The research in my laboratory focuses on the elucidation of the role of the 2',5'-oliogadenylate [2-5A] synthetase/RNase L and p68 kinase [PKR] pathways in the development of antiviral and antiproliferative states in mammalian cells. The 2-5A synthetase/RNase L and PKR pathways are part of the antiviral defense mechanism in mammalian cells. These double-stranded RNA-dependent, interferon-inducible enzyme pathways are also involved in the regulation of cell growth and differentiation.

The understanding and restructuring of the mechanisms by which antiviral host defense pathways control HIV-1 replication is a major research effort in Dr. Suhadolnik's laboratory. HIV-1 infection results in the downregulation of the antiviral cellular defense pathways. Two strategies are being employed to control HIV-1 infection and to explore the basic biology that occurs between 2-5A synthetase, RNase L, PKR and HIV-1. First, a group of nuclease-resistant, non-toxic, biologically active 2-5A derivatives has been developed. These structurally and stereochemically modified 2-5A derivatives are being used as biological probes to explore the molecular mechanisms of development of the antiviral state in HIV-1 infected cells. Second, the principle of intracellular immunization of cells against virus infection is being applied to HIV-1 replication. Cellular regulatory genes, including 2-5A synthetase and PKR, are put under control of a promoter that is selectively expressed in HIV-1 infected cells. Current anti-HIV-1 therapies have been limited by the emergence of drug-resistant HIV-1 variants and the reactivation of latent HIV-1 reservoirs. As pivotal components of the natural antiviral pathway in mammalian cells, the 2-5OAS and PKR gene products are less likely to be subject to host immune surveillance or to be affected by mutations of HIV-1. We have designed and tested a HIV-based lentiviral vector encoding the PKR transgene as an anti-HIV-1 approach with promising results. This vector effectively transduced PKR into CD34+ hematopoietic stem cells (HSC) and resulted in inhibition of HIV-1 replication in CD34+-derived T lymphocyte progeny. Expression and activity of PKR in the transduced HSC had no adverse effects on T cell differentiation. Our results demonstrate the feasibility of delivery of the PKR transgene to HSC by a lentiviral vector as a gene therapy approach for HIV-1 disease. This approach is being continued with self-inactivating lentiviral vectors. Studies are underway to determine the effects of transduction of 2-5OAS and PKR cDNA-containing constructs and mutants on the reactivation of HIV-1 in latently-infected cells. In a related project, studies are underway to define the capacity of opioid agonists to modulate host-pathogen interactions during HIV-1 infection.

In another project, Dr. Suhadolnik seeks to further define the capacity of opioid agonists to modulate host-pathogen interactions during HIV-1 infection. Certain opioids act to increase the replication of HIV-1 in infected cells. The opioids exert a significant impact on the expression of the very critical chemokines and chemokine receptor genes and have the capacity to regulate, not only trafficking of immune cells to sites of infection, but also the regulation of chemokine receptors which serve as critical HIV-1 co-receptors. The working hypothesis is that the opioid-induced increase in HIV-1 replication can be reversed by natural cellular antiviral defense mechanisms. Studies are underway to evaluate the impact of opioids on the antiviral pathways of target cells and to define the capacity of opioids to modulate the expression of critical cytokines/cytokine receptors. Overall, these studies should provide a rational basis for strategies to control the disease process at the level of replication and the level of cellular mobilization/uptake of the virus.

Another major focus of research in Dr. Suhadolnik's laboratory concerns chronic fatigue syndrome (also known as CFS or CFIDS). Chronic fatigue syndrome is an illness of unknown etiology that is associated with sudden onset, flu-like symptoms, debilitating fatigue, low-grade fever, myalgia and neurocognitive dysfunction. Dr. Suhadolnik has reported biochemical abnormalities in the 2-5A synthetase/RNase L and PKR antiviral defense mechanisms in peripheral blood mononuclear cells from individuals with CFS. These findings are consistent with the reactivation of one or more viruses in individuals with CFS and subsequent dysregulation of the antiviral defense pathways. A major research effort in the laboratory focuses on the isolation and characterization of a novel low molecular weight (37-kDa) form of 2-5A-dependent RNase L from CFS peripheral blood mononuclear cells. In recent studies, we have demonstrated that the 37-kDa form of RNase L shares structural and functional features with the native 80-kDa RNase L, in particular, at the 2-5A binding and catalytic domains. Even though the 80-kDa and the 37-kDa RNase L both bind 2-5A and hydrolyze single-stranded RNA in a 2-5A-dependent manner, these two forms of RNase L have different kinetic parameters for 2-5A-dependent activation. We have recently reported enzyme kinetic data demonstrating that the rate of RNA hydrolysis by the 37-kDa RNase L is three times faster than that of the 80-kDa RNase L. Continuing characterization of the structural and functional relationship between the 80-kDa and 37-kDa forms of RNase L is designed to provide insight into the role that alterations in the 2-5OAS/RNase L pathway play in the pathogenesis of CFS.

A recent study examined a number of attributes of clinical presentation, functional status, immune function and the RNase L pathway in a cohort of CFS patients and two well-defined control populations. Our results are consistent with the immune activation model of CFS and do not support the contention that CFS is simply a form of depression. The abnormalities demonstrated in the RNase L pathway, i.e., increased 37/80 kDa RNase L ratio, can reliably identify a relatively homogeneous subset of patients within the larger group that comply with the working definition of CFS. Observation of an elevated 37/80 kDa RNase L ratio is a quantitative measure that correlates well with the severity of CFS symptoms and with low natural killer (NK) cell function.