Showing papers by "Brian Wigdahl published in 2017"

Designing broad-spectrum anti-HIV-1 gRNAs to target patient-derived variants

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9), including specific guide RNAs (gRNAs), can excise integrated human immunodeficiency virus type 1 (HIV-1) provirus from host chromosomes. To date, anti-HIV-1 gRNAs have been designed to account for off-target activity, however, they seldom account for genetic variation in the HIV-1 genome within and between patients, which will be crucial for therapeutic application of this technology. This analysis tests the ability of published anti-HIV-1 gRNAs to cleave publicly available patient-derived HIV-1 sequences to inform gRNA design and provides basic computational tools to researchers in the field.

Specific amino acids in HIV-1 Vpr are significantly associated with differences in patient neurocognitive status

Abstract: Even in the era of combination antiretroviral therapies used to combat human immunodeficiency virus type 1 (HIV-1) infection, up to 50 % of well-suppressed HIV-1-infected patients are still diagnosed with mild neurological deficits referred to as HIV-associated neurocognitive disorders (HAND). The multifactorial nature of HAND likely involves the HIV-1 accessory protein viral protein R (Vpr) as an agent of neuropathogenesis. To investigate the effect of naturally occurring variations in Vpr on HAND in well-suppressed HIV-1-infected patients, bioinformatic analyses were used to correlate peripheral blood-derived Vpr sequences with patient neurocognitive performance, as measured by comprehensive neuropsychological assessment and the resulting Global Deficit Score (GDS). Our studies revealed unique associations between GDS and the presence of specific amino acid changes in peripheral blood-derived Vpr sequences [neuropsychological impairment Vpr (niVpr) variants]. Amino acids N41 and A55 in the Vpr sequence were associated with more pronounced neurocognitive deficits (higher GDS). In contrast, amino acids I37 and S41 were connected to measurably lower GDS. All niVpr variants were also detected in DNA isolated from HIV-1-infected brain tissues. The implication of these results is that niVpr variants alter the genesis and/or progression of HAND through differences in Vpr-mediated effects in the peripheral blood and/or the brain.

Human Immunodeficiency Virus (HIV) Infection and Cancer

Abstract: Nearly 37 million people were living worldwide with human immunodeficiency virus (HIV) infection in 2016, and about 2 million become infected every year. Acquired immunodeficiency syndrome (AIDS) is defined as HIV infection associated with an absolute CD4 count below 200/uL (age 6 and older) and/or an AIDS-defining opportunistic infection or AIDS-defined cancer, including Kaposi sarcoma (KS), non-Hodgkin lymphoma, and cervical cancer. About 40% of HIV-associated malignancies are associated with oncogenic viruses, including human papillomavirus (HPV), Epstein-Barr virus (EBV), human herpesvirus 8(HHV-8), and hepatitis B and C viruses. KS is an angioproliferative tumor that originates when HHV-8 infects cells of endothelial lineage and maintains replication during host cell division. The use of combination antiretroviral therapy is a cornerstone of therapy for patients with HIV-associated KS. Other key considerations in the management of KS include the location, extent, and type of skin lesions, and immune function. HIV infection itself appears not directly involved in carcinogenesis but results in disrupted immune surveillance of tumor antigens and oncogenic viruses. It also provides a milieu of chronic antigen stimulation, cytokine dysregulation, and genetic alterations, which is permissive for lymphomagenesis. EBV and HHV-8 are involved in lymphoma pathogenesis. The most common anogenital cancers in HIV-infected individuals include squamous cell carcinoma of the cervix (women) and anal canal (men and women). Both are almost always associated with HPV infection, are potentially curable when disease is localized, may be detected at precancerous stages and possibly prevented with local therapy, and potentially could be prevented with HPV vaccination.

Myelomonocytic Cell Lines in Modeling HIV-1 Infection of the Bone Marrow

Abstract: Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of acquired immunodeficiency syndrome (AIDS), primarily infects T cells and cells of the monocyte-macrophage lineage. This is due to the presence of the cell surface receptor CD4 and the coreceptors, CXCR4, and CCR5. While the T-cell has classically been the cell type associated with HIV-1 disease progression, cells of the monocyte-macrophage lineage have also been shown to play a major role in this viral pathologic process. Classically, this has involved monocytic cells in the peripheral blood and tissue macrophages, however, over the course of HIV disease, the promyelomonocytic cells of the bone marrow (BM) have also been shown to play a role in pathogenesis retroviral disease in that they play an integral role in the reseeding of the periphery and end-organ tissues. This has involved an initial infection of the bone marrow hematopoietic progenitor cells. Given this observation, over the years there have been a number of cell lines that have been developed and provided valuable insights into research questions surrounding HIV-1 infection of the monocyte-macrophage cell lineage. In this regard, we will examine the biological and immunological properties of these BM-derived cell lines with respect to their utility in exploring the pathogenesis of HIV-1 in humans.

cAMP Signaling Enhances HIV-1 Long Terminal Repeat (LTR)-directed Transcription and Viral Replication in Bone Marrow Progenitor Cells.

Abstract: CD34+ hematopoietic progenitor cells have been shown to be susceptible to HIV-1 infection, possibly due to a low-level expression of CXCR4, a coreceptor for HIV-1 entry. Given these observations, we have explored the impact of forskolin on cell surface expression of CXCR4 in a cell line model (TF-1). The elevation of intracellular cyclic adenosine monophosphate (cAMP) by forskolin through adenylyl cyclase (AC) resulted in transcriptional upregulation of CXCR4 with a concomitant increase in replication of the CXCR4-utilizing HIV-1 strain IIIB. Transient expression analyses also demonstrated an increase in CXCR4-, CCR5-, and CXCR4-/CCR5-utilizing HIV-1 (LAI, YU2, and 89.6, respectively) promoter activity. Studies also implicated the protein kinase A (PKA) pathway and the downstream transcription factor CREB-1 in interfacing with cAMP response elements located in the CXCR4 and viral promoter. These observations suggest that the cAMP signaling pathway may serve as a regulator of CXCR4 levels and concomitantly of HIV-1 replication in bone marrow (BM) progenitor cells.

Evidence of Divergent Amino Acid Usage in Comparative Analyses of R5- and X4-Associated HIV-1 Vpr Sequences

Abstract: Vpr is an HIV-1 accessory protein that plays numerous roles during viral replication, and some of which are cell type dependent To test the hypothesis that HIV-1 tropism extends beyond the envelope into the vpr gene, studies were performed to identify the associations between coreceptor usage and Vpr variation in HIV-1-infected patients Colinear HIV-1 Env-V3 and Vpr amino acid sequences were obtained from the LANL HIV-1 sequence database and from well-suppressed patients in the Drexel/Temple Medicine CNS AIDS Research and Eradication Study (CARES) Cohort Genotypic classification of Env-V3 sequences as X4 (CXCR4-utilizing) or R5 (CCR5-utilizing) was used to group colinear Vpr sequences To reveal the sequences associated with a specific coreceptor usage genotype, Vpr amino acid sequences were assessed for amino acid diversity and Jensen-Shannon divergence between the two groups Five amino acid alphabets were used to comprehensively examine the impact of amino acid substitutions involving side chains with similar physiochemical properties Positions 36, 37, 41, 89, and 96 of Vpr were characterized by statistically significant divergence across multiple alphabets when X4 and R5 sequence groups were compared In addition, consensus amino acid switches were found at positions 37 and 41 in comparisons of the R5 and X4 sequence populations These results suggest an evolutionary link between Vpr and gp120 in HIV-1-infected patients

Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site:

Abstract: Previous studies have identified a CCAAT/enhancer binding protein (C/EBP) site located downstream of the transcriptional start site (DS3). The role of the DS3 element with respect to HIV-1 transactivation by Tat and viral replication has not been characterized. We have demonstrated that DS3 was a functional C/EBPβ binding site and mutation of this site to the C/EBP knockout DS3-9C variant showed lower HIV-1 long terminal repeat (LTR) transactivation by C/EBPβ. However, it was able to exhibit similar or even higher transcription levels by Tat compared to the parental LTR. C/EBPβ and Tat together further enhanced the transcription level of the parental LAI-LTR and DS3-9C LTR, with higher levels in the DS3-9C LTR. HIV molecular clone viruses carrying the DS3-9C variant LTR demonstrated a decreased replication capacity and delayed rate of replication. These results suggest that DS3 plays a role in virus transcriptional initiation and provides new insight into C/EBP regulation of HIV-1.