A facile quantitative assay for viral particle genesis reveals cooperativity in virion assembly and saturation of an antiviral protein

Conventional assays of viral particle assembly and release are time consuming and laborious. We have developed an enzymatic virus-like particle (VLP) genesis assay that rapid and quantitative and is also versatile and applicable to diverse viruses including HIV-1 and Ebola virus. Using this assay, which has a dynamic range of several orders of magnitude, we show that the efficiency of VLP assembly and release, i.e., the fraction of the expressed protein that is assembled into extracellular particles, is dependent on the absolute level of expression of either HIV-1 Gag or Ebola virus VP40. We also demonstrate that the activity of the antiviral factor tetherin is dependent on the level of HIV-1 Gag expression and the numbers of VLPs generated, and appears to become saturated as these parameters are increased

Deficiency of Leishmania phosphoglycans influences the magnitude but does not affect the quality of secondary (memory) anti-Leishmania immunity

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8(+) T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals

Degradation of Host Sphingomyelin Is Essential for Leishmania Virulence

In eukaryotes, sphingolipids (SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN), a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid phospholipase C-Like). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that seen with IPC. Null mutants of ISCL (iscl−) showed modest accumulation of IPC, but grew and differentiated normally in vitro. Interestingly, iscl− mutants did not induce lesion pathology in the susceptible BALB/c mice, yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl− was completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of host-derived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the manifestation of acute disease pathology

Differential impact of LPG-and PG-deficient Leishmania major mutants on the immune response of human dendritic cells

<div><p>Background</p><p><i>Leishmania major</i> infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of <i>Leishmania</i> parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.</p><p>Methodology/Principal Findings</p><p>Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating <i>L</i>. <i>major</i> Friedlin V1 mutants defective in LPG alone, (FV1 <i>lpg1-</i>), or generally deficient for all PGs, (FV1 <i>lpg2-</i>). Infection with metacyclic, infective stage, <i>L</i>. <i>major</i> or purified LPG induced high levels of <i>IL12B</i> subunit gene transcripts in hDCs, which was abrogated with FV1 <i>lpg1-</i> infections. In contrast, hDC infections with FV1 <i>lpg2-</i> displayed increased <i>IL12B</i> expression, suggesting other PG-related/<i>LPG2</i> dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 <i>lpg1-</i>, FV1 <i>lpg2-</i> infections revealed that FV1 <i>lpg1-</i> mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription.</p><p>Conclusions/Significance</p><p>These data suggest that <i>L</i>. <i>major</i> LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring <i>Leishmania</i> surface glycoconjugates that result in modulation of host cellular IL12.</p></div

Transcriptional and genomic parallels between the monoxenous parasite Herpetomonas muscarum and Leishmania

Trypanosomatid parasites are causative agents of important human and animal diseases such as sleeping sickness and leishmaniasis. Most trypanosomatids are transmitted to their mammalian hosts by insects, often belonging to Diptera (or true flies). These are called dixenous trypanosomatids since they infect two different hosts, in contrast to those that infect just insects (monoxenous). However, it is still unclear whether dixenous and monoxenous trypanosomatids interact similarly with their insect host, as fly-monoxenous trypanosomatid interaction systems are rarely reported and under-studied–despite being common in nature. Here we present the genome of monoxenous trypanosomatid Herpetomonas muscarum and discuss its transcriptome during in vitro culture and during infection of its natural insect host Drosophila melanogaster. The H. muscarum genome is broadly syntenic with that of human parasite Leishmania major. We also found strong similarities between the H. muscarum transcriptome during fruit fly infection, and those of Leishmania during sand fly infections. Overall this suggests Drosophila-Herpetomonas is a suitable model for less accessible insect-trypanosomatid host-parasite systems such as sand fly-Leishmania

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

The virus family Arenaviridae includes several hemorrhagic fever causing agents such as Lassa, Guanarito, Junin, Machupo, and Sabia virus that pose a major public health concern to the human population in West African and South American countries. Current treatment options to control fatal outcome of disease are limited to the ribonucleoside analogue ribavirin, although its use has some significant limitations. The lack of effective treatment alternatives emphasizes the need for novel antiviral therapeutics to counteract these life-threatening infections. Maturation cleavage of the viral envelope glycoprotein by the host cell proprotein convertase site 1 protease (S1P) is critical for infectious virion production of several pathogenic arenaviruses. This finding makes this protease an attractive target for the development of novel anti-arenaviral therapeutics. We demonstrate here that highly selective S1P-adapted α1-antitrypsins have the potential to efficiently inhibit glycoprotein processing, which resulted in reduced Lassa virus replication. Our findings suggest that S1P should be considered as an antiviral target and that further optimization of modified α1-antitrypsins could lead to potent and specific S1P inhibitors with the potential for treatment of certain viral hemorrhagic fevers