Biomedical subjects

Christopher S Sullivan

Publications and source records attributed to Christopher S Sullivan.

8 recordsLinked to original sources

PubMed2025

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.

Animals↗

PubMed2006

Coordinate suppression of ERBB2 and ERBB3 by enforced expression of micro-RNA miR-125a or miR-125b.

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.

Breast Neoplasms↗

PubMed2006

A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses.

We have developed an approach to identify microRNAs (miRNAs) that is based on bioinformatics and array-based technologies, without the use of cDNA cloning. The approach, designed for use on genomes of small size (<2 Mb), was tested on cells infected by either of two lymphotropic herpesviruses, KSHV and EBV. The viral genomes were scanned computationally for pre-miRNAs using an algorithm (VMir) we have developed. Candidate hairpins suggested by this analysis were then synthesized as oligonucleotides on microarrays, and the arrays were hybridized with small RNAs from infected cells. Candidate miRNAs that scored positive on the arrays were then subjected to confirmatory Northern blot analysis. Using this approach, 10 of the known KSHV pre-miRNAs were identified, as well as a novel pre-miRNA that had earlier escaped detection. This method also led to the identification of seven new EBV-encoded pre-miRNAs; by using additional computational approaches, we identified a total of 18 new EBV pre-miRNAs that produce 22 mature miRNA molecules, thereby more than quadrupling the total number of hitherto known EBV miRNAs. The advantages and limitations of the approach are discussed.

Algorithms↗

PubMed2005

MicroRNAs and viral infection.

MicroRNAs (miRNAs) play a pivotal role in the regulation of genes involved in diverse processes such as development, differentiation, and cellular growth control. Recently, many viral-encoded miRNAs have been discovered, for the most part in viruses transcribed from double-stranded DNA genomes. As with their cellular counterparts, the functions of most viral-derived miRNAs are unknown; however, functions have been documented or proposed for viral miRNAs from three different viral families-herpesviruses, polyomaviruses, and retroviruses. Several virus-encoded miRNAs have unique aspects to their biogenesis, such as the polymerase that transcribes them or their location within the precursor transcript. Additionally, viral interactions with cellular miRNAs have also been identified, and these have substantially expanded our appreciation of miRNA functions.

Animals↗

PubMed2005

SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells.

MicroRNAs (miRNAs) are small (approximately 22-nucleotide) RNAs that in lower organisms serve important regulatory roles in development and gene expression, typically by forming imperfect duplexes with target messenger RNAs. miRNAs have also been described in mammalian cells and in infections with Epstein-Barr virus (EBV), but the function of most of them is unknown. Although one EBV miRNA probably altered the processing of a viral mRNA, the regulatory significance of this event is uncertain, because other transcripts exist that can supply the targeted function. Here we report the identification of miRNAs encoded by simian virus 40 (SV40) and define their functional significance for viral infection. SVmiRNAs accumulate at late times in infection, are perfectly complementary to early viral mRNAs, and target those mRNAs for cleavage. This reduces the expression of viral T antigens but does not reduce the yield of infectious virus relative to that generated by a mutant lacking SVmiRNAs. However, wild-type SV40-infected cells are less sensitive than the mutant to lysis by cytotoxic T cells, and trigger less cytokine production by such cells. Thus, viral evolution has taken advantage of the miRNA pathway to generate effectors that enhance the probability of successful infection.

Animals↗

PubMed2005

A virus-encoded inhibitor that blocks RNA interference in mammalian cells.

Nodamura virus (NoV) is a small RNA virus that is infectious for insect and mammalian hosts. We have developed a highly sensitive assay of RNA interference (RNAi) in mammalian cells that shows that the NoV B2 protein functions as an inhibitor of RNAi triggered by either short hairpin RNAs or small interfering RNAs. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RNA-induced silencing complex (RISC)-processed RNAs and inhibits the Dicer cleavage reaction and, potentially, one or more post-Dicer activities. In vitro, NoV B2 inhibits Dicer-mediated RNA cleavage in the absence of any other host factors and specifically binds double-stranded RNAs corresponding in structure to Dicer substrates and products. Its abilities to bind to Dicer precursor and post-Dicer RISC-processed RNAs suggest a mechanism of inhibition that is unique among known viral inhibitors of RNAi.

Cell Line↗

PubMed2005

Complete nucleotide sequence of polyomavirus SA12.

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.

Amino Acid Sequence↗

PubMed2002

T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis.

Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen-a most amazing molecule.

Amino Acid Sequence↗