All ETDs from UAB

Advisory Committee Chair

Elena I Frolova

Advisory Committee Members

Allan J Zajac

Hubert M Tse

Ilya V Frolov

Qiana L Matthews

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Evolution of vertebrates has resulted in the development of a wide range of cellular pattern recognition receptors (PRRs), which detect the presence of virus-specific molecules termed pathogen-associated molecular patterns (PAMPs). Sensing of the incoming viruses and/or their replication by PRRs activates the cascade of cellular pathways that ultimately result in activation of antiviral response. Such response is determined by hundreds of cellular genes, whose products either directly interfere with virus replication or are released from the cells and activate antiviral state in yet uninfected cells. Consequently, the latter cell signaling prevents development of the next rounds of infection both in vivo and in vitro. Viruses, in turn, have developed numerous mechanisms of interfering with PRR-mediated response to sustain infection at the levels required for their transmission to next susceptible vertebrate hosts or insect vectors. Further understanding of virus-induced diseases, and designing of efficient and highly attenuated vaccines and new therapeutic means requires detailed understanding of both viral and cellular components in virus-host interactions. The rationale of our study was i) to dissect the mechanism(s) of PRR-mediated activation of cellular response by replicating alphaviruses, ii) to define particular PRRs and virus-specific PAMPs involved in this process and iii) to identify activated cellular pathway(s). On the other hand, we intended to advance our knowledge of the mechanism of alphavirus interference with development of antiviral response and to define the means of its inactivation. Our results have unambiguously demonstrated that retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated protein 5 (MDA5) are the only PRRs that sense replicating alphavirus RNAs. Expression of either of them is sufficient for alphavirus detection and mounting the antiviral response and cell signaling. However, the knock out of expression of both genes makes the cell no longer capable of inducing type I IFN. Stable knock-in (KI) cell lines, which produced different, biologically relevant levels of RIG-I and/or MDA5, revealed that the efficiency of alphavirus sensing and activation of type I IFN response are directly correlated with the intracellular concentrations of these PRRs at the time of infection. Continuous lines of the murine fibroblasts contain very low levels of RIG-I and MDA5, while in the primary cells they are present at higher concentration. These data provided a plausible explanation for often detected discrepancies between the results of in vivo and in vitro studies of the mechanism of induction of the innate immune response. Despite encoding only a handful of proteins, alphaviruses have developed the means of interfering with cellular antiviral response and cell signaling. The main components of these virus-specific countermeasures are transcriptional and translational shutoffs, which blocks activation and expression of cellular genes. These alphavirus-specific inhibitory functions appear to also be major contributors to the development of cytopathic effect (CPE) during replication in vertebrate cells. In the second line of our research, we applied a variety of newly developed experimental systems to define the molecular mechanism of alphavirus-induced transcription and translation inhibition and CPE development and dissected the roles of virus-specific proteins in these processes. We identified particular fragments in the specific domains of Sindbis virus (SINV) nonstructural protein 2 (nsP2) and nsP3 proteins, which play critical roles in inhibition of transcription and translation, respectively. Defined mutations in these peptides had deleterious effects on the development of transcriptional and translational shutoffs without affecting virus replication rates. These structural and functional data are applicable to other Old World (OW) alphaviruses and are currently being used for designing attenuated, noncytopathic alphaviruses that are incapable of interfering with the innate immune response. These viral mutants will serve as a platform for future studies of alphavirus-host interactions and development of attenuated vaccine candidates.

Share

COinS