Publications by authors named "Eva M Medina"

5 Publications

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Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III promoters and enhances non-coding RNA expression.

J Virol 2021 Apr 28. Epub 2021 Apr 28.

Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses (γHVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during γHV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 (γHV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with γHV68 increased the transcription from multiple host and viral pol III promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis.Gammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.
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http://dx.doi.org/10.1128/JVI.00079-21DOI Listing
April 2021

Optimized Detection of Acute MHV68 Infection With a Reporter System Identifies Large Peritoneal Macrophages as a Dominant Target of Primary Infection.

Front Microbiol 2021 9;12:656979. Epub 2021 Mar 9.

Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.

Investigating the dynamics of virus-host interactions remains an important challenge, often limited by the ability to directly identify virally infected cells. Here, we utilize a beta-lactamase activated fluorescent substrate to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages (LPMs) by 8 days post-infection, and a concordant increase in small peritoneal macrophages (SPMs) and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC II LPMs emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 and 3 days post-infection, MHV68 infects ∼5-10% of peritoneal cells, with >75% being LPMs. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in SPMs, with few infected dendritic cells and B cells. Importantly, limiting dilution analysis indicates that at 3 days post-infection, the majority of MHV68-infected cells harbor latent rather than lytic virus at frequencies consistent with those identified based on reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection.
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http://dx.doi.org/10.3389/fmicb.2021.656979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985543PMC
March 2021

Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68.

Cell Rep 2019 06;27(13):3988-4002.e5

Department of Molecular Genetics & Microbiology, UF Health Cancer Center, University of Florida, Gainesville, FL, USA. Electronic address:

The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, γHV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome.
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http://dx.doi.org/10.1016/j.celrep.2019.05.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071827PMC
June 2019

Host Tumor Suppressor p18 Functions as a Potent Cell-Intrinsic Inhibitor of Murine Gammaherpesvirus 68 Reactivation and Pathogenesis.

J Virol 2018 03 26;92(6). Epub 2018 Feb 26.

Immunology and Microbiology Department, University of Colorado Denver School of Medicine, Aurora, Colorado, USA

Gammaherpesviruses are common viruses associated with lifelong infection and increased disease risk. Reactivation from latency aids the virus in maintaining infection throughout the life of the host and is responsible for a wide array of disease outcomes. Previously, we demonstrated that the virus-encoded cyclin (v-cyclin) of murine gammaherpesvirus 68 (γHV68) is essential for optimal reactivation from latency in normal mice but not in mice lacking the host tumor suppressor p18 (p18). Whether p18 plays a cell-intrinsic or -extrinsic role in constraining reactivation remains unclear. Here, we generated recombinant viruses in which we replaced the viral cyclin with the cellular p18 gene (p18KI) for targeted expression of p18, specifically within infected cells. We find that the p18KI virus is similar to the cyclin-deficient virus (cycKO) in lytic infection, establishment of latency, and infected cell reservoirs. While the cycKO virus is capable of reactivation in p18-deficient mice, expression of p18 from the p18KI virus results in a profound reactivation defect. These data demonstrate that p18 limits reactivation within latently infected cells, functioning in a cell-intrinsic manner. Further, the p18KI virus showed greater attenuation of virus-induced lethal pneumonia than the cycKO virus, indicating that p18 could further restrict γHV68 pathogenesis even in p18-sufficient mice. These studies demonstrate that host p18 imposes the requirement for the viral cyclin to reactivate from latency by functioning in latently infected cells and that p18 expression is associated with decreased disease, thereby identifying p18 as a compelling host target to limit chronic gammaherpesvirus pathogenesis. Gammaherpesviruses are ubiquitous viruses associated with multiple malignancies. The propensity to cycle between latency and reactivation results in an infection that is never cleared and often difficult to treat. Understanding the balance between latency and reactivation is integral to treating gammaherpesvirus infection and associated disease outcomes. This work characterizes the role of a novel inhibitor of reactivation, host p18, thereby bringing more clarity to a complex process with significant outcomes for infected individuals.
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http://dx.doi.org/10.1128/JVI.01604-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827403PMC
March 2018

Gammaherpesvirus small noncoding RNAs are bifunctional elements that regulate infection and contribute to virulence in vivo.

mBio 2015 Feb 17;6(1):e01670-14. Epub 2015 Feb 17.

Department of Immunology & Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA

Unlabelled: Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (γHVs), including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine γHV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes in vitro, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to γHV infection, we have used γHV68, a small-animal model of γHV pathogenesis. γHV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the γHV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to γHV acute infection and pathogenesis, we generated and characterized a recombinant γHV68 strain devoid of all eight TMERs. TMER-deficient γHV68 has wild-type levels of lytic replication in vitro and normal establishment of latency in B cells early following acute infection in vivo. In contrast, during acute infection of immunodeficient mice, TMER-deficient γHV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that γHV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate γHV-induced infection and pathogenesis.

Importance: The gammaherpesviruses (γHVs) are a subfamily of viruses associated with chronic inflammatory diseases and cancer, particularly in immunocompromised individuals. These viruses uniformly encode multiple types of noncoding RNAs (ncRNAs) that are not translated into proteins. It remains unclear how virus-expressed ncRNAs influence the course and outcome of infection in vivo. Here, we generated a mouse γHV that lacks the expression of multiple ncRNAs. Notably, this mutant virus is critically impaired in the ability to cause disease in immunocompromised hosts yet shows a paradoxical increase in infected cells early during infection in these hosts. While the original mouse virus encodes multiple ncRNAs, the expression of a single domain of one ncRNA can partially reverse the defects of the mutant virus. These studies demonstrate that γHV ncRNAs can directly contribute to virus-induced disease in vivo and that these RNAs may be multifunctional, allowing the opportunity to specifically interfere with different functional domains of these RNAs.
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http://dx.doi.org/10.1128/mBio.01670-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337559PMC
February 2015