Biology subjects

Thornhill, D.

Publications and source records attributed to Thornhill, D..

3 recordsLinked to original sources

bioRxiv2026

The specific interface for selenocysteine-decoding tRNA in HIV-1 Gag MAdomain regulates progeny virion infectivity

Localization of HIV-1 particle assembly at the plasma membrane (PM) is mediated by the HIV-1 Gag matrix (MA) domain. Competing binding of PM acidic phospholipids and cellular tRNAs to MA-highly basic region (MA-HBR) likely regulate the PM-specific binding of Gag; however, the specificity of the MA-tRNA interaction and its role in viral replication remain poorly understood. Here, using a modified crosslinking-immunoprecipitation (CLIP)-sequencing workflow for identifying MA-bound tRNAs, we determined that most MA-bound tRNAs were also abundantly present in cells. However, selenocysteine-tRNA (SeCTCA) is overrepresented among Gag-bound tRNAs despite its paucity. The binding of SeCTCA to Gag is entirely dependent on MA-HBR, whereas non-SeCTCA tRNAs can bind Gag through both MA and nucleocapsid domains. Structure prediction of MA-tRNA complexes revealed the binding interface unique to the MA-SeCTCA complex, which was verified experimentally. CLIP experiments demonstrated that this interface is specifically needed for the MA binding to SeCTCA but not to other tRNAs. Notably, disruption of this interface altered Gag localization pattern, reduced infectivity of virions, and impaired virus replication in CD4+ T cells. Collectively, our results indicate that SeCTCA tRNA binds the HIV-1 Gag MA domain with high specificity and promotes virion formation and efficient replication in T cells.

microbiology↗

bioRxiv2021

Toxoplasma gondii subverts the host ESCRT machinery for parasite uptake of host cytosolic proteins

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole, the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of GRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PVM. These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission. Author summaryIntracellular pathogens exploit their host to gain the resources necessary to sustain infection; however, precisely how the intracellular parasite Toxoplasma gondii acquires essential nutrients from its host remains poorly understood. Previous work showed that T. gondii is capable of internalizing host derived cytosolic proteins and delivering them to its lysosome-like compartment for degradation. However, the mechanism by which the material is trafficked across the membrane delimiting the replicative vacuole in which the parasite resides remained unclear. Here, we report a role for the parasite effector protein TgGRA14 in the recruitment of the host ESCRT machinery for the uptake of host cytosolic proteins. Important human pathogens have developed strategies for exploiting the host ESCRT machinery for intracellular subsistence. Our study sheds lights on the strategy used by a eukaryotic pathogen in subverting the host ESCRT machinery for the internalization of resources from its host cells.

microbiology↗

bioRxiv2019

Relationships between MA-RNA binding in cells and suppression of HIV-1 Gag mislocalization to intracellular membranes

The HIV-1 Gag matrix (MA) domain mediates localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25/26 or 29/31: Lys->Arg (KR) (25/26KR and 29/31KR) and Lys->Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently unlike the corresponding KR mutants. Both 25/26KT Gag-YFP and 29/31KT Gag-YFP bound non-specifically to PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of non-specific membrane binding.\n\nIMPORTANCEThe PM-specific localization of HIV-1 Gag is a crucial early step in the infectious progeny production. The interaction between the MA highly basic region (HBR) of Gag and the PM-specific lipid PI(4,5)P2 is critical for Gag localization to the PM. Additionally, in vitro evidence has indicated that MA-RNA binding prevents non-specific binding of Gag to non-PI(4,5)P2-containing membranes. However, cell-based evidence supporting a role for HIV-1 MA-RNA binding in PM-specific subcellular localization has been scarce; thus, it remained possible that in cells, just the high basic charge or the PI(4,5)P2-binding ability is sufficient for MA to direct Gag specifically to the PM. The current study revealed for the first time an excellent correlation between RNA binding of MA-HBR and inhibition of promiscuous Gag localization, both within the cells, and thereby provided cell-based evidence supporting a mechanism in which HIV-1 MA binding to RNA ensures specific localization of Gag to the PM.

microbiology↗