A Histoplasma capsulatum Lipid Metabolic Map Identifies Antifungal Targets.

Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here, we built a comprehensive map of the Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide, and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low-micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development. IMPORTANCE It is estimated that 150 people die per hour due to the insufficient therapeutic treatments to combat fungal infections. A major hurdle to developing antifungal therapies is the scarce knowledge on the fungal metabolic pathways and mechanisms of virulence. In this context, fungal lipid metabolism is an excellent candidate for developing drugs due to its essential roles in cellular scaffolds, energy storage, and signaling transductors. Here, we provide a detailed map of Histoplasma capsulatum lipid metabolism. The map revealed points of this fungus lipid metabolism that can be targeted for developing antifungal drugs.

Nipah Virus-Like Particle Egress Is Modulated by Cytoskeletal and Vesicular Trafficking Pathways: a Validated Particle Proteomics Analysis.

Classified as a biosafety level 4 (BSL4) select agent, Nipah virus (NiV) is a deadly henipavirus in the Paramyxoviridae family, with a nearly 75% mortality rate in humans, underscoring its global and animal health importance. Elucidating the process of viral particle production in host cells is imperative both for targeted drug design and viral particle-based vaccine development. However, little is understood concerning the functions of cellular machinery in paramyxoviral and henipaviral assembly and budding. Recent studies showed evidence for the involvement of multiple NiV proteins in viral particle formation, in contrast to the mechanisms understood for several paramyxoviruses as being reliant on the matrix (M) protein alone. Further, the levels and purposes of cellular factor incorporation into viral particles are largely unexplored for the paramyxoviruses. To better understand the involvement of cellular machinery and the major structural viral fusion (F), attachment (G), and matrix (M) proteins, we performed proteomics analyses on virus-like particles (VLPs) produced from several combinations of these NiV proteins. Our findings indicate that NiV VLPs incorporate vesicular trafficking and actin cytoskeletal factors. The involvement of these biological processes was validated by experiments indicating that the perturbation of key factors in these cellular processes substantially modulated viral particle formation. These effects were most impacted for NiV-F-modulated viral particle formation either autonomously or in combination with other NiV proteins, indicating that NiV-F budding relies heavily on these cellular processes. These findings indicate a significant involvement of the NiV fusion protein, vesicular trafficking, and actin cytoskeletal processes in efficient viral particle formation.IMPORTANCE Nipah virus is a zoonotic biosafety level 4 agent with high mortality rates in humans. The genus to which Nipah virus belongs, Henipavirus, includes five officially recognized pathogens; however, over 20 species have been identified in multiple continents within the last several years. As there are still no vaccines or treatments for NiV infection, elucidating its process of viral particle production is imperative both for targeted drug design as well as for particle-based vaccine development. Developments in high-throughput technologies make proteomic analysis of isolated viral particles a highly insightful approach to understanding the life cycle of pathogens such as Nipah virus.

Coupled transcriptome and proteome analysis of human lymphotropic tumor viruses: insights on the detection and discovery of viral genes.

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. RESULTS: The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. CONCLUSIONS: This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.

Sialic acid on herpes simplex virus type 1 envelope glycoproteins is required for efficient infection of cells.

Herpes simplex virus type 1 (HSV-1) envelope proteins are posttranslationally modified by the addition of sialic acids to the termini of the glycan side chains. Although gC, gD, and gH are sialylated, it is not known whether sialic acids on these envelope proteins are functionally important. Digestion of sucrose gradient purified virions for 4 h with neuraminidases that remove both alpha2,3 and alpha2,6 linked sialic acids reduced titers by 1,000-fold. Digestion with a alpha2,3-specific neuraminidase had no effect, suggesting that alpha2,6-linked sialic acids are required for infection. Lectins specific for either alpha2,3 or alpha2,6 linkages blocked attachment and infection to the same extent. In addition, the mobility of gH, gB, and gD in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was altered by digestion with either alpha2,3 specific neuraminidase or nonspecific neuraminidases, indicating the presence of both linkages on these proteins. The infectivity of a gC-1-null virus, DeltagC2-3, was reduced to the same extent as wild-type virus after neuraminidase digestion, and attachment was not altered. Neuraminidase digestion of virions resulted in reduced VP16 translocation to the nucleus, suggesting that the block occurred between attachment and entry. These results show for the first time that sialic acids on HSV-1 virions play an important role in infection and suggest that targeting virion sialic acids may be a valid antiviral drug development strategy.