Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SS Bsa ) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SS Bsa activity. Although intracellular movement was essential for cell-cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell-cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SS Bsa -mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.

Zoonotic transmission of lethal Henipaviruses (HNV) from their natural fruit bat reservoirs to humans has only been reported in Australia and South/Southeast Asia. However, a recent study discovered numerous HNV clades in African bat samples. To determine the potential for HNV spillover events among humans in Africa, here we examine well-curated sets of bat (Eidolon helvum, n=44) and human (n=497) serum samples from Cameroon for Nipah virus (NiV) cross-neutralizing antibodies (NiV-X-Nabs). Using a VSV-based pseudoparticle seroneutralization assay, we detect NiV-X-Nabs in 48% and 3-4% of the bat and human samples, respectively. Seropositive human samples are found almost exclusively in individuals that reported butchering bats for bushmeat. Seropositive human sera also neutralize Hendra virus and Gh-M74a (an African HNV) pseudoparticles, as well as live NiV. Butchering bat meat and living in areas undergoing deforestation are the most significant risk factors associated with seropositivity. Evidence for HNV spillover events warrants increased surveillance efforts.

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype.

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G-ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus-host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structurefunction analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptorinduced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations.emerging virus | viral attachment | glycoprotein | henipavirus | structure