Doctor of Philosophy / Biology / Stefan Rothenburg / Poxviruses are large double-stranded DNA viruses that collectively exhibit a broad host range. Whereas many members of the poxvirus family are capable of infecting various host species, others are restricted to only one or a very limited numbers of species, such as variola virus, which is the causative agent of smallpox and is restricted to humans. Since the entry of poxviruses is not dependent upon any specific receptors, the cell tropism is therefore fully determined by the virus’ ability to manipulate the cellular signaling networks that are responsible for antagonizing viral infections. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a unique antiviral protein found in most vertebrates, which serves both as a virus sensor by detecting the presence of dsRNA and an antiviral effector by suppressing cap-dependent translation during virus infection. Many viruses, including poxviruses, have therefore evolved genes that encode for PKR inhibitors, such as vaccinia virus K3L, which shows sequence homology to the N-terminal region of the eukaryotic translation initiation factor 2α (eIF2α), the substrate of PKR. K3L is able to inhibit PKR-mediated eIF2α phosphorylation in vitro and in vivo. Because K3L was shown to be indispensable for virus replication in Syrian hamster cells but not in human cells, it was categorized as a host range factor. However, the molecular basis for K3L’s host range function is not fully understood. We examined the interactions of poxvirus K3L orthologs, especially vaccinia virus K3L and M156R, the K3L ortholog in the rabbit-specific myxoma virus, and PKR from a variety of host species in multiple assays, and found that K3L and M156R inhibit PKR in a species-specific manner, which likely contributes to the cell tropism and host range for both viruses. Inactivation of M156R or K3L led to virus attenuation in cells, which could be rescued by ectopic expression of viral PKR inhibitors. We also identified the helix αG region as the main molecular determinant for PKR’s sensitivity to inhibition by K3L orthologs. In conclusion, the research summarized here indicates that the interactions of PKR and poxvirus pseudosubstrate inhibitors play important roles in virus host range and virulence.

Doctor of Philosophy / Department of Biology / Stefan Rothenburg / Vaccinia virus is the prototypic poxvirus that has been widely used as a model for investigating poxvirus biology and genetics. Like several members of the Poxviridae family, vaccinia virus can infect several different species including mice, cows and humans. Because the entry of poxviruses into a host cell relies on ubiquitously expressed surface molecules, which are found in many species, the ability of poxviruses to infect and replicate in different host cells primarily depends on their ability to subvert the host’s innate immune response. One critical barrier to infection is overcoming the general shutdown of protein translation initiated by the cellular protein kinase PKR. PKR detects cytoplasmic double-stranded (ds) RNA generated during infection by the replicating virus, which activates it to phosphorylate the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2) and suppress general translation. Poxviruses are large viruses with dsDNA genomes that encode around 200 genes. Several of these genes are known as host range genes and are important for replication in different host species and many interact with components of the host immune response to promote viral replication. Two genes in vaccinia virus, called E3L and K3L, are known inhibitors of PKR and have previously been shown to be important for virus replication in cells from different species. The molecular explanation behind their host range function, however, is unknown. The main goal of the research presented in this thesis is to determine the molecular mechanisms for the host range function of vaccinia virus E3L and K3L, particularly in different hamster host cells. Along with an analysis of vaccinia virus host range genes, we have used genome-wide comparisons between host-restricted poxviruses in the Leporipoxvirus genus to parse out the potential genomic determinants of host range restriction in this clade of poxviruses. The overarching aim of this thesis work is to better understand the molecular mechanisms for host range in poxviruses.

Tuberculosis (TB) is a deadly infectious lung disease caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb). The identification of macrophage signaling proteins exploited by Mtb during infection will enable the development of alternative host-directed therapies (HDT) for TB. HDT strategies will boost host immunity to restrict the intracellular replication of Mtb and therefore hold promise to overcome antimicrobial resistance, a growing crisis in TB therapy. Protein Kinase R (PKR) is a key host sensor that functions in the cellular antiviral response. However, its role in defense against intracellular bacterial pathogens is not clearly defined. Herein, we demonstrate that expression and activation of PKR is upregulated in macrophages infected with Mtb. Immunological profiling of human THP-1 macrophages that overexpress PKR (THP-PKR) showed increased production of IP-10 and reduced production of IL-6, two cytokines that are reported to activate and inhibit IFNy-dependent autophagy, respectively. Indeed, sustained expression and activation of PKR reduced the intracellular survival of Mtb, an effect that could be enhanced by IFNy treatment. We further demonstrate that the enhanced anti-mycobacterial activity of THP-PKR macrophages is mediated by a mechanism dependent on selective autophagy as indicated by increased levels of LC3-II that colocalize with intracellular Mtb. Consistent with this mechanism, inhibition of autophagolysosome maturation with bafilomycin A1 abrogated the ability of THP-PKR macrophages to limit replication of Mtb, whereas pharmacological activation of autophagy enhanced the anti-mycobacterial effect of PKR overexpression. As such, PKR represents a novel and attractive host target for development of HDT for TB, and our data suggest value in the design of more specific and potent activators of PKR.