Upon infection of its host cell, human immunodeficiency virus (HIV) establishes a quiescent and non-productive state capable of spontaneous reactivation. Diverse cell types harboring the provirus form a latent reservoir, constituting a major obstacle to curing HIV. Here, we investigate the effects of latency reversal agents (LRAs) in an HIV-infected THP-1 monocyte cell line in vitro. We demonstrate that leading drug treatments synergize activation of the HIV long terminal repeat (LTR) promoter. We establish a latency model in THP-1 monocytes using a replication incompetent HIV reporter vector with functional Tat, and show that chromatin modifiers synergize with a potent transcriptional activator to enhance HIV reactivation, similar to T-cells. Furthermore, leading reactivation cocktails are shown to differentially affect latency reactivation and surface expression of chemokine receptor type 4 (CXCR4), leading to altered host cell migration. This study investigates the effect of chromatin-modifying LRA treatments on HIV latent reactivation and cell migration in monocytes. As previously reported in T-cells, epigenetic mechanisms in monocytes contribute to controlling the relationship between latent reactivation and cell migration. Ultimately, advanced “Shock and Kill” therapy needs to successfully target and account for all host cell types represented in a complex and composite latency milieu.
more »
« less
Cell size dependent migration of T-cells latently infected with HIV
Human immunodeficiency virus (HIV) preferentially infects T-lymphocytes by integrating into host DNA and forming a latent transcriptionally silent provirus. As previously shown, HIV-1 alters migration modes of T-lymphocytes by co-regulating viral gene expression with human C-X-C chemokine receptor-4 (CXCR4). Here, we show that motility of infected T-lymphocytes is cell size dependent. In cell migration assays, migrating cells are consistently larger than non-migrating cells. This effect is drug-treatment independent. The cell size dependent motility observed in a previously generated Jurkat latency model correlates with the motility of primary human CD4+ T-cells containing a modified HIV-1 full-length construct JLatd2GFP. In addition, large migrating T-cells, latently infected with HIV, show a slightly decreased rate of reactivation from latency. these results demonstrate that HIV reactivation is cell migration-dependent, where host cell size acts as a catalyst for altered migration velocity. We believe that host cell size controlled migration uncovers an additional mechanism of cellular controlled viral fate determination important for virus dissemination and reactivation from latency. This observation may provide more insights into viral-host interactions regulating cell migration and reactivation from latency and helps in the design and implementation of novel therapeutic strategies.
more »
« less
- Award ID(s):
- 1943740
- NSF-PAR ID:
- 10203866
- Date Published:
- Journal Name:
- Journal of life sciences
- Volume:
- 2
- Issue:
- 1
- ISSN:
- 2688-1020
- Page Range / eLocation ID:
- 1-8
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
ABSTRACT Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.more » « less
-
more » « less
Abstract HIV is difficult to eradicate due to the persistence of a long-lived reservoir of latently infected cells. Previous studies have shown that natural killer cells are important to inhibiting HIV infection, but it is unclear whether the administration of natural killer cells can reduce rebound viremia when anti-retroviral therapy is discontinued. Here we show the administration of allogeneic human peripheral blood natural killer cells delays viral rebound following interruption of anti-retroviral therapy in humanized mice infected with HIV-1. Utilizing genetically barcoded virus technology, we show these natural killer cells efficiently reduced viral clones rebounding from latency. Moreover, a kick and kill strategy comprised of the protein kinase C modulator and latency reversing agent SUW133 and allogeneic human peripheral blood natural killer cells during anti-retroviral therapy eliminated the viral reservoir in a subset of mice. Therefore, combinations utilizing latency reversal agents with targeted cellular killing agents may be an effective approach to eradicating the viral reservoir.
-
Upon treatment removal, spontaneous reactivation of latently infected T cells remains a major barrier toward curing HIV. Therapies that reactivate and clear the latent reservoir are only partially effective, while latency-promoting agents (LPAs) used to suppress reactivation and stabilize latency are understudied and lack diversity in their mechanisms of action. Here, we identify additional LPAs using a screen for gene-expression fluctuations (or “noise”) that drive cell-fate specification and control HIV reactivation from latency. Single-cell protein dynamics of a minimal HIV gene circuit were monitored with time-lapse fluorescence microscopy. We screened 1,806 drugs, out of which 279 modulate noise magnitude or half autocorrelation time. Next, we tested the strongest noise modulators in a Jurkat T cell latency model and discovered three LPAs that would be overlooked by quantifying their mean expression levels alone. The LPAs reduced reactivation of latency in both Jurkat and primary cell models when challenged by synergistic and potent combinations of HIV activators. The two strongest LPAs, NSC 401005 and NSC 400938, are structurally and functionally related to inhibitors of thioredoxin reductase, a protein involved in maintaining redox balance in host cells. Experiments with multiple functional analogs revealed two additional LPAs, PX12 and tiopronin, and suggest a potential LPA family, within which some are commercially available and Food and Drug Administration–approved. The LPAs presented here may provide new strategies to complement antiretroviral treatments. Screening for gene expression noise holds the potential for drug discovery in other diseases.more » « less
-
more » « less
Abstract In α1-antitrypsin–deficient HIV patients, an accelerated decline of CD4+ T cell numbers is observed, suggesting that α1-antitrypsin is a potential endogenous HIV inhibitor. In infected T lymphocytes, α1-antitrypsin potently blocks NF-κB activation and HIV-1 replication by directly interacting with IκBα in the cytosol, thereby altering its ubiquitination pattern. However, the mechanism of α1-antitrypsin entry into the cytosol, where IκBα locates, remains unclear. In the present study, we investigated the mechanism of α1-antitrypsin internalization in CD4+ T cells. Thus, primary CD4+ T cells were infected with HIV-1 and then incubated with α1-antitrypsin to detect its internalization. We found that CD4+ T cells internalized α1-antitrypsin through a clathrin-dependent endocytosis process. Next, intracellular α1-antitrypsin exerted the inhibitory effect on NF-κB activation and HIV-1 replication. On primary CD4+ T cells, α1-antitrypsin interacted with low-density lipoprotein receptor-related protein 1 to initiate the internalization. Inside CD4+ T lymphocytes, α1-antitrypsin was transported from the endosome to the lysosome and then released into the cytosol, where it is possible for α1-antitrypsin to directly interact with IκBα. These results together suggest that α1-antitrypsin internalization is a clathrin-dependent and low-density lipoprotein receptor-related protein 1–mediated endocytosis process. Internalized α1-antitrypsin is transported through the endosome–lysosome–cytosol routine to interact with cytosolic IκBα and block NF-κB activation and HIV-1 replication.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- The DOI is not currently available.
