Browsing by Subject "HIV-1"

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ADAP1 Promotes Latent HIV-1 Reactivation by Tuning the KRAS-ERK-AP-1 Signaling-Transcriptional Axis
(December 2021) Ramirez, Nora-Guadalupe Piña; Schoggins, John W.; D'Orso, Iván; Pfeiffer, Julie K.; Alto, Neal
Immune stimulation fuels cell signaling-transcriptional programs that induce biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states. However, many regulatory mechanisms are still unfolding. As such, here I take advantage of the unique intrinsic reliance HIV-1 has on host cell signaling-transcriptional programs to discover undescribed cell signaling regulators. Specifically, I implemented a functional screening platform, given HIV-1 gene expression relies on CD4+ T cell activation state, to identify host factors modulating CD4+ T cell signaling-transcriptional axes and consequently HIV-1 fate. Among the hits, I focus on ADAP1 (ArfGAP with Dual PH Domains 1), a previously thought neuro-restricted factor, and discover it is an amplifier of select human CD4+ T cell signaling programs. Using physiological models, I characterize ADAP1 expression is low in naïve and memory CD4+ T cells, but largely induced upon immune stimulation where it interacts with the immune signalosome. Using complementary biochemical and cellular assays, I demonstrate ADAP1 directly stimulates the GTPase activity of KRAS to amplify CD4+ T cell signaling through targeted activation of ERK-AP-1 axis. In primary CD4+ T cells which I have genetically ablated ADAP1, I show loss of ADAP1 function blunts gene expression programs in response to stimulation thereby reducing CD4+ T cell expansion and dampening latent HIV-1 reactivation. Supporting the impact of these findings, I propose the reduced CD4+ T cell programs and proliferation upon ADAP1 loss validates Genome-wide Association Studies linking ADAP1 single nucleotide polymorphisms in non-coding enhancers to an altered T lymphocyte count trait, potentially attributed to ADAP1 haploinsufficiency. Through these combined experimental approaches, I was able to define ADAP1 as an unexpected tuner of CD4+ T cell activation programs and co-opted by HIV-1 to escape latency.
Anti-retroviral therapy of HIV-1 infection
(2003-10-30) Keiser, Philip
Does HIV-1 infection always cause AIDS?: long-term survivors and exposed-uninfected individuals
(1997-03-06) Kazi, Salahuddin
Eradicating the latent reservoir of HIV: finding light at the end of the tunnel
(2005-01-13) Margolis, David M.
Functional Characterization of the HIV-1 NEF Acidic Cluster
(2005-05-12) Baugh, Laura Louise; Garcia-Martinez, J. Victor
The human immunodeficiency virus (HIV) infects over 39.5 million people worldwide (UNAIDS/WHO 2006 Epidemic Report). Unfortunately, various socioeconomic factors have hastened the epidemic spread of HIV in Africa and in third world countries where many individuals do not have access to appropriate healthcare. Vaccines have, as of yet, been unsuccessful as a preventive measure; however, new measures, including peptide inhibitors and microbicides, are currently being developed and studied for their efficacy. The potential of finding new targets for controlling or preventing infection relies heavily on developments relating to the basic science of the virus as well as the host environment. HIV and simian immunodeficiency virus (SIV) encode an accessory protein Nef proposed to promote pathogenesis based on evidence from HIV-infected longterm nonprogressors and SIV studies in non-human primates. The pathogenic potential of Nef has been studied in relation to some of its in vitro effects including: (1) the downregulation of major histocompatibility complex I (MHC-I), (2) the downregulation of CD4, (3) infectivity enhancement, and (4) Pak2 activation. These in vitro effects, with the exception of CD4 downregulation, rely in part upon Nef's acidic cluster which has been reported to affect Nef's redirection of cell surface MHC-I to the trans-Golgi network by binding to the cytosolic adapter phosphofurin acidic cluster sorting-1 (PACS-1) (19, 147). Because Nef is potentially significant to the course of disease progression, correlations between the amino acid composition of the Nef acidic cluster and Nef activity were characterized. Nef acidic cluster mutants reducing the acidic residues in the cluster from 4 to 2, 1, and 0 were developed. Phenotypes for these mutants indicate at least one or more of the four acidic cluster glutamates are involved not only in MHC-I downregulation, but also Pak2 activation and infectivity enhancement. However, because partial activity is observed with one acidic residue and full activity with only two acidic residues, these data lead to the following conclusion: Although the Nef acidic cluster contributes to a conformational integrity important for Nef-mediated MHC-I downregulation as well as other Nef activities, it is not a prototypical acidic cluster determinant involved in PACS-1-mediated trafficking.
HIV associated lymphomas
(2013-09-13) Naina, Harris V.
New paradigms in the history of highly active antiretroviral therapy for HIV-1 infection
(2009-09-11) Sinclair, Gary
Omnia retrorsum: the other human retroviruses
(1987-07-30) Baine, William B.
Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2
(2009-06-19) Kuo, Lillian S.; Garcia-Martinez, J. Victor
Nef is an accessory protein encoded by HIV-1 that activates the host cellular p21 activated protein kinase 2 (PAK-2). Previous work has characterized the structural plasticity of Nef with regard to PAK-2 activation. Residues 89 and 191 were identified to be components of an effector domain required for Nef mediating PAK-2 activation with lesser contributions from position 85 and 188. H89 and F191 are highly conserved in subtype B Nefs (LHKF), however in subtype E Nef F89 and R191 predominate. Subtype E Nefs also activate PAK-2, therefore it appeared at least two different structural variants are present in HIV-1 Nefs. Substitution of all four residues in a subtype B Nef with subtype E-like residues (F85, F89, A188 and R191, FFAR) generated a fully functional subtype E PAK-2 effector domain in a subtype B background. A third effector domain found in subtype C Nefs (F85, F89, H188, and H191, FFHH) was also investigated. The contribution of residues 187 and 188 in these alternative Nef structural variants (LHKF, FFAR, and FFHH) to activate PAK-2 was determined. Surprisingly, the L188 substitution in the LHKF structure resulted in PAK-2 hyperactivation. While the I187 substitution in LHKF completely ablated PAK-2 activity. In stark contrast, I187 in the FFHH variant resulted in hyperactivation. Thus, subtle changes in amino acid composition can dramatically affect kinase activation levels. The work in this thesis has characterized a PAK-2 effector domain on Nef constituted by amino acid position 85, 89, 187, 188 and 191. The results indicate that this is not the only Nef region mediating PAK-2 activation. The highly conserved polyproline helix also plays a role in the activation of PAK-2. Conservative mutations of this SH3 binding region completely abrogated PAK-2 activation suggesting SH3 binding is necessary, however this binding appears to be weak. My data suggest a model where activation of PAK-2 by Nef requires a ternary, or higher order, complex containing SH3/Nef/PAK-2. Synergistic interactions between the two Nef effector domains investigated here and a host cell protein, or proteins, could explain the specific activation of PAK-2 by Nef.
Toward the Rational Design of Better Antivirals: The Development of cGAMP as an HIV-1 Anti-Retroviral and the Genetic Surveillance of WNV Evolution
(2017-04-17) Aroh, Chukwuemika Nnabuike; Pasare, Chandrashekar; Wakeland, Edward K.; Yan, Nan; Schoggins, John W.; Pfeiffer, Julie K.
The innate immune response is the first line of defense against pathogens and thus represents the first hurdle viruses must overcome to cause severe disease in humans. Understanding the consequences of viral evolution can give insights to mechanisms of viral pathogenesis as well as the development of novel therapeutics. Here I studied two clinically important viruses: Human Immunodeficiency Virus (HIV) and West Nile Virus (WNV). HIV-1 has evolved several mechanisms to evade immune detection by the cGAS-STING cytosolic DNA sensing pathway. A small cyclic di-nucleotide, cGAMP, activates the same pathway by directly binding STING. Treatment with cGAMP, delivered by ultra-pH sensitive nanoparticles or by liposomes, in human peripheral blood mononuclear cells (PBMCs) induced potent and long-acting protection against replication of several laboratory-adapted and clinical HIV-1 isolates in contrast to the short-lasting effect of current anti-retroviral therapy (ARTs). These results present the first evidence for potentially developing cGAMP or other STING agonists as a long-acting antiretroviral immunotherapy. West Nile Virus (WNV) is a mosquito-borne Flavivirus which was introduced to North America in 1999 and is currently the leading cause of viral encephalitis. The lack of specific therapeutics or human vaccines makes WNV an ongoing public health threat. Now endemic, WNV is steadily evolving, but the contribution of positively-selected mutations to human disease remains unclear. In 2012 the second largest outbreak of human West Nile disease occurred in the U.S., with one-third of the cases happening in Texas. The outbreak was associated with groups of WNV carrying positively-selected mutations. By sequencing WNV in Texas from 2012-2015, we show that positively-selected mutations in WNV mediate increased circulation and over-wintering in the environment, which may promote increases of human disease. Additionally, we show evidence that the WNV population is still evolving new alleles. These results advance our understanding of the impact of WNV evolution to human disease, and may afford insights to the evolution of other invading flaviviruses, such as Dengue and Zika virus. Altogether, these results show that understanding the consequences of viral evolution can be harnessed towards overcoming challenges to the development of more effective therapeutics.