Mateusz Kudelko

H5-Type Influenza Virus Hemagglutinin Is Functionally Recognized by the Natural Killer-Activating Receptor NKp44

ABSTRACT Antiviral immune defenses involve natural killer (NK) cells. We previously showed that the NK-activating receptor NKp44 is involved in the functional recognition of H1-type influenza virus strains by NK cells. In the present study, we investigated the interaction of NKp44 and the hemagglutinin of a primary influenza virus H5N1 isolate. Here we show that recombinant NKp44 recognizes H5-expressing cells and specifically interacts with soluble H5 hemagglutinin. H5-pseudotyped lentiviral particles bind to NK cells expressing NKp44. Following interaction with target cells expressing H5, pseudotyped lentiviral particles, or membrane-associated H5, NK cells show NKp44-mediated induced activity. These findings indicate that NKp44-H5 interactions induce functional NK activation.

Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins

Background Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E. Methodology/Principal Findings We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant. Conclusions/Significance Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.

Class II ADP-ribosylation Factors Are Required for Efficient Secretion of Dengue Viruses

Background: To date, very few cellular factors required for secretion of flaviviruses have been described. Results: Simultaneous depletion of class II Arf (Arf4 and Arf5) blocks dengue flavivirus secretion, without altering the constitutive secretory pathway. Dengue glycoprotein prM interacts with Arf4 and Arf5. Conclusion: Arf4 and Arf5 play a crucial role in dengue flavivirus secretion. Significance: Our findings reveal a molecular mechanism of dengue flavivirus secretion. Identification and characterization of virus-host interactions are very important steps toward a better understanding of the molecular mechanisms responsible for disease progression and pathogenesis. To date, very few cellular factors involved in the life cycle of flaviviruses, which are important human pathogens, have been described. In this study, we demonstrate a crucial role for class II Arf proteins (Arf4 and Arf5) in the dengue flavivirus life cycle. We show that simultaneous depletion of Arf4 and Arf5 blocks recombinant subviral particle secretion for all four dengue serotypes. Immunostaining analysis suggests that class II Arf proteins are required at an early pre-Golgi step for dengue virus secretion. Using a horseradish peroxidase protein fused to a signal peptide, we show that class II Arfs act specifically on dengue virus secretion without altering the secretion of proteins through the constitutive secretory pathway. Co-immunoprecipitation data demonstrate that the dengue prM glycoprotein interacts with class II Arf proteins but not through its C-terminal VXPX motif. Finally, experiments performed with replication-competent dengue and yellow fever viruses demonstrate that the depletion of class II Arfs inhibits virus secretion, thus confirming their implication in the virus life cycle, although data obtained with West Nile virus pointed out the differences in virus-host interactions among flaviviruses. Our findings shed new light on a molecular mechanism used by dengue viruses during the late stages of the life cycle and demonstrate a novel function for class II Arf proteins.

The interaction of flavivirus M protein with light chain Tctex-1 of human dynein plays a role in late stages of virus replication

The role of the membrane protein (prM/M) in flavivirus life cycle remains unclear. Here, we identified a cellular interactor to the 40-residue-long ectodomain of prM/M (ectoM) using a yeast two-hybrid screen against a human cDNA library and GST pull-down assays. We showed that dynein light chain Tctex-1 interacts with the ectoM of dengue 1-4, West Nile, and Japanese encephalitis flaviviruses. No interaction was found with yellow fever and tick-borne flaviviruses. This interaction is highly specific since a single amino-acid change in the ectoM abrogates the interaction with Tctex-1. To understand the role of this interaction, silencing of Tctex-1 using siRNA was performed prior to infection. A significant decrease in progeny production was observed for dengue and West Nile viruses. Silencing Tctex-1 inhibited the production of recombinant dengue subviral particles (RSPs). Thus Tctex-1 may play a role in late stages of viral replication through its interaction with the membrane protein.

Label-Free Quantitative Proteomics Reveals Survival Mechanisms Developed by Hypertrophic Chondrocytes under ER Stress

Emerging evidence implicates ER stress caused by unfolded mutant proteins in chondrocytes as the underlying pathology of chondrodysplasias. ER stress is triggered in hypertrophic chondrocytes (HCs) in a mouse model (13del) of metaphyseal chondrodysplasia type Schmid (MCDS) caused by misfolded mutant collagen X proteins, but the HCs do not undergo apoptosis; rather chondrocyte differentiation is altered, causing skeletal abnormality. How 13del HCs can escape from apoptosis and survive ER stress is not understood. Here we compared the proteomes of HCs isolated from 13del growth plates with normal HCs using a label-free quantitative mass spectrometry approach. Pathway enrichment analyses of differentially expressed proteins showed significant changes in glycolysis and ER-mitochondria pathways in 13del HCs as well as in ATDC5 cell lines expressing wt and 13del collagen X. In vivo, we showed expression of mitochondrial calcium channels was reduced while mitochondrial membrane polarity was maintained in 13del chondrocytes, while in vitro, glucose uptake was maintained. We propose 13del HCs survive by a mechanism whereby changes in ER-mitochondria communication reduce import of calcium coupled to maintenance of mitochondrial membrane polarity. These findings provide the initial insights into our understanding of growth plate changes caused by protein misfolding in the pathogenesis of chondrodysplasias.

Identification of cellular enhancing and restricting factors of dengue virus egress

Dengue has emerged as the most important life-threatening illness in the world, especially in Asian countries around Hong Kong, where the incidence of dengue hemorrhagic fever (DHF) is much greater than other continents. However, little is known about molecular and cellular processes sustaining egress of dengue virus in the host cell. To better understand the viral and cellular determinants of dengue virus egress, we have established a dengue VLP producing stable cell line (HeLa-prME) and demonstrated that dengue VLP was able to mimic the budding and egress process of dengue viruses so that it constitutes a safe and convenient tool for the study of egress of dengue virus. Under the support of RFCID grant, HeLa-prME cells were used to screen a siRNA library that included 122 cellular membrane trafficking genes. Our screen results revealed that knockdown of ADP-ribosylation factor (ARF) 1 and ARF6 had significant effects on VLP production by HeLa-prME cell. Experiments with other ARF proteins, which were not included in the siRNA library, showed that the ARF4/ARF5 double knockdown could inhibit VLP production but had no effect on secretion of other proteins such as soluble dengue E protein, suggesting the specificity of their involvement in VLP production. Further experiments using real virus showed that the depletion of ARF4/5 by siRNA could significantly reduced the replication of dengue 1 virus, dengue 4 virus and yellow fever virus, confirming the important role of ARF4 and ARF5 for not only dengue viruses but also other flaviviruses. Our study uncovered the importance of class II ARFs in the egress of dengue virus. Results from this project provided information on mechanism of dengue virus assembly and its dependence on cellular machineries.

Comparison of proteomic datasets from hypertrophic chondrocytes in response to ER stress.

Cartilage proteomics is challenging due to the dominance of poorly soluble matrix components and limited available tissue. Using a “spatial” strategy coupled to MS/MS analysis we have specifically labeled and extracted hypertrophic chondrocytes within the growth plate providing thus a comprehensive proteomic map of normal hypertrophic chondrocytes. Furthermore our established 13del mouse model in which the activation of ER stress did not lead to apoptosis of the hypertrophic cells allowed us to address the natural consequences of ER stress in vivo. Thus our data provide also an overview of proteomic changes occurring in cells under ER stress. Associated with the published study [1] this dataset article provided the detailed information of experimental designing, methods, features as well as the raw data of mass spectrometry (MS) identification. Furthermore the data presented here allow the reader to assert the extent of proteomic changes occurring under ER stress in hypertrophic chondrocytes as well as address the data technical reproducibility in both wild type and stress condition. The mass spectrometry proteomics data can be fully accessed from the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD002125.