Novel Tsg101 Binding Partners Regulate Viral L Domain Trafficking

Two decades ago, Tsg101, a component of the Endosomal Sorting Complexes Required for Transport (ESCRT) complex 1, was identified as a cellular factor recruited by the human immunodeficiency virus type 1 (HIV-1) to facilitate budding of viral particles assembled at the cell periphery. A highly conserved Pro-(Thr/Ser)-Ala-Pro [P(T/S)AP] motif in the HIV-1 structural polyprotein, Gag, engages a P(T/S)AP-binding pocket in the Tsg101 N-terminal domain. Since the same domain in Tsg101 that houses the pocket was found to bind mono-ubiquitin (Ub) non-covalently, Ub binding was speculated to enhance P(T/S)AP interaction. Within the past five years, we found that the Ub-binding site also accommodates di-Ub, with Lys63-linked di-Ub exhibiting the highest affinity. We also identified small molecules capable of disrupting Ub binding and inhibiting budding. The structural similarity of these molecules, prazoles, to nucleosides prompted testing for nucleic acid binding and led to identification of tRNA as a Tsg101 binding partner. Here, we discuss these recently identified interactions and their contribution to the viral assembly process. These new partners may provide additional insight into the control and function of Tsg101 as well as identify opportunities for anti-viral drug design.

SNX-3 mediates retromer-independent tubular endosomal recycling by opposing EEA-1-facilitated trafficking

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.

Final Exon Frameshift Biallelic PTPN23 Variants Are Associated with Microcephalic Complex Hereditary Spastic Paraplegia

The hereditary spastic paraplegias (HSPs) are a large clinically heterogeneous group of genetic disorders classified as ‘pure’ when the cardinal feature of progressive lower limb spasticity and weakness occurs in isolation and ‘complex’ when associated with other clinical signs. Here, we identify a homozygous frameshift alteration occurring in the last coding exon of the protein tyrosine phosphatase type 23 (PTPN23) gene in an extended Palestinian family associated with autosomal recessive complex HSP. PTPN23 encodes a catalytically inert non-receptor protein tyrosine phosphatase that has been proposed to interact with the endosomal sorting complex required for transport (ESCRT) complex, involved in the sorting of ubiquitinated cargos for fusion with lysosomes. In view of our data, we reviewed previously published candidate pathogenic PTPN23 variants to clarify clinical outcomes associated with pathogenic gene variants. This determined that a number of previously proposed candidate PTPN23 alterations are likely benign and revealed that pathogenic biallelic PTPN23 alterations cause a varied clinical spectrum comprising of complex HSP associated with microcephaly, which may occur without intellectual impairment or involve more severe neurological disease. Together, these findings highlight the importance of the inclusion of the PTPN23 gene on HSP gene testing panels globally.

Ilaprazole and other novel prazole-based compounds that bind Tsg101 inhibit viral budding of HSV-1/2 and HIV from cells

In many enveloped virus families, including HIV and HSV, a crucial, yet unexploited, step in the viral life cycle is releasing particles from the infected cell membranes. This release process is mediated by host ESCRT complex proteins, which are recruited by viral structural proteins and provides the mechanical means for membrane scission and subsequent viral budding. The prazole drug, tenatoprazole, was previously shown to bind to ESCRT complex member Tsg101 and to quantitatively block the release of infectious HIV-1 from cells in culture. In this report we show that tenatoprazole and a related prazole drug, ilaprazole, effectively block infectious Herpes Simplex Virus (HSV)-1/2 release from Vero cells in culture. By electron microscopy, we found that both prazole drugs block the transit of HSV particles through the cell nuclear membrane resulting in their accumulation in the nucleus. Ilaprazole also quantitatively blocks the release of HIV-1 from 293T cells with an EC50 of 0.8-1.2 μM, which is much more potent than tenatoprazole. Our results indicate that prazole-based compounds may represent a class of drugs with potential to be broad-spectrum antiviral agents against multiple enveloped viruses, by interrupting cellular Tsg101 interaction with maturing virus, thus blocking the budding process that releases particles from the cell. Importance These results provide the basis for the development of drugs that target enveloped virus budding that can be used ultimately to control multiple virus infections in humans.

Vtc5 is localized to the vacuole membrane by the conserved AP-3 complex to regulate polyphosphate synthesis in budding yeast

Polyphosphates (polyP) are energy-rich polymers of inorganic phosphates assembled into chains ranging from 3-1000s of residues in length. They are thought to exist in all cells on earth and play roles in an eclectic mix of functions ranging from phosphate homeostasis to cell signaling, infection control, and blood clotting. In the budding yeast Saccharomyces cerevisiae, polyP chains are synthesized by the vacuole-bound VTC complex, which synthesizes polyP while simultaneously translocating it into the vacuole lumen where it is stored at high concentrations. VTC's activity is promoted by an accessory subunit called Vtc5. In this work, we find that the conserved AP-3 complex is required for proper Vtc5 localization to the vacuole membrane. In human cells, previous work has demonstrated that mutation of AP-3 subunits gives rise to Hermansky-Pudlak Syndrome, a rare disease with molecular phenotypes that include decreased polyP accumulation in platelet dense granules. In yeast AP-3 mutants, we find that Vtc5 is rerouted to the vacuole lumen by the ESCRT complex, where it is degraded by the vacuolar protease Pep4. Cells lacking functional AP-3 have decreased levels of polyP, demonstrating that membrane localization of Vtc5 is required for its VTC stimulatory activity in vivo. Our work provides insight into the molecular trafficking of a critical regulator of polyP metabolism in yeast. We speculate that AP-3 may also be responsible for the delivery of polyP regulatory proteins to platelet dense granules in higher eukaryotes.

Interplay of TRIM2 E3 Ubiquitin Ligase and ALIX/ESCRT Complex: Control of Developmental Plasticity During Early Neurogenesis

Tripartite motif 2 (TRIM2) drives neurite outgrowth and polarization, is involved in axon specification, and confers neuroprotective functions during rapid ischemia. The mechanisms controlling neuronal cell fate determination and differentiation are fundamental for neural development. Here, we show that in Xenopus, trim2 knockdown affects primary neurogenesis and neural progenitor cell survival. Embryos also suffer from severe craniofacial malformation, a reduction in brain volume, and the loss of motor sensory function. Using a high-throughput LC-MS/MS approach with GST-Trim2 as bait, we pulled down ALG-2 interacting protein X (Alix) from Xenopus embryonic lysates. We demonstrate that the expression of trim2/TRIM2 and alix/ALIX overlap during larval development and on a cellular level in cell culture. Interestingly, trim2 morphants showed a clustering and apoptosis of neural progenitors, which are phenotypic hallmarks that are also observed in Alix KO mice. Therefore, we propose that the interaction of Alix and Trim2 plays a key role in the determination and differentiation of neural progenitors via the modulation of cell proliferation/apoptosis during neurogenesis.

Ilaprazole and other novel prazole-based compounds that bind Tsg101 inhibit viral budding of HSV-1/2 and HIV from cells

In many enveloped virus families, including HIV and HSV, a crucial, yet unexploited, step in the viral life cycle is releasing particles from the infected cell membranes. This release process is mediated by host ESCRT complex proteins, which is recruited by viral structural proteins and provides the mechanical means for membrane scission and subsequent viral budding. The prazole drug, tenatoprazole, was previously shown to bind to ESCRT complex member Tsg101 and quantitatively block the release of infectious HIV-1 from cells in culture. In this report we show that tenatoprazole and a related prazole drug, ilaprazole, effectively block infectious Herpes Simplex Virus (HSV)-1/2 release from Vero cells in culture. By electron microscopy, we found that both prazole drugs block the release of HSV particles from the cell nuclear membrane resulting in their accumulation in the nucleus. Ilaprazole also quantitatively blocks the release of HIV-1 from 293T cells with an EC50 of 0.8 μM, which is more potent than tenatoprazole. Finally, we synthesized and tested multiple novel prazole-based analogs that demonstrate both binding to Tsg101 and inhibition of viral egress in the nanomolar range of HIV-1 from 293T cells. Our results indicate that prazole-based compounds may represent a class of drugs with potential to be broad-spectrum antiviral agents against multiple enveloped viruses, by interrupting cellular Tsg101 interaction with maturing virus, thus blocking the budding process that releases particles from the cell.ImportanceThese results provide the basis for the development of drugs that target enveloped virus budding that can be used ultimately to control multiple virus infections in humans.