Showing papers by "Brian Wigdahl published in 2019"

Genetic variation and function of the HIV-1 Tat protein

Abstract: Human immunodeficiency virus type 1 (HIV-1) encodes a transactivator of transcription (Tat) protein, which has several functions that promote viral replication, pathogenesis, and disease. Amino acid variation within Tat has been observed to alter the functional properties of Tat and, depending on the HIV-1 subtype, may produce Tat phenotypes differing from viruses’ representative of each subtype and commonly used in in vivo and in vitro experimentation. The molecular properties of Tat allow for distinctive functional activities to be determined such as the subcellular localization and other intracellular and extracellular functional aspects of this important viral protein influenced by variation within the Tat sequence. Once Tat has been transported into the nucleus and becomes engaged in transactivation of the long terminal repeat (LTR), various Tat variants may differ in their capacity to activate viral transcription. Post-translational modification patterns based on these amino acid variations may alter interactions between Tat and host factors, which may positively or negatively affect this process. In addition, the ability of HIV-1 to utilize or not utilize the transactivation response (TAR) element within the LTR, based on genetic variation and cellular phenotype, adds a layer of complexity to the processes that govern Tat-mediated proviral DNA-driven transcription and replication. In contrast, cytoplasmic or extracellular localization of Tat may cause pathogenic effects in the form of altered cell activation, apoptosis, or neurotoxicity. Tat variants have been shown to differentially induce these processes, which may have implications for long-term HIV-1-infected patient care in the antiretroviral therapy era. Future studies concerning genetic variation of Tat with respect to function should focus on variants derived from HIV-1-infected individuals to efficiently guide Tat-targeted therapies and elucidate mechanisms of pathogenesis within the global patient population.

Novel gRNA design pipeline to develop broad-spectrum CRISPR/Cas9 gRNAs for safe targeting of the HIV-1 quasispecies in patients.

Abstract: The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.