scholarly journals Identifying HIV-1 RNA splice variant protein interactomes using HyPR-MSSV

2020 ◽  
Author(s):  
Rachel A. Knoener ◽  
Edward L. Evans ◽  
Jordan T. Becker ◽  
Mark Scalf ◽  
Bayleigh E. Benner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Splice Variant ◽  
Rna Stability ◽  
Mass Spectrometric ◽  
Host Protein ◽  
Single Population ◽  
Mass Spectrometric Identification ◽  
Variant Protein ◽  
Hiv 1

ABSTRACTHIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs; each playing distinct roles in viral replication. Elucidating their host protein “interactomes” is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes; including, preferential association of regulators of RNA stability with US- and PS-transcripts and, unexpectedly, mitochondria-linked proteins with US-transcripts. Remarkably, >80 of these factors screened by siRNA knock-down impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.

scholarly journals Identification of host proteins differentially associated with HIV-1 RNA splice variants

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Rachel Knoener ◽  
Edward Evans ◽  
Jordan T Becker ◽  
Mark Scalf ◽  
Bayleigh Benner ◽  
...  
Keyword(s):  
Splice Variants ◽  
Rna Stability ◽  
Host Protein ◽  
Host Proteins ◽  
Single Population ◽  
Mass Spectrometric Identification ◽  
Sirna Knockdown ◽  
Variant Protein ◽  
Hiv 1

HIV-1 generates unspliced (US), partially spliced (PS), and completely spliced (CS) classes of RNAs, each playing distinct roles in viral replication. Elucidating their host protein ‘interactomes’ is crucial to understanding virus-host interplay. Here, we present HyPR-MSSV for isolation of US, PS, and CS transcripts from a single population of infected CD4+ T-cells and mass spectrometric identification of their in vivo protein interactomes. Analysis revealed 212 proteins differentially associated with the unique RNA classes, including preferential association of regulators of RNA stability with US and PS transcripts and, unexpectedly, mitochondria-linked proteins with US transcripts. Remarkably, >80 of these factors screened by siRNA knockdown impacted HIV-1 gene expression. Fluorescence microscopy confirmed several to co-localize with HIV-1 US RNA and exhibit changes in abundance and/or localization over the course of infection. This study validates HyPR-MSSV for discovery of viral splice variant protein interactomes and provides an unprecedented resource of factors and pathways likely important to HIV-1 replication.

scholarly journals Identification of a Novel Human Immunodeficiency Virus Type 1 Integrase Interactor, Gemin2, That Facilitates Efficient Viral cDNA Synthesis In Vivo

2006 ◽  
Vol 80 (12) ◽  
pp. 5670-5677 ◽  
Author(s):  
Seiji Hamamoto ◽  
Hironori Nishitsuji ◽  
Teruo Amagasa ◽  
Mari Kannagi ◽  
Takao Masuda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Host Protein ◽  
Cdna Synthesis ◽  
Immunodeficiency Virus ◽  
Viral Cdna ◽  
Hiv 1

ABSTRACT Retroviral integrase (IN) catalyzes the integration of viral cDNA into a host chromosome. Additional roles have been suggested for IN, including uncoating, reverse transcription, and nuclear import of the human immunodeficiency virus type 1 (HIV-1) genome. However, the underlying mechanism is largely unknown. Here, using a yeast two-hybrid system, we identified a survival motor neuron (SMN)-interacting protein 1 (Gemin2) that binds to HIV-1 IN. Reduction of Gemin2 with small interfering RNA duplexes (siGemin2) dramatically reduced HIV-1 infection in human primary monocyte-derived macrophages and also reduced viral cDNA synthesis. In contrast, siGemin2 did not affect HIV-1 expression from the integrated proviral DNA. Although Gemin2 was undetectable in cell-free viral particles, coimmunoprecipitation experiments using FLAG-tagged Gemin2 strongly suggested that Gemin2 interacts with the incoming viral genome through IN. Further experiments reducing SMN or other SMN-interacting proteins suggested that Gemin2 might act on HIV-1 either alone or with unknown proteins to facilitate efficient viral cDNA synthesis soon after infection. Thus, we provide the evidence for a novel host protein that binds to HIV-1 IN and facilitates viral cDNA synthesis and subsequent steps that precede integration in vivo.

scholarly journals Intracellular HIV-1 Tat protein represses constitutive LMP2 transcription increasing proteasome activity by interfering with the binding of IRF-1 to STAT1

2006 ◽  
Vol 396 (2) ◽  
pp. 371-380 ◽  
Author(s):  
Anna L. Remoli ◽  
Giulia Marsili ◽  
Edvige Perrotti ◽  
Eleonora Gallerani ◽  
Ramona Ilari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mhc Class I ◽  
Consensus Sequence ◽  
Class I ◽  
Tat Protein ◽  
Cellular Functions ◽  
Infected Cells ◽  
Cryptic Epitopes ◽  
Hiv 1

The Tat protein is the transcriptional activator of HIV-1 gene expression, which is not only essential for viral replication, but also important in the complex HIV-induced pathogenesis of AIDS, as both an intracellular and an extracellular released protein. Accordingly, Tat is able to profoundly affect cellular gene expression, regulating several cellular functions, also in non-infected cells. We showed recently that Tat induces modification of immunoproteasomes in that it up-regulates LMP7 (low-molecular-mass polypeptide 7) and MECL1 (multicatalytic endopeptidase complex-like 1) subunits and down-modulates the LMP2 subunit, resulting in a change in the generation and presentation of epitopes in the context of MHC class I. In particular, Tat increases presentation of subdominant and cryptic epitopes. In the present study, we investigated the molecular mechanism responsible for the Tat-induced LMP2 down-regulation and show that intracellular Tat represses transcription of the LMP2 gene by competing with STAT1 (signal transducer and activator of transcription 1) for binding to IRF-1 (interferon-regulatory factor-1) on the overlapping ICS-2 (interferon consensus sequence-2)–GAS (γ-interferon-activated sequence) present in the LMP2 promoter. This element is constitutively occupied in vivo by the unphosphorylated STAT1–IRF-1 complex, which is responsible for the basal transcription of the gene. Sequestration of IRF-1 by intracellular Tat impairs the formation of the complex resulting in lower LMP2 gene transcription and LMP2 protein expression, which is associated with increased proteolytic activity. On the other hand, extracellular Tat induces the expression of LMP2. These effects of Tat provide another effective mechanism by which HIV-1 affects antigen presentation in the context of the MHC class I complex and may have important implications in the use of Tat for vaccination strategies.

scholarly journals Acetylation of cytidine residues boosts HIV-1 gene expression by increasing viral RNA stability

2020 ◽  
Author(s):  
Kevin Tsai ◽  
Ananda Ayyappan Jaguva Vasudevan ◽  
Cecilia Martinez Campos ◽  
Ann Emery ◽  
Ronald Swanstrom ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Stability ◽  
Antiviral Drug ◽  
Viral Gene Expression ◽  
Mrna Translation ◽  
Viral Gene ◽  
Viral Mrnas ◽  
Cellular Target ◽  
Covalent Modifications ◽  
Hiv 1

AbstractCovalent modifications added to individual nucleotides on mRNAs, called epitranscriptomic modifications, have recently emerged as key regulators of both cellular and viral mRNA function1,2 and RNA methylation has now been shown to enhance the replication of human immunodeficiency virus 1 (HIV-1) and several other viruses3–11. Recently, acetylation of the N4 position of cytidine (ac4C) was reported to boost cellular mRNA function by increasing mRNA translation and stability12. We therefore hypothesized that ac4C and N-acetyltransferase 10 (NAT10), the cellular enzyme that adds ac4C to RNAs, might also have been subverted by HIV-1 to increase viral gene expression. We now confirm that HIV-1 transcripts are indeed modified by addition of ac4C at multiple discreet sites and demonstrate that silent mutagenesis of a subset of these ac4C addition sites inhibits HIV-1 gene expression in cis. Moreover, reduced expression of NAT10, and the concomitant decrease in the level of ac4C on viral RNAs, inhibits HIV-1 replication by reducing HIV-1 RNA stability. Interestingly Remodelin, a previously reported inhibitor of NAT10 function13,14, also inhibits HIV-1 replication without affecting cell viability, thus raising the possibility that the addition of ac4C to viral mRNAs might emerge as a novel cellular target for antiviral drug development.

scholarly journals Derivation of simian tropic HIV-1 infectious clone reveals virus adaptation to a new host

2019 ◽  
Vol 116 (21) ◽  
pp. 10504-10509 ◽  
Author(s):  
Fabian Schmidt ◽  
Brandon F. Keele ◽  
Gregory Q. Del Prete ◽  
Dennis Voronin ◽  
Christine M. Fennessey ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Infectious Clone ◽  
Host Protein ◽  
Peripheral Blood Mononuclear ◽  
New Host ◽  
Species Specific ◽  
Hiv 1 ◽  
Pigtail Macaques

To replicate in a new host, lentiviruses must adapt to exploit required host factors and evade species-specific antiviral proteins. Understanding how host protein variation drives lentivirus adaptation allowed us to expand the host range of HIV-1 to pigtail macaques. We have previously derived a viral swarm (in the blood of infected animals) that can cause AIDS in this new host. To further exploit this reagent, we generated infectious molecular clones (IMCs) from the viral swarm. We identified clones with high replicative capacity in pigtail peripheral blood mononuclear cells (PBMC) in vitro and used in vivo replication to select an individual IMC, named stHIV-A19 (for simian tropic HIV-1 clone A19), which recapitulated the phenotype obtained with the viral swarm. Adaptation of HIV-1 in macaques led to the acquisition of amino acid changes in viral proteins, such as capsid (CA), that are rarely seen in HIV-1–infected humans. Using stHIV-A19, we show that these CA changes confer a partial resistance to the host cell inhibitor Mx2 from pigtail macaques, but that complete resistance is associated with a fitness defect. Adaptation of HIV-1 to a new host will lead to a more accurate animal model and a better understanding of virus–host interactions.

scholarly journals Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability

2020 ◽  
Vol 28 (2) ◽  
pp. 306-312.e6 ◽  
Author(s):  
Kevin Tsai ◽  
Ananda Ayyappan Jaguva Vasudevan ◽  
Cecilia Martinez Campos ◽  
Ann Emery ◽  
Ronald Swanstrom ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Stability ◽  
Viral Rna ◽  
Hiv 1

scholarly journals Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability

2020 ◽  
Author(s):  
Kevin Tsai ◽  
Ananda Ayyappan Jaguva Vasudevan ◽  
Cecilia Martinez Campos ◽  
Ann Emery ◽  
Ronald Swanstrom ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Stability ◽  
Viral Rna ◽  
Hiv 1
Sign in / Sign up

Export Citation Format

Share Document