Human immunodeficiency virus infection is a chronic condition causing significant changes in the immune system, which are reflected in the altered gene expression patterns of the immune cells. By studying these patterns through gene expression profiling it is possible to describe not only the current states the cells are in, but also to extrapolate the proximal signals that resulted in the observed patterns. In the studies described herein, we have applied this approach to better understand the alterations in the immune function that occur in HIV infection. First, we have obtained transcriptional profiles of CD4+ and CD8+ T cells from patients in early infection, in chronic infection, and in non-progressive infection, and we compared these profiles to each other and to the profiles from uninfected donors. The analyses of the profiles revealed no discernable changes in the T cells of the non-progressive patients when compared to the uninfected individuals. On the other hand, T cells from patients with progressive infection, both early and late, showed patterns characteristic of type I interferon (IFN) exposure. We next examined experimentally the possible proximal causes of the observed transcriptional profiles. We analyzed the gene expression patterns induced by TGFβ, 13 type I interferons, as well as recombinant HIV Tat protein, in T cells and peripheral blood mononuclear cells. The TGFβ responses were inconsistent with the transcriptional profiles seen in HIV-infected patients, whereas both type I IFNs and HIV Tat induced genes in patterns consistent with those seen in patients. In fact, the thirteen IFN-induced patterns were indistinguishable from each other. Tat treatments induced interferon-stimulated genes (ISGs) as well as other genes and the response was not dependent on the presence of plasmacytoid dendritic cells (pDCs), suggesting monocytes as the possible source of the interferon response. In the last study, we examined the responses of plasmacytoid dendritic cells (pDCs) to HIV and other stimuli in healthy and HIV-infected subjects. We observed induction of IFN genes in pDCs of all subjects in response to influenza virus and TLR7 agonist imiquimod, but not to HIV virus. In summary, HIV infection results in chronic induction of type I IFN response in cells of the immune system. The source of this response is likely to be type I IFNs produced by monocytes/macrophages rather than plasmacytoid cells. The monocytic production of type I IFN may be a Tat-dependent response.

Human immunodeficiency virus infection is a chronic condition causing significant changes in the immune system, which are reflected in the altered gene expression patterns of the immune cells. By studying these patterns through gene expression profiling it is possible to describe not only the current states the cells are in, but also to extrapolate the proximal signals that resulted in the observed patterns. In the studies described herein, we have applied this approach to better understand the alterations in the immune function that occur in HIV infection. First, we have obtained transcriptional profiles of CD4+ and CD8+ T cells from patients in early infection, in chronic infection, and in non-progressive infection, and we compared these profiles to each other and to the profiles from uninfected donors. The analyses of the profiles revealed no discernable changes in the T cells of the non-progressive patients when compared to the uninfected individuals. On the other hand, T cells from patients with progressive infection, both early and late, showed patterns characteristic of type I interferon (IFN) exposure. We next examined experimentally the possible proximal causes of the observed transcriptional profiles. We analyzed the gene expression patterns induced by TGFβ, 13 type I interferons, as well as recombinant HIV Tat protein, in T cells and peripheral blood mononuclear cells. The TGFβ responses were inconsistent with the transcriptional profiles seen in HIV-infected patients, whereas both type I IFNs and HIV Tat induced genes in patterns consistent with those seen in patients. In fact, the thirteen IFN-induced patterns were indistinguishable from each other. Tat treatments induced interferon-stimulated genes (ISGs) as well as other genes and the response was not dependent on the presence of plasmacytoid dendritic cells (pDCs), suggesting monocytes as the possible source of the interferon response. In the last study, we examined the responses of plasmacytoid dendritic cells (pDCs) to HIV and other stimuli in healthy and HIV-infected subjects. We observed induction of IFN genes in pDCs of all subjects in response to influenza virus and TLR7 agonist imiquimod, but not to HIV virus. In summary, HIV infection results in chronic induction of type I IFN response in cells of the immune system. The source of this response is likely to be type I IFNs produced by monocytes/macrophages rather than plasmacytoid cells. The monocytic production of type I IFN may be a Tat-dependent response.

Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...

Oncolytic viruses (OVs) have made tremendous strides as a viable cancer therapeutic in recent years; however, variable infectivity rates have since limited clinical efficacy. Residual type 1 interferon (IFN-1) responses are integral to the tumour’s innate antiviral defense and confer resistance to OVs. To combat this, small molecules with viral sensitizing ability can be used in combination to transiently knockdown IFN-1 responses, allowing OVs to gain a foothold for increased infectivity and therapeutic efficacy. Accordingly, we hypothesize that some chemical or genetic manipulations of cellular processes can indirectly antagonize antiviral IFN-1 responses and modulate pro-inflammatory pathways to potentiate oncolytic virotherapy. In this thesis, we identify several avenues to modify cell signalling events to increase OV therapeutic efficacy through IFN-1 inhibition. Firstly, with respect to the demonstrated OV-enhancing effects of vanadium, a pan-phosphatase (PP) inhibitor, we elucidate that its IFN-1 suppressing activity involves activation of the epidermal growth factor receptor (EGFR) pathway via STAT1/2 and NF-κB. Pharmacological inhibition of EGFR abrogated vanadium’s viral sensitizing ability in vivo. Secondly, using high-throughput screening methodology, we identify protein phosphatases that inherently regulate the IFN-1 response as targets for oncolytic vesicular stomatitis virus (VSV∆51) potentiation. Indeed, cloning interfering RNA against one of these PP targets, acid phosphatase 2 (ACP2), into the VSV∆51 platform demonstrated superior infectivity and cancer cell cytotoxicity compared to the non-targeting VSV∆51 control. Thirdly, we characterize pevonedistat, a first in-class neddylation activating enzyme inhibitor, to potentiate OV therapeutic efficacy across several in vitro and in vivo contexts. We demonstrate pevonedistat’s ability to inhibit IFN-1 signalling and pro-inflammatory cytokine production using both neddylation independent and dependent mechanisms. Taken altogether, we dissect multiple signaling mechanisms by which the IFN-1 response can be modulated for the purposes of improving OV therapeutic efficacy. This knowledge can subsequently be directly translated into designing optimized OV strategies for clinical testing.