SummaryThe Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met. Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond. In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N-terminal LRR region and C-terminal GW domains. The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans. Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor-mediated mode, as opposed to the ligand-mediated mode observed for the LRR fragment. Surprisingly, we find that gC1q-R, a host protein implicated in InlB-mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q-R is unnecessary for bacterial invasion. Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone-like mimicry of HGF.

The rapid global spread of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has created an unprecedented healthcare crisis. The treatment for the severe respiratory illness caused by this virus is primarily symptomatic at this point, although the usage of a broad antiviral drug Remdesivir has been allowed on emergency basis by the Food and Drug Administration (FDA). The ever-increasing death toll highlights an urgent need for development of specific antivirals. In this work, we have utilized docking and simulation methods to identify small molecule inhibitors of SARS-CoV-2 Membrane (M) and Envelope (E) proteins, which are essential for virus assembly and budding. A total of 70 compounds from an Indian medicinal plant source (Azadirachta indica or Neem) were virtually screened against these two proteins and further analyzed with molecular dynamics simulations, which resulted in the identification of a few common compounds with strong binding to both structural proteins. The compounds bind to biologically critical regions of M and E, indicating their potential to inhibit the functionality of these components. We hope that our computational approach may result in the identification of effective inhibitors of SARS-CoV-2 assembly.

The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic ␥ peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the ␥ peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of ␥ peptides from the virus particle, and (iii) the exposed/released ␥ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.Flock House virus (FHV), a nonenveloped, positive-sense RNA virus, has been employed as a model system in several important studies to address a wide range of biological questions (reviewed in reference 55). FHV has been instrumental in understanding virus structure and assembly (17,19,45), RNA replication (2,3,37), and specific packaging of the genome (33,44,53,54). Studies of FHV infection in Drosophila melanogaster flies have provided valuable information about the antiviral innate immune response in invertebrate hosts (29,57). FHV is also used in nanotechnology applications as an epitope-presenting platform to develop novel vaccines and medical therapies (31,48). In this report, we use FHV as a model system to further elucidate the means by which nonenveloped viruses enter host cells and traverse cellular membranes.

The mitochondrial F-type ATP synthase, a multisubunit nanomotor, is critical for maintaining cellular ATP levels. In T. gondii and other apicomplexan parasites, many subunit components necessary for proper assembly and functioning of this enzyme appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (approximately 600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits a, b, and d can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid, and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel antiparasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.