Project description:Dengue virus (DENV) is a flavivirus transmitted by mosquitoes, and is the most prevalent arboviral pathogen in the world. The extent of acute DENV hepatitis has been linked to severe outcomes such as vascular leakage and hemorrhage, although the means by which hepatic responses to infection contribute to virus replication and disease are not well understood. We conducted an integrated transcriptomic, proteomic, and phosphoproteomic survey of hepatic cellular responses to DENV infection to identify host processes associated with increased inflammation, reduced coagulation factor synthesis, regulation of cell cycle progression and apoptosis, and translational control. Integration of these data sets into a comprehensive network model revealed modulation of several critical host signaling pathways, including PI3K/Akt, Ras/Raf/MAPK, Hsp90, and mTOR signaling. Experimental testing of these pathways demonstrated that Hsp90 inhibition impairs DENV replication, suggesting that heat shock responses may contribute significantly to hepatic infection.
Project description:The goal of this study was to compare the transcriptional profile (RNA-seq) of Dengue virus 2 and mock infected cells at 24 and 36 hours post infection. Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. A549 cells where infected with Dengue virus 2 or mock and after 24 and 36 hours post infection mRNA was purified. Then the transcriptional profile of these cells was analyzed using RNA-seq.
Project description:Lipid metabolism is an intricate yet crucial cellular process co-opted by multiple viruses for replication and biogenesis. Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two recently identified ER-resident lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. Importantly, TMEM41B is also a newly validated host dependency factor required for productive infection of several medically important enveloped RNA viruses, such as flaviviruses and human coronaviruses. However, the exact underlying mechanism of TMEM414B in modulating viral infections remains an open question. Here, we uncovered that TMEM41B and VMP1 deficiencies severely impaired replication of flavivirus and human coronavirus via multiple parallel cellular mechanisms. In accordance with previous reports, we validated that both TMEM41B and VMP1 are indispensable for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43) to infect human cells, but not chikungunya virus, an alphavirus. Impaired dengue virus replication in TMEM41B and VMP1 deficient cells could induce a robust activation of innate immune RNA sensing as evidenced by hyperactivation of RIG-I and MDA5. However, this phenomenon was a consequence but not the root cause of the diminished viral replication. Notably, the impact of TMEM41B deficiency on DENV replication could be reversed by complementing the cells using exogenous unsaturated fatty acids, indicating a metabolic role for TMEM41B in flavivirus infection. Furthermore, we found that derailed cellular energy metabolism could be a contributing factor to block DENV infection as TMEM41B and VMP1 deficient cells harbored higher levels of compromised mitochondria that exhibited aberrant functions in facilitating beta-oxidation. Using lipidome and metabolome profiling of TMEM41B and VMP1 deficient cells, we further revealed that each of these genetic deficiencies result in distinctive cellular metabolic dysregulations, underlining their necessity for a balanced metabolic landscape, and strengthening the metabolic role of these ER membrane proteins in facilitating virus infection. Our results highlighted that TMEM41B and VMP1 are required for homeostasis of cellular metabolism, and this metabolic role contributes to their essentiality in facilitating DENV infection.
Project description:Dengue virus is an + strand RNA virus. We have carried our infections of human cells with Dengue and analyzed the translation, replication, and localization of the Dengue RNA. This allowed for clear definition of the life cycle of the Dengue virus inside a host cell. We also assessed the host response to Dengue virus, finding that a large fraction of the translational response is due to Interferon function. Translational and transcriptional analysis of the cellular response to Dengue virus infection
Project description:The goal of this study was to compare the transcriptional profile (RNA-seq) of Dengue virus 2 and mock infected cells at 24 and 36 hours post infection. Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.
Project description:Dengue virus is an + strand RNA virus. We have carried our infections of human cells with Dengue and analyzed the translation, replication, and localization of the Dengue RNA. This allowed for clear definition of the life cycle of the Dengue virus inside a host cell. We also assessed the host response to Dengue virus, finding that a large fraction of the translational response is due to Interferon function.
Project description:The objective of this study was to understand the shared and unique elements of the host transcriptional response to different viral pathogens. We identified 162 subjects in the US and Sri Lanka with infections due to influenza, enterovirus/rhinovirus, human metapneumovirus, dengue virus, cytomegalovirus, Epstein Barr Virus, or adenovirus. Our dataset allowed us to identify common pathways at the molecular level as well as virus-specific differences in the host immune response. Conserved elements of the host response to these viral infections high-lighted the importance of interferon pathway activation. However, the magnitude of the re-sponses varied between pathogens. We also identified virus-specific responses to influenza, enterovirus/rhinovirus, and dengue infections.
Project description:Efficient virus replication in its vector, Aedes mosquitoes, is essential for the transmission of arboviral diseases like dengue virus (DENV) in populations. In order to identify RNA-independent host factors involved in DENV replication in mosquitoes, we established a system expressing all non-structural proteins within the context of the macro protein complex as observed during viral infections. We GFP-tagged Loqs to purify it's interactors by label-free mass spectrometry.
Project description:Efficient virus replication in its vector, Aedes mosquitoes, is essential for the transmission of arboviral diseases like dengue virus (DENV) in populations. In order to identify RNA-independent host factors involved in DENV replication in mosquitoes, we established a system expressing all non-structural proteins within the context of the macro protein complex as observed during viral infections. Mosquito host factors interacting with 3xFLAGED-tagged DENV non-structural proteins NS1 or NS5 proteins were identified by label-free mass spectrometry.
Project description:Microarray data was used to study the gene expression changes following dengue infection at various time points to identify host genes and pathways responsible for dengue virus replication Keywords: time course