Disruption of LEDGF/p75-directed integration derepresses antisense transcription of the HIV-1 genome.

Disruption of HIV-1 Integrase (IN) interactions with the host-factor Lens Epithelium-Derived Growth Factor (LEDGF)/p75 leads to decreased, random integration, increased latent infection, and described here, accumulation of HIV-1 antisense RNA (asRNA). asRNA increase was observed following interruptions of IN-LEDGF/p75 interactions either through pharmacologic perturbations of IN-LEDGF/p75 by treatment with allosteric HIV-1 integrase inhibitors (ALLINIs) or in cell lines with LEDGF genetic knockout. Additionally, by impairing Tat-dependent HIV transcription, asRNA abundance markedly increases.

Potent broadly neutralizing antibodies mediate efficient antibody-dependent phagocytosis of HIV-infected cells.

Antibody-dependent cellular phagocytosis (ADCP) has been implicated in protection against HIV-1. However, methods for measuring ADCP currently rely on the phagocytosis of gp120- or gp41-coated beads that do not reflect physiologically relevant conformations of the viral envelope glycoprotein or the size of a virus-infected cell. We therefore developed a novel approach for measuring ADCP of HIV-infected cells expressing natural conformations of Env.

Human lysyl-tRNA synthetase phosphorylation promotes HIV-1 proviral DNA transcription.

Human lysyl-tRNA synthetase (LysRS) was previously shown to be re-localized from its normal cytoplasmic location in a multi-aminoacyl-tRNA synthetase complex (MSC) to the nucleus of HIV-1 infected cells. Nuclear localization depends on S207 phosphorylation but the nuclear function of pS207-LysRS in the HIV-1 lifecycle is unknown. Here, we show that HIV-1 replication was severely reduced in a S207A-LysRS knock-in cell line generated by CRISPR/Cas9; this effect was rescued by S207D-LysRS.

Retroviral hijacking of host transport pathways for genome nuclear export.

Recent advances in the study of virus-cell interactions have improved our understanding of how viruses that replicate their genomes in the nucleus (e.g., retroviruses, hepadnaviruses, herpesviruses, and a subset of RNA viruses) hijack cellular pathways to export these genomes to the cytoplasm where they access virion egress pathways.

Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells.

Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells.

HIV RGB: Automated Single-Cell Analysis of HIV-1 Rev-Dependent RNA Nuclear Export and Translation Using Image Processing in KNIME.

Single-cell imaging has emerged as a powerful means to study viral replication dynamics and identify sites of virus−host interactions. Multivariate aspects of viral replication cycles yield challenges inherent to handling large, complex imaging datasets. Herein, we describe the design and implementation of an automated, imaging-based strategy, “Human Immunodeficiency Virus Red-Green-Blue” (HIV RGB), for deriving comprehensive single-cell measurements of HIV-1 unspliced (US) RNA nuclear export, translation, and bulk changes to viral RNA and protein (HIV-1 Rev and Gag) subcellular distribution over time.

Substitutions in Nef That Uncouple Tetherin and SERINC5 Antagonism Impair Simian Immunodeficiency Virus Replication in Primary Rhesus Macaque Lymphocytes.

Most simian immunodeficiency viruses (SIVs) use Nef to counteract restriction by the tetherin proteins of their nonhuman primate hosts. In addition to counteracting tetherin, SIV Nef has a number of other functions, including the downmodulation of CD3, CD4, and major histocompatibility complex class I (MHC I) molecules from the surface of SIV-infected cells and the enhancement of viral infectivity by preventing the incorporation of SERINC5 into virions. Although these activities require different surfaces of Nef, they can be difficult to separate because of their dependence on similar interactions with AP-1 or AP-2 for clathrin-mediated endocytosis.

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies.

The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association.

HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

Unlabelled: HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores.

Cooperativity among Rev-associated nuclear export signals regulates HIV-1 gene expression and is a determinant of virus species tropism.

Unlabelled: Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions.