In-Vitro Subtype-Specific Modulation of HIV-1 Trans-Activator of Transcription (Tat) on RNAi Silencing Suppressor Activity and Cell Death

Human immunodeficiency virus (HIV) is a global health concern affecting millions of individuals with a wide variety of currently circulating subtypes affecting various regions of the globe. HIV relies on multiple regulatory proteins to modify the host cell to promote replication in infected T cells, and these regulatory proteins can have subtle phenotypic differences between subtypes. One of these proteins, HIV-1 Trans-Activator of Transcription (Tat), is capable of RNA interference (RNAi) Silencing Suppressor (RSS) activity and induction of cell death in T cells. However, the subtype-specific RSS activity and induction of cell death have not been explored. We investigated the ability of Tat subtypes and variants to induce RSS activity and cell death. TatB, from HIV-1 subtype B, was found to be a potent RSS activator by 40% whereas TatC, from HIV-1 subtype C, showed 15% RSS activity while subtype TatC variants exhibited varying levels. A high level of cell death (50–53%) was induced by subtype TatB when compared to subtype TatC (25–28%) and varying levels were observed with subtype TatC variants. These differential activities could be due to variations in the functional domains of Tat. These observations further our understanding of subtype-specific augmentation of Tat in HIV-1 replication and pathogenesis

In silico Analyses of Subtype Specific HIV-1 Tat-TAR RNA Interaction Reveals the Structural Determinants for Viral Activity

HIV-1 Tat transactivates viral genes through strong interaction with TAR RNA. The stem-loop bulged region of TAR consisting of three nucleotides at the position 23–25 and the loop region consisting of six nucleotides at the position 30–35 are essential for viral transactivation. The arginine motif of Tat (five arginine residues on subtype TatC) is critically important for TAR interaction. Any mutations in this motif could lead to reduce transactivation ability and pathogenesis. Here, we identified structurally important residues (arginine and lysine residues) of Tat in this motif could bind to TAR via hydrogen bond interactions which is critical for transactivation. Natural mutant Ser46Phe in the core motif could likely led to conformational change resulting in more hydrogen bond interactions than the wild type Tat making it highly potent transactivator. Importantly, we report the possible probabilities of number of hydrogen bond interactions in the wild type Tat and the mutants with TAR complexes. This study revealed the differential transactivation of subtype B and C Tat could likely be due to the varying number of hydrogen bonds with TAR. Our data support that the N-terminal and the C-terminal domains of Tat is involved in the TAR interactions through hydrogen bonds which is important for transactivation. This study highlights the evolving pattern of structurally important determinants of Tat in the arginine motif for viral transactivation

Autophagy Intertwines with Different Diseases—Recent Strategies for Therapeutic Approaches

Autophagy is a regular and substantial “clear-out process„ that occurs within the cell and that gets rid of debris that accumulates in membrane-enclosed vacuoles by using enzyme-rich lysosomes, which are filled with acids that degrade the contents of the vacuoles. This machinery is well-connected with many prevalent diseases, including cancer, HIV, and Parkinson’s disease. Considering that autophagy is well-known for its significant connections with a number of well-known fatal diseases, a thorough knowledge of the current findings in the field is essential in developing therapies to control the progression rate of diseases. Thus, this review summarizes the critical events comprising autophagy in the cellular system and the significance of its key molecules in manifesting this pathway in various diseases for down- or upregulation. We collectively reviewed the role of autophagy in various diseases, mainly neurodegenerative diseases, cancer, inflammatory diseases, and renal disorders. Here, some collective reports on autophagy showed that this process might serve as a dual performer: either protector or contributor to certain diseases. The aim of this review is to help researchers to understand the role of autophagy-regulating genes encoding functional open reading frames (ORFs) and its connection with diseases, which will eventually drive better understanding of both the progression and suppression of different diseases at various stages. This review also focuses on certain novel therapeutic strategies which have been published in the recent years based on targeting autophagy key proteins and its interconnecting signaling cascades

Genetic characterization of natural variants of Vpu from HIV-1 infected individuals from Northern India and their impact on virus release and cell death.

BackgroundGenetic studies reveal that vpu is one of the most variable regions in HIV-1 genome. Functional studies have been carried out mostly with Vpu derived from laboratory adapted subtype B pNL 4-3 virus. The rationale of this study was to characterize genetic variations that are present in the vpu gene from HIV-1 infected individuals from North-India (Punjab/Haryana) and determine their functional relevance.MethodsFunctionally intact vpu gene variants were PCR amplified from genomic DNA of HIV-1 infected individuals. These variants were then subjected to genetic analysis and unique representative variants were cloned under CMV promoter containing expression vector as well as into pNL 4-3 HIV-1 virus for intracellular expression studies. These variants were characterized with respect to their ability to promote virus release as well as cell death.ResultsBased on phylogenetic analysis and extensive polymorphisms with respect to consensus Vpu B and C, we were able to arbitrarily assign variants into two major groups (B and C). The group B variants always showed significantly higher virus release activity and exhibited moderate levels of cell death. On the other hand, group C variants displayed lower virus release activity but greater cell death potential. Interestingly, Vpu variants with a natural S61A mutation showed greater intracellular stability. These variants also exhibited significant reduction in their intracellular ubiquitination and caused greater virus release. Another group C variant that possessed a non-functional β-TrcP binding motif due to two critical serine residues (S52 and S56) being substituted with isoleucine residues, showed reduced virus release activity but modest cytotoxic activity.ConclusionsThe natural variations exhibited by our Vpu variants involve extensive polymorphism characterized by substitution and deletions that contribute toward positive selection. We identified two major groups and an extremely rare β-TrcP binding motif mutant that show widely varying biological activities with potential implications for conferring subtype-specific pathogenesis

The S61A mutation conferred enhanced intracellular stability.

<p>A) Equal amounts of various pCMV HA Vpu expression constructs were transfected into HEK-293T cells by Lipofectamine 2000 for forty eight hours. Thereafter cell lysate were subjected to immunoblotting to measure relative abundance of various Vpu variants. B) CHX-chase assay was performed to check kinetic stability of various Vpu variants by transfecting HEK-293T cells with various Vpu constructs and thereafter treated with 100 µg/ml of CHX for indicated time period and harvested for immunoblotting. C) HEK-293T cells were co-transfected with His-ubiquitin (His-Ub), and various Vpu constructs. After thirty six hours, cells were treated with MG132 for eight hours followed by lysis in denaturation buffer and then total ubiquitinated proteins were pulled down using Ni-NTA beads and Vpu ubiquitination was checked by immunoblotting using anti-HA antibody. These results are representative of three independent experiments.</p

Phenotypic characterization of natural Vpu variants with respect to induction of cell death upon infection.

<p>A) Infected MOLT-4 T Cells were harvested, stained with Propidium iodide (10 µg/ml) and analyzed by flow cytometry to determine the extent of cell death. The extent of cell death is indicated in upper right corner of each panel. The FACS data were analyzed by WinMDI 2.9 software. B) Equal amounts of lysate from infected cells were subjected to immunoblotting to measure Vpu expression. These results are representative of three independent experiments.</p

The clinical data for HIV-1 infected individuals from North India.

<p>The clinical data for HIV-1 infected individuals from North India.</p

The rate of accumulation of non-synonymous and synonymous substitution.

<p>The rate of accumulation of non-synonymous and synonymous substitution.</p

Genetic variation in HIV-1 <i>vpu</i> gene from North Indian HIV-1 infected individuals.

<p><b>A)</b>The phylogenetic tree was constructed using the Neighbor Joining (NJ) method with the Kimura two-parameter distance matrix. The first letter of the reference sequence denotes the type or subtype or CRF, the second letter denotes the country from sequence sampled and the third letter denotes the accession number. Filled triangle mark the subtype B variants and filled circles mark the subtype C variants sampled from North India. The accession numbers for Indian samples (NII-PGI-IND-vpu sequences) were marked within the brackets. The main supported clades were marked with asterisk (*) along the branch represents the bootstrap support >70%. The scale bar represents the evolutionary distance of 0.1 nucleotides per position in the sequence. <b>B)</b> Multiple sequence alignment of primary isolates of HIV-1 Vpu collected from HIV-1 infected individuals from North India<b>.</b> The color coding generated by software represents difference in color for amino acid with different physiochemical properties. Identical residues are represented as dots.</p