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Browsing by Author "Wilusz, Carol, advisor"

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    Characterization of big brown bat (Eptesicus fuscus) rabies virus in a mouse model
    (Colorado State University. Libraries, 2011) Ndaluka, Christina, author; Bowen, Richard A., advisor; Wilusz, Carol, advisor; Webb, Colleen, committee member; Mason, Gary, committee member
    A majority of human rabies cases in the United States are either imported from countries where dog rabies is endemic or classified as cryptic human cases, where a route of exposure is not known. Notably, essentially all rabies virus (RV) variants associated with cryptic cases of human rabies are maintained in bats. Understanding how RV is maintained in populations of bats and characterizing the diversity of bat RV is thus a high priority problem for public health. Among the knowledge gaps related to bat rabies are understanding the variation in virulence within the population of a single variant and explaining the observation that a substantial number of healthy wild bats have neutralizing antibodies to RV, but no apparent clinical illness. The work described here was designed to address both of those issues. Nine RV isolates were isolated from big brown bats in Colorado and low-passage stocks of each were prepared. These isolates were evaluated for virulence, immunogenicity and salivary gland dissemination to investigate whether there were major differences in these characteristics within this virus population. Inoculated mice were maintained for 12 weeks after virus inoculation to assess mortality and were bled regularly to evaluate their humoral immune responses. Salivary glands from mice that developed clinical rabies were evaluated for virus replication as an indication for potential for further transmission. The dose of RV inoculated had a greater influence on the incubation period and mortality than the individual RV isolate. There was no difference in the humoral immune response in mice between those that were protected and those that succumbed to infection. The only salivary glands that were positive for RV replication were observed from mice in the high dose inoculation groups. Collectively, the results of this experiment indicated that there was low diversity in biologic behavior within the sample of Epitesicus fuscus viruses tested. The humoral immune response of mice to a big brown bat RV variant was explored to address the hypothesis that dose, route or frequency of inoculation may explain the prevalence of neutralizing rabies antibody seen in wild bat populations. Mice were inoculated via intramuscular, intradermal and intranasal routes, with two different low doses of virus and two inoculation schedules. The highest frequency of seroconversion was seen in mice inoculated intramuscularly with the higher of the two doses of RV. Mice that were inoculated intranasally experienced the highest mortality. Mice were rechallenged 3 months following the initial challenge with a high dose of virus intramuscularly to determine if the neutralizing rabies antibodies were protective and if priming of the immune system to RV had occurred in those that failed to seroconvert. The results of this experiment indicate that inoculation of low doses of virus by any of several routes can elicit a detectable humoral immune response without development of disease, which supports the hypothesis that exposure of wild bats to low doses of RV results in seroconversion without clinical disease.
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    Inhibition of the host 5'-3' RNA decay pathway is a novel mechanism by which flaviviruses influence cellular gene expression
    (Colorado State University. Libraries, 2014) Moon, Stephanie L., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor; Schenkel, Alan, committee member; Curthoys, Norman, committee member
    Host gene expression is an intricate process that requires many levels of regulation to allow the cell to react properly to a given stimulus or maintain homeostasis. One mechanism by which RNA viruses perturb host gene expression and potentially favor the allocation of host cell resources for viral proliferation is through interfering with cellular post-transcriptional processes. Furthermore, because viral RNAs must persist in the host cell cytoplasm to allow translation of viral proteins and ultimately viral replication, the same post-transcriptional processes that regulate host messenger RNAs (mRNAs) likely act on viral RNAs as well. The general RNA decay machinery in the cell serves as an important regulatory step for proper gene expression at the post-transcriptional level. Many RNA viruses have evolved unique mechanisms for dealing with the cellular RNA decay machinery to preserve their transcripts and ensure a productive infection. Flaviviruses contain positive-sense, single-stranded RNA genomes that are not polyadenylated. Therefore, these viral RNAs are likely recognized by the host cell as deadenylated, incongruous mRNAs and are likely substrates for the general cellular RNA decay machinery. Remarkably, flaviviruses including the dengue viruses (DENV) and West Nile virus (WNV) produce an abundant non-coding subgenomic RNA (sfRNA) during infection that is generated through incomplete degradation of the viral genome by the host 5'-3' exoribonuclease 1 (XRN1). We demonstrate that human and mosquito XRN1 stalls on highly structured, conserved elements in the 3' untranslated region of flaviviral RNAs, resulting in sfRNA formation. Furthermore, we determined that these sfRNAs act as competitive, reversible inhibitors of XRN1. Infected cells display several signs of sfRNA-dependent XRN1 dysfunction, including the accumulation of uncapped transcripts and an overall stabilization of host mRNAs. Additionally, sfRNA acts as a weak inhibitor of the host cell RNA interference (RNAi) pathway. We propose that sfRNA likely acts as a sponge for Argonaute-2 (AGO2) and DICER, and have determined that siRNA-mediated decay is suppressed in an sfRNA-dependent fashion in flavivirus-infected human cells. This suppression of the RNAi pathway appears to alter host gene expression to a limited extent, and may be especially important for viral replication in the mosquito vector. Other flaviviruses, including hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) do not form an sfRNA from their 3' untranslated regions, but they do contain highly structured 5' untranslated regions. Herein we show that aside from acting as internal ribosome entry sites, the 5' UTRs of HCV and BVDV also stall and inhibit XRN1. Therefore, flaviviruses, pestiviruses and hepaciviruses appear to inhibit a major mRNA decay pathway by suppressing XRN1 activity via highly structured viral RNAs. Consequences of XRN1 suppression during viral infection include the stabilization and upregulation of short-lived transcripts including those encoding oncogenes, angiogenic factors, and pro-inflammatory factors. Furthermore, we present evidence that WNV sfRNA may dysregulate the coordination between mRNA stability and transcription. Therefore, the suppression of XRN1 may potentially act as an important mechanism by which diverse viruses in the Flaviviridae induce pathogenesis by dysregulating cellular gene expression.
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    Repeated sequences encoding Cys2His2 zinc finger motifs influence mRNA polyadenylation and localization
    (Colorado State University. Libraries, 2017) Jalkanen, Aimee L., author; Wilusz, Carol, advisor; Wilusz, Jeffrey, advisor; Bailey, Susan, committee member; Bouma, Gerrit, committee member; Thamm, Douglas, committee member
    The Cysteine2 Histidine2 zinc finger (C2H2-ZNF) proteins are a vast family with over 700 members in primates, many of which are transcription factors with important roles in development, differentiation, cell cycle progression, and tumor suppression. Due to the sheer number of C2H2-ZNF proteins and their roles in modulating expression of other genes, any mechanism for coordinating their expression could have wide-ranging impacts on cell function and phenotype. Previously, a large subset of C2H2-ZNF transcripts were determined to have significant populations with short poly(A) tails. Here, we show that multiple C2H2-ZNF mRNAs accumulate with very short or undetectable poly(A) tails, even when newly transcribed. Furthermore, these C2H2-ZNF mRNAs are restricted to the nucleus. Reporter mRNAs with sequences from the ZNF12 open reading frame (ORF) and/or the 3' untranslated region (3' UTR) have short poly(A) tails and are retained in the nucleus. Deletion analysis suggests that repeated sequence elements in the ZNF12 mRNA that code for zinc finger protein motifs are important in controlling both poly(A) tail length and nuclear localization. Remnants of C2H2-ZNF motif sequences found in the ZNF12 3' UTR are also able to confer short poly(A) tails and nuclear retention. Finally, we use RNA-fluorescence in situ hybridization (RNA-FISH) to reveal that ZNF12 reporter transcripts are found in foci within the nucleus that could represent sites for storage or processing. Overall, our findings suggest repeated sequence elements encoding C2H2-ZNF protein motifs play a dual role as regulatory elements that may coordinate expression of the C2H2-ZNF protein family by controlling post-transcriptional events.
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    Sequential exposure to manganese and encephalitic viral infection causes a Parkinsonian phenotype likely mediated by astrogliosis
    (Colorado State University. Libraries, 2021) Hampton, Lucas, author; Wilusz, Carol, advisor; Vigh, Jozsef, committee member; Bouma, Gerrit, committee member
    Developmental exposure to environmental toxins increases neuronal susceptibility to injury from subsequent viral challenges. Neurodegenerative diseases such as Parkinson's Disease (PD) present with a neuroinflammatory component often linked to environmental risk factors— including toxic metals, chemicals, physical injury, and viral infection. While many of these risk factors are sufficient to cause a Parkinsonian disease state, none have been shown to be necessary, suggesting an underlying shared mechanism. Furthermore, for any individual risk factor, there is high variability regarding development and severity of the disease. One way to address these issues employs multiple risk factors to model the disease more accurately, although it is unclear how or why multiple unrelated insults have a compounding effect. Neuroinflammation is a shared consequence of the known environmental risk factors. Increased susceptibility to one insult following a challenge with another environmental toxicant may therefore be mediated by neuroinflammatory signaling cascades, a process largely regulated by glial cells, primarily astrocytes. Reactive astrocytes produce neuroinflammatory cytokines, the expression of which is governed by the transcription factor complex NF-κB, and its regulatory kinase, IKK2. The current study uses a two-hit model of environmental neurodegeneration, juvenile exposure to manganese (Mn) followed by adult infection with western equine encephalitic virus (WEEV). We found that WEEV alone produced a significant PD effect, evident by immunohistological staining of pathogenic markers and behavioral analyses; and that WEEV and Mn exposure partially enhanced this effect. These exposures were conducted in both wildtype mice and in astrocyte-specific knockout mice lacking nuclear factor Ikappa-B kinase subunit beta (IKK-KO), hypothesizing that innate immune inflammatory signaling in reactive astrocytes modulates neuroinflammation and neuronal injury following combined exposure to Mn and WEEV. In multi-hit and single exposure treatment groups, IKK-KO mice displayed reduced viral replication and had decreased α-synuclein protein aggregation, astrogliosis and neuronal loss in multiple brain regions including the substantia nigra pars compacta, suggesting that astrocyte-mediated neuroinflammation may be one mechanism by which developmental toxin exposure can potentiate vulnerability to subsequent viral infections. Given the relevance of metal toxicity and viral infection to public health, these results provide insight into disease etiology and support further exploration of neuroinflammation as a mechanism of neurodegenerative pathologies.
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    Sindbis virus usurps the cellular HUR protein to stabilize its transcripts and promote infections of mammalian and mosquito cells
    (Colorado State University. Libraries, 2010) Sokoloski, Kevin J., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor; Blair, Carol, committee member; Peersen, Olve, committee member; Quackenbush, Sandra, committee member
    Members of the genus Alphavirus are recognized as significant human pathogens. Infection of vertebrate hosts often results in febrile illness and occasionally severe encephalitis. The archetypical alphavirus is Sindbis virus, which we have utilized in these studies. The genomic and subgenomic RNAs of Sindbis virus strongly resemble cellular mRNAs as they are capped at their 5’ ends and polyadenylated at their 3’ termini. These features allow the viral RNAs to act like cellular mRNAs and make them prime substrates for the cellular mRNA decay machinery. Sindbis virus RNAs are indeed subject to degradation by the cellular mRNA decay machinery in cell culture models of infection. Nevertheless, they decay by a mechanism that is different from the majority of cellular mRNAs as the decay of Sindbis virus transcripts is predominantly deadenylation-independent. As cellular mRNAs are often regulated by elements present in their 3’ untranslated regions (UTR), we hypothesized that these viral 3’UTR elements were functioning similarly to cellular mRNA stability elements resulting in the enhancement of viral infection. The primary goal of the research described in this dissertation was to characterize in mechanistic detail how the Sindbis virus 3’UTR represses deadenylation. To this end we used both cell free extracts and tissue culture systems to assay the effects of the viral 3’UTR on transcript stability. Interestingly, multiple elements were found to be independently repressing deadenylation in mosquito cytoplasmic extracts. Further examination revealed that a major stability determinant was the U-rich element (URE) observed in the 3’UTR of many alphaviruses. The ability to repress deadenylation in our cell free extract system was similarly observed with the UREs of Venezuelan equine, eastern equine, western equine and Semliki Forest viruses. Taken together, these data strongly assert that the repression of deadenylation via the URE is evolutionarily conserved. Prior to this study, the URE had no ascribed function. The repression of deadenylation imparted by the URE correlated with the binding of a cellular 38kDa factor. This 38kDa factor was determined to be the cellular HuR protein. Both the human and mosquito HuR proteins were found to bind with high affinity to the Sindbis virus 3’UTR. Reduction of cellular HuR protein levels using RNAi resulted in an increase in the rate of viral RNA decay. Furthermore, a significant decrease in the titer of progeny virus was observed. A similar effect on viral titer was observed when the predominant HuR binding site, the URE, was deleted from the viral 3’UTR. Taken together these observations identify a novel Alphavirus/ host interface that significantly impacts viral biology. Furthermore these studies have confirmed our hypothesis that the members of genus Alphavirus have indeed evolved RNA stability elements that resemble cellular mRNA stability elements for the purpose of enhancing viral infection. Furthermore these studies identify a potential therapeutic anti-viral target - the cellular HuR protein.
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    The role of cellular RNA decay pathways in Sindbis virus infection
    (Colorado State University. Libraries, 2009) Garneau, Nicole L., author; Wilusz, Jeffrey, advisor; Wilusz, Carol, advisor
    Sindbis virus is the prototypic species of the Alphavirus genus. Members of this genus can cause febrile illness, arthritic pain and potentially fatal encephalitis. The alphaviral lifecycle generates single-stranded, positive-sense genomic and subgenomic RNAs which are capped on the 5' terminus, contain 5' and 3' untranslated regions (UTRs), and are polyadenylated at the 3' terminus. These characteristics make alphaviral RNAs similar in structure to cellular mRNAs. Such features allow alphaviruses, such as Sindbis, to benefit from the host cell translation process; however, they also could make the viral transcript vulnerable to the cellular mRNA decay enzymes. mRNA decay is a form of post-transcriptional regulation of gene expression found in both mammalian and mosquito hosts of Sindbis virus. The interaction between Sindbis viral RNAs and mRNA decay pathways was investigated in this dissertation. Using a novel in vivo viral RNA decay assay to accurately assess the rate of alphavirus RNA decay during infection, we found a correlation between Sindbis viral RNA stability and viral replication efficiency, demonstrating mRNA decay potentially represents a novel host cell restriction factor. We established that the RNAi pathway likely plays a dominant role in the decay of the viral RNAs during infection in mammalian cells. These data represent a novel demonstration that the RNAi pathway is potentially an effective antiviral response in the mammalian host as it is in the mosquito host. With the development of a highly sensitive method to assess poly(A) tail length, we were able to demonstrate the importance of the viral 3'UTR as a repressor of deadenylation of viral RNAs in vivo. Lastly, we found that Repeat Sequence Element 3 (RSE 3), the third and final in a series of three RSEs within the viral 3'UTR, hinders the processivity of the cellular deadenylases on viral RNAs in vitro, providing the first evidence for a function of this conserved alphaviral genome element. Taken together, these results shed light on the much understudied area of viral RNA decay. Our data support the notion that the interaction between viral RNAs and the cellular RNA decay machinery is very important to the biology of the virus.