Host Cell Redox Alterations Promote Latent HIV-1 Reactivation through Atypical Transcription Factor Cooperativity.

Immune cell state alterations rewire HIV-1 gene expression, thereby influencing viral latency and reactivation, but the mechanisms are still unfolding. Here, using a screen approach on CD4+ T cell models of HIV-1 latency, we revealed Small Molecule Reactivators (SMOREs) with unique chemistries altering the CD4+ T cell state and consequently promoting latent HIV-1 transcription and reactivation through an unprecedented mechanism of action. SMOREs triggered rapid oxidative stress and activated a redox-responsive program composed of cell-signaling kinases (MEK-ERK axis) and atypical transcription factor (AP-1 and HIF-1α) cooperativity. SMOREs induced an unusual AP-1 phosphorylation signature to promote AP-1/HIF-1α binding to the latent HIV-1 proviral genome for its activation. Consistently, latent HIV-1 reactivation was compromised with pharmacologic inhibition of oxidative stress sensing or of cell-signaling kinases, and transcription factor's loss of expression, thus functionally linking the host redox-responsive program to viral transcriptional rewiring. Notably, SMOREs induced the redox program in primary CD4+ T cells and reactivated latent HIV-1 in aviremic patient samples alone and in combination with known latency-reversing agents, thus providing physiological relevance. Our findings suggest that manipulation of redox-sensitive pathways could be exploited to alter the course of HIV-1 latency, thus rendering host cells responsive to help achieve a sterilizing cure.

ADAP1 promotes latent HIV-1 reactivation by selectively tuning KRAS-ERK-AP-1 T cell signaling-transcriptional axis.

Immune stimulation fuels cell signaling-transcriptional programs inducing biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states; however, the regulatory mechanisms are still unfolding. Here, we implemented a functional screen leveraging HIV-1's dependence on CD4+ T cell signaling-transcriptional programs and discovered ADAP1 is an undescribed modulator of HIV-1 proviral fate. Specifically, we report ADAP1 (ArfGAP with dual PH domain-containing protein 1), a previously thought neuronal-restricted factor, is an amplifier of select T cell signaling programs. Using complementary biochemical and cellular assays, we demonstrate ADAP1 inducibly interacts with the immune signalosome to directly stimulate KRAS GTPase activity thereby augmenting T cell signaling through targeted activation of the ERK-AP-1 axis. Single cell transcriptomics analysis revealed loss of ADAP1 function blunts gene programs upon T cell stimulation consequently dampening latent HIV-1 reactivation. Our combined experimental approach defines ADAP1 as an unexpected tuner of T cell programs facilitating HIV-1 latency escape.

HIV-1 Proviral Transcription and Latency in the New Era.

Three decades of extensive work in the HIV field have revealed key viral and host cell factors controlling proviral transcription. Various models of transcriptional regulation have emerged based on the collective information from in vitro assays and work in both immortalized and primary cell-based models. Here, we provide a recount of the past and current literature, highlight key regulatory aspects, and further describe potential limitations of previous studies. We particularly delve into critical steps of HIV gene expression including the role of the integration site, nucleosome positioning and epigenomics, and the transition from initiation to pausing and pause release. We also discuss open questions in the field concerning the generality of previous regulatory models to the control of HIV transcription in patients under suppressive therapy, including the role of the heterogeneous integration landscape, clonal expansion, and bottlenecks to eradicate viral persistence. Finally, we propose that building upon previous discoveries and improved or yet-to-be discovered technologies will unravel molecular mechanisms of latency establishment and reactivation in a "new era".

Transcriptional Circuit Fragility Influences HIV Proviral Fate.

Transcriptional circuit architectures in several organisms have been evolutionarily selected to dictate precise given responses. Unlike these cellular systems, HIV is regulated through a complex circuit composed of two successive phases (host and viral), which create a positive feedback loop facilitating viral replication. However, it has long remained unclear whether both phases operate identically and to what extent the host phase influences the entire circuit. Here, we report that, although the host phase is regulated by a checkpoint whereby KAP1 mediates transcription activation, the virus evolved a minimalist system bypassing KAP1. Given the complex circuit's architecture, cell-to-cell KAP1 fluctuations impart heterogeneity in the host transcriptional responses, thus affecting the feedback loop. Mathematical modeling of a complete circuit reveals how these oscillations ultimately influence homogeneous reactivation potential of a latent virus. Thus, although HIV drives molecular innovation to fuel robust gene activation, it experiences transcriptional fragility, thereby influencing viral fate and cure efforts.

The RNA uridyltransferase Zcchc6 is expressed in macrophages and impacts innate immune responses.

Alveolar macrophages orchestrate pulmonary innate immunity and are essential for early immune surveillance and clearance of microorganisms in the airways. Inflammatory signaling must be sufficiently robust to promote host defense but limited enough to prevent excessive tissue injury. Macrophages in the lungs utilize multiple transcriptional and post-transcriptional mechanisms of inflammatory gene expression to delicately balance the elaboration of immune mediators. RNA terminal uridyltransferases (TUTs), including the closely homologous family members Zcchc6 (TUT7) and Zcchc11 (TUT4), have been implicated in the post-transcriptional regulation of inflammation from studies conducted in vitro. In vivo, we observed that Zcchc6 is expressed in mouse and human primary macrophages. Zcchc6-deficient mice are viable and born in Mendelian ratios and do not exhibit an observable spontaneous phenotype under basal conditions. Following an intratracheal challenge with S. pneumoniae, Zcchc6 deficiency led to a modest but significant increase in the expression of select cytokines including IL-6, CXCL1, and CXCL5. These findings were recapitulated in vitro whereby Zcchc6-deficient macrophages exhibited similar increases in cytokine expression due to bacterial stimulation. Although loss of Zcchc6 also led to increased neutrophil emigration to the airways during pneumonia, these responses were not sufficient to impact host defense against infection.

Access of HIV-2 to CD169-dependent dendritic cell-mediated trans infection pathway is attenuated.

The mechanisms behind the low viral loads and lower mortality rates of HIV-2(+) individuals remain unknown. We hypothesized that reduced interaction of HIV-2 with CD169, the primary HIV-1 attachment factor on monocyte-derived dendritic cells (DCs) that targets captured virus particles to the trans infection pathway, contributes to its diminished pathogenic phenotype in vivo. We observed a significant decrease in capture of HIV-2 Gag-eGFP virus-like particles (VLPs) and infectious GFP-containing HIV-2 particles compared to corresponding HIV-1 particles by CD169(+) mature DCs. Interestingly, there was decreased co-localization of HIV-2 with HIV-1 Gag at plasma membrane microdomains in virus producer cells which correlated with reduced incorporation of GM3, the CD169 ligand, in HIV-2 virions, and reduction in mature DC-mediated HIV-2 trans infection compared to HIV-1. We conclude that limited interaction of HIV-2 with CD169 diminishes virus access to the mature DC-mediated trans infection pathway and might result in attenuated HIV-2 dissemination in vivo.

Dressing up Nanoparticles: A Membrane Wrap to Induce Formation of the Virological Synapse.

Next-generation nanoparticle-based drug delivery systems require the ability to target specific organelles or subcellular regions in selected target cells. Human immunodeficiency virus type I (HIV-1) particles are evolutionarily optimized nanocarriers that have evolved to avoid intracellular degradation and achieve enrichment at the synapse between mature dendritic cells (mDCs) and T cells by subverting cellular trafficking mechanisms. This study demonstrates that integration of the glycosphingolipid, GM3, in a membrane around a solid nanoparticle (NP) core is sufficient to recapitulate key aspects of the virus particle trafficking in mDCs. GM3-presenting artificial virus NPs (GM3-AVNs) accumulate in CD169(+) and CD81(+) nonlysosomal compartments in an actin-dependent process that mimics the sequestration of HIV-1. Live-cell optical tracking studies reveal a preferential recruitment and arrest of surface scanning CD4(+) T cells in direct vicinity to the AVN-enriched compartments. The formed mDC-T cell conjugates exhibit strong morphological similarities between the GM3-AVN-containing mDC-T cell synapse and the HIV-1 virological synapse, indicating that GM3-CD169 interactions alone are sufficient for establishing the mDC-T cell virological synapse. These results emphasize the potential of the GM3-AVN approach for providing therapeutic access to a key step of the host immune response--formation of the synaptic junction between an antigen-presenting cell (mDC) and T cells--for modulating and controlling immune responses.

Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells.

Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

Virus particle release from glycosphingolipid-enriched microdomains is essential for dendritic cell-mediated capture and transfer of HIV-1 and henipavirus.

UNLABELLED: Human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DCs) to promote its transmission to T cells. We recently reported that the capture of HIV-1 by mature dendritic cells (MDCs) is mediated by an interaction between the glycosphingolipid (GSL) GM3 on virus particles and CD169/Siglec-1 on MDCs. Since HIV-1 preferentially buds from GSL-enriched lipid microdomains on the plasma membrane, we hypothesized that the virus assembly and budding site determines the ability of HIV-1 to interact with MDCs. In support of this hypothesis, mutations in the N-terminal basic domain (29/31KE) or deletion of the membrane-targeting domain of the HIV-1 matrix (MA) protein that altered the virus assembly and budding site to CD63(+)/Lamp-1-positive intracellular compartments resulted in lower levels of virion incorporation of GM3 and attenuation of virus capture by MDCs. Furthermore, MDC-mediated capture and transmission of MA mutant viruses to T cells were decreased, suggesting that HIV-1 acquires GSLs via budding from the plasma membrane to access the MDC-dependent trans infection pathway. Interestingly, MDC-mediated capture of Nipah and Hendra virus (recently emerged zoonotic paramyxoviruses) M (matrix) protein-derived virus-like particles that bud from GSL-enriched plasma membrane microdomains was also dependent on interactions between virion-incorporated GSLs and CD169. Moreover, capture and transfer of Nipah virus envelope glycoprotein-pseudotyped lentivirus particles by MDCs were severely attenuated upon depletion of GSLs from virus particles. These results suggest that GSL incorporation into virions is critical for the interaction of diverse enveloped RNA viruses with DCs and that the GSL-CD169 recognition nexus might be a conserved viral mechanism of parasitization of DC functions for systemic virus dissemination. IMPORTANCE: Dendritic cells (DCs) can capture HIV-1 particles and transfer captured virus particles to T cells without establishing productive infection in DCs, a mechanism of HIV-1 trans infection. We have recently identified CD169-mediated recognition of GM3, a host-derived glycosphingolipid (GSL) incorporated into the virus particle membrane, as the receptor and ligand for the DC-HIV trans infection pathway. In this study, we have identified the matrix (MA) domain of Gag to be the viral determinant that governs incorporation of GM3 into HIV-1 particles, a previously unappreciated function of the HIV-1 MA. In addition, we demonstrate that the GSL-CD169-dependent trans infection pathway is also utilized as a dissemination mechanism by henipaviruses. GSL incorporation in henipaviruses was also dependent on the viral capsid (M) protein-directed assembly and budding from GSL-enriched lipid microdomains. These findings provide evidence of a conserved mechanism of retrovirus and henipavirus parasitization of cell-to-cell recognition pathways for systemic virus dissemination.