massively parallel sequencing technology, and novel computational approaches to track long-term infections in mice. We expect these methods to be of use not only to the study of DNA viruses but also for understanding persitent infection of diverse microbes. The study revealed organ-specific virus reservoirs and two distinct shedding patterns: constant low-level shedding of numerous barcodes and episodic high-level shedding of few barcodes. Over time, the diversity of shed barcodes decreased substantially. These findings suggest a persistent low-level infection in multiple reservoirs, with occasional bursts of replication in a small subset of infected cells. This combination of broad reservoirs and varied shedding mechanisms may contribute to polyomavirus success in transmission and maintaining long-term infections.

pathology↗

bioRxiv2024

Viral piracy of host RNA phosphatase DUSP11 by avipoxviruses

Proper recognition of viral pathogens is an essential part of the innate immune response. A common viral replicative intermediate and chemical signal that cells use to identify pathogens is the presence of a triphosphorylated 5 end (5ppp) RNA, which activates the cytosolic RNA sensor RIG-I and initiates downstream antiviral signaling. While 5pppRNA generated by viral RNA-dependent RNA polymerases (RdRps) can be a potent activator of the immune response, endogenous RNA polymerase III (RNAPIII) transcripts can retain the 5pppRNA generated during transcription and induce a RIG-I-mediated immune response. We have previously shown that host RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) can act on both host and viral RNAs, altering their levels and reducing their ability to induce RIG-I activation. Our previous work explored how artificially altered DUSP11 can impact immune activation, prompting further exploration into natural contexts of altered DUSP11. Here, we have identified viral DUSP11 homologs (vDUSP11s) present in some avipoxviruses. Consistent with the known functions of endogenous DUSP11, we have shown that expression of vDUSP11s: 1) reduces levels of endogenous RNAPIII transcripts, 2) reduces a cells sensitivity to 5pppRNA-mediated immune activation, and 3) restores virus infection defects seen in the absence of DUSP11. Our results identify a virus-relevant context where DUSP11 activity has been co-opted to alter RNA metabolism and influence the outcome of infection.

molecular biology↗

bioRxiv2024

PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-{beta} and IFN-{lambda} responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14s antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1), a DNA virus, vesicular stomatitis virus (VSV), a negative-sense RNA virus, and lymphocytic choriomeningitis virus (LCMV), an ambisense RNA virus, to infect A549 PARP14 knockout (KO) cells. While LCMV infection was unaffected, HSV-1 replication was increased in PARP14 KO cells and VSV replication was decreased. These results indicate that PARP14 restricts HSV-1 replication but enhances the replication of VSV. A PARP14 active site inhibitor had no impact on HSV-1 or VSV replication, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both proviral and antiviral functions targeting multiple viruses. IMPORTANCEThe antiviral response is largely regulated by post-translation modifications (PTM), including ADP-ribosylation. PARP14 is an ADP-ribosyltransferase that is upregulated by interferon and is under positive selection, indicating that it is involved in host-pathogen conflict. However, no anti-viral function has been described for PARP14. Here, we found that PARP14 represses both coronavirus and HSV-1 replication, demonstrating that PARP14 has antiviral functions. Surprisingly, we also found that PARP14 also has pro-viral functions, as it was critical for the efficient replication of VSV. These data indicate that PARP14 has both proviral and antiviral functions. Defining the mechanisms used by PARP14 to both repress and promote virus replication will provide new insights into how PARPs regulate virus infection.

microbiology↗

bioRxiv2022

Interpreting and de-noising genetically engineered barcodes in a DNA virus

The concept of a nucleic acid barcode applied to pathogen genomes is easy to grasp and the many possible uses are straightforward. But implementation may not be easy, especially when growing through multiple generations or assaying the pathogen long-term. The potential problems include: the barcode might alter fitness, the barcode may accumulate mutations, and construction of the marked pathogens may result in unintended barcodes that are not as designed. Here, we generate approximately 5000 randomized barcodes in the genome of the prototypic small DNA virus murine polyomavirus. We describe the challenges faced with interpreting the barcode sequences obtained from the library. Our Illumina NextSeq sequencing recalled much greater variation in barcode sequencing reads than the expected 5000 barcodes - necessarily stemming from the Illumina library processing and sequencing error. Using data from defined control virus genomes cloned into plasmid backbones we develop a vetted post-sequencing method to cluster the erroneous reads around the true virus genome barcodes. These findings may foreshadow problems with randomized barcodes in other microbial systems and provide a useful approach for future work utilizing nucleic acid barcoded pathogens.

microbiology↗

bioRxiv2020

Human Polyomavirus-Encoded Circular RNAs

Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset of circRNAs are translated into peptides. Here we describe circular RNAs encoded by human polyomaviruses (HPyVs), including circular forms of RNAs encoding variants of the previously described alternative large T antigen open reading frame (ALTO) gene. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. MCPyV circALTOs produce ALTO protein in cultured cells. MCPyV ALTO promotes the transcription of co-transfected reporter genes. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and tissues, encode for proteins, and may contribute to the infectious and tumorigenic properties of these viruses.

molecular biology↗

bioRxiv2020

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

Gene deserts are defined as genomic regions devoid of protein coding genes and spanning more than 500 kilobases, collectively encompassing about 25% of the human genome. Approximately 30% of all gene deserts are enriched for conserved elements with cis-regulatory signatures. These are located predominantly near developmental transcription factors (TFs) but despite predicted critical functions, the transcriptional contributions and biological necessity of most gene deserts remain elusive. Here, we explore the cis-regulatory impact of a gene desert flanking the Shox2 gene, a TF indispensable for proximal limb, craniofacial and cardiac pacemaker development. Using a functional genomics approach in mouse embryos we identify the gene desert as a hub for numerous Shox2-overlapping enhancers arranged in a globular chromatin domain with tissue-specific features. In accordance, using endogenous CRISPR deletion, we demonstrate that the gene desert interval is essential for Shox2 transcriptional control in developing limbs, craniofacial compartments, and the heart. Phenotypically, gene desert ablation leads to pacemaker-related embryonic lethality due to Shox2 depletion in the cardiac sinus venosus. We show that this role is partially mediated through a distal gene desert enhancer, providing evidence for intra-gene desert regulatory robustness. Finally, we uncover a multi-layered functional role of the gene desert by revealing an additional requirement for stylopod morphogenesis, mediated through an array of proximal limb enhancers (PLEs). In summary, our study establishes the Shox2 gene desert as a fundamental genomic unit that controls pleiotropic gene expression through modular arrangement and coordinated dynamics of tissue-specific enhancers.

developmental biology↗

bioRxiv2017

Murine polyomavirus microRNAs promote viuria during the acute phase of infection

Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs during in vivo infections are mostly unknown. Studies presented here use murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis reveals that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection, indicating that during infection of a natural host, PyV miRNAs function to reduce viral replication during the persistent phase of infection. Additionally, MuPyV miRNAs promote viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2-/-mice. These findings implicate miRNA activity in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response.\n\nImportanceMicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of their in vivo function. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of possible clinical relevance. Here we show a novel activity for a viral miRNA in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA in long-term persistence, that it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an effective adaptive immune response, our work also connects the in vivo activity of a PyV miRNA to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings may help to better understand how the balance between PyV and the immune response becomes altered in pathogenic states.

microbiology↗

bioRxiv2017

Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes

Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, the U6 and H1 promoters encompass the majority of the [~]270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by greater than two-fold as compared to the U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting.

molecular biology↗