A Molecular Determinant of West Nile Virus Secretion and Morphology as a Target for Viral Attenuation.

West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains.IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.

Innovative in cellulo method as an alternative to in vivo neurovirulence test for the characterization and quality control of human live Yellow Fever virus vaccines: A pilot study.

Live attenuated vaccines have proved to be mostly valuable in the prevention of infectious diseases in humans, especially in developing countries. The safety and potency of vaccine, and the consistency of vaccine batch-to-batch manufacturing, must be proven before being administrated to humans. For now, the tests used to control vaccine safety largely involve animal testing. For live viral vaccines, regulations require suppliers to demonstrate the absence of neurovirulence in animals, principally in non-human primates and mice. In a search to reduce the use of animals and embracing the 3Rs principles (Replacement, Reduction, Refinement in the use of laboratory animals), we developed a new Blood-Brain Barrier Minibrain (BBB-Minibrain) in cellulo device to evaluate the neuroinvasiveness/neurovirulence of live Yellow Fever virus (YFV) vaccines. A pilot study was performed using the features of two distinct YFV strains, with the ultimate goal of proposing a companion test to characterize YFV neurovirulence. Here, we demonstrate that the BBB-Minibrain model is a promising alternative to consider for future replacement of YFV vaccine in vivo neurovirulence testing (see graphical abstract).

Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses.

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCE: RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.

Identifying Attenuating Mutations: Tools for a New Vaccine Design against Flaviviruses.

Emerging Flaviviruses pose an increasing threat to global human health. To date, human vaccines against yellow fever virus (YFV), Japanese encephalitis virus (JEV), dengue virus (DV), and tick-borne encephalitis virus (TBEV) exist. However, there is no human vaccine against other Flaviviruses such as Zika virus (ZIKV) and West Nile virus (WNV). In order to restrict their spread and to protect populations against the diseases they induce, vaccines against these emerging viruses must be designed. Obtaining new live attenuated Flavivirus vaccines using molecular biology methods is now possible. Molecular infectious clones of the parental viruses are relatively easy to generate. Key mutations present in live attenuated vaccines or mutations known to have a key role in the Flavivirus life cycle and/or interactions with their hosts can be identified by sequencing, and are then inserted in infectious clones by site-directed mutagenesis. More recently, the use of chimeric viruses and large-scale reencoding and introduction of microRNA target sequences have also been tested. Indeed, a combination of these methods will help in designing new generations of vaccines against emerging and reemerging Flaviviruses.

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts.

UNLABELLED: Japanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuation in vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virus in vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts. IMPORTANCE: JEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.

Class II ADP-ribosylation factors are required for efficient secretion of dengue viruses.

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.

Evaluation of NS4A, NS4B, NS5 and 3'UTR Genetic Determinants of WNV Lineage 1 Virulence in Birds and Mammals.

West Nile virus (WNV) is amplified in an enzootic cycle involving birds as amplifying hosts. Because they do not develop high levels of viremia, humans and horses are considered to be dead-end hosts. Mosquitoes, especially from the Culex genus, are vectors responsible for transmission between hosts. Consequently, understanding WNV epidemiology and infection requires comparative and integrated analyses in bird, mammalian, and insect hosts. So far, markers of WNV virulence have mainly been determined in mammalian model organisms (essentially mice), while data in avian models are still missing. WNV Israel 1998 (IS98) is a highly virulent strain that is closely genetically related to the strain introduced into North America in 1999, NY99 (genomic sequence homology > 99%). The latter probably entered the continent at New York City, generating the most impactful WNV outbreak ever documented in wild birds, horses, and humans. In contrast, the WNV Italy 2008 strain (IT08) induced only limited mortality in birds and mammals in Europe during the summer of 2008. To test whether genetic polymorphism between IS98 and IT08 could account for differences in disease spread and burden, we generated chimeric viruses between IS98 and IT08, focusing on the 3' end of the genome (NS4A, NS4B, NS5, and 3'UTR regions) where most of the non-synonymous mutations were detected. In vitro and in vivo comparative analyses of parental and chimeric viruses demonstrated a role for NS4A/NS4B/5'NS5 in the decreased virulence of IT08 in SPF chickens, possibly due to the NS4B-E249D mutation. Additionally, significant differences between the highly virulent strain IS98 and the other three viruses were observed in mice, implying the existence of additional molecular determinants of virulence in mammals, such as the amino acid changes NS5-V258A, NS5-N280K, NS5-A372V, and NS5-R422K. As previously shown, our work also suggests that genetic determinants of WNV virulence can be host-dependent.

Human Claudin-Derived Peptides Block the Membrane Fusion Process of Zika Virus and Are Broad Flavivirus Inhibitors.

Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged in the Pacific islands in 2007 and spread to the Americas in 2015. The infection remains asymptomatic in most cases but can be associated with severe neurological disorders. Despite massive efforts, no specific drug or vaccine against ZIKV infection is available to date. Claudins are tight-junction proteins that favor the entry of several flaviviruses, including ZIKV. In this study, we identified two peptides derived from the N-terminal sequences of claudin-7 and claudin-1, named CL7.1 and CL1.1, respectively, that inhibited ZIKV infection in a panel of human cell lines. Using cell-to-cell fusion assays, we demonstrated that these peptides blocked the ZIKV E-mediated membrane fusion. A comparison of the antiviral efficacy of CL1.1 and CL7.1 pointed to the importance of the peptide amphipathicity. Electron microscopic analysis revealed that CL1.1 altered the ultrastructure of the viral particles likely by binding the virus lipid envelope. However, amphipathicity could not fully explain the antiviral activity of CL1.1. In silico docking simulations suggested that CL1.1 may also interact with the E protein, near its stem region. Overall, our data suggested that claudin-derived peptides inhibition may be linked to simultaneous interaction with the E protein and the viral lipid envelope. Finally, we found that CL1.1 also blocked infection by yellow fever and Japanese encephalitis viruses but not by HIV-1 or SARS-CoV-2. Our results provide a basis for the future development of therapeutics against a wide range of endemic and emerging flaviviruses. IMPORTANCE Zika virus (ZIKV) is a flavivirus transmitted by mosquito bites that have spread to the Pacific Islands and the Americas over the past decade. The infection remains asymptomatic in most cases but can cause severe neurological disorders. ZIKV is a major public health threat in areas of endemicity, and there is currently no specific antiviral drug or vaccine available. We identified two antiviral peptides deriving from the N-terminal sequences of claudin-7 and claudin-1 with the latter being the most effective. These peptides block the envelope-mediated membrane fusion. Our data suggested that the inhibition was likely achieved by simultaneously interacting with the viral lipid envelope and the E protein. The peptides also inhibited other flaviviruses. These results could provide the basis for the development of therapies that might target a wide array of flaviviruses from current epidemics and possibly future emergences.

Comparative analysis of neuroinvasion by Japanese encephalitis virulent and vaccine viral strains in an in vitro model of human blood-brain barrier.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.

Zika Virus Requires the Expression of Claudin-7 for Optimal Replication in Human Endothelial Cells.

Zika virus (ZIKV) infection has been associated with a series of neurological pathologies. In patients with ZIKV-induced neurological disorders, the virus is detectable in the central nervous system. Thus, ZIKV is capable of neuroinvasion, presumably through infection of the endothelial cells that constitute the blood-brain barrier (BBB). We demonstrate that susceptibility of BBB endothelial cells to ZIKV infection is modulated by the expression of tight-junction protein claudin-7 (CLDN7). Downregulation of CLDN7 reduced viral RNA yield, viral protein production, and release of infectious viral particles in several endothelial cell types, but not in epithelial cells, indicating that CLDN7 implication in viral infection is cell-type specific. The proviral activity of CLDN7 in endothelial cells is ZIKV-specific since related flaviviruses were not affected by CLDN7 downregulation. Together, our data suggest that CLDN7 facilitates ZIKV infection in endothelial cells at a post-internalization stage and prior to RNA production. Our work contributes to a better understanding of the mechanisms exploited by ZIKV to efficiently infect and replicate in endothelial cells and thus of its ability to cross the BBB.

Role of PDZ-binding motif from West Nile virus NS5 protein on viral replication.

West Nile virus (WNV) is a Flavivirus, which can cause febrile illness in humans that may progress to encephalitis. Like any other obligate intracellular pathogens, Flaviviruses hijack cellular protein functions as a strategy for sustaining their life cycle. Many cellular proteins display globular domain known as PDZ domain that interacts with PDZ-Binding Motifs (PBM) identified in many viral proteins. Thus, cellular PDZ-containing proteins are common targets during viral infection. The non-structural protein 5 (NS5) from WNV provides both RNA cap methyltransferase and RNA polymerase activities and is involved in viral replication but its interactions with host proteins remain poorly known. In this study, we demonstrate that the C-terminal PBM of WNV NS5 recognizes several human PDZ-containing proteins using both in vitro and in cellulo high-throughput methods. Furthermore, we constructed and assayed in cell culture WNV replicons where the PBM within NS5 was mutated. Our results demonstrate that the PBM of WNV NS5 is important in WNV replication. Moreover, we show that knockdown of the PDZ-containing proteins TJP1, PARD3, ARHGAP21 or SHANK2 results in the decrease of WNV replication in cells. Altogether, our data reveal that interactions between the PBM of NS5 and PDZ-containing proteins affect West Nile virus replication.

Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts.

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.

Comparison of a human neuronal model proteome upon Japanese encephalitis or West Nile Virus infection and potential role of mosquito saliva in neuropathogenesis.

Neurotropic flavivirus Japanese encephalitis virus (JEV) and West Nile virus (WNV) are amongst the leading causes of encephalitis. Using label-free quantitative proteomics, we identified proteins differentially expressed upon JEV (gp-3, RP9) or WNV (IS98) infection of human neuroblastoma cells. Data are available via ProteomeXchange with identifier PXD016805. Both viruses were associated with the up-regulation of immune response (IFIT1/3/5, ISG15, OAS, STAT1, IRF9) and the down-regulation of SSBP2 and PAM, involved in gene expression and in neuropeptide amidation respectively. Proteins associated to membranes, involved in extracellular matrix organization and collagen metabolism represented major clusters down-regulated by JEV and WNV. Moreover, transcription regulation and mRNA processing clusters were also heavily regulated by both viruses. The proteome of neuroblastoma cells infected by JEV or WNV was significantly modulated in the presence of mosquito saliva, but distinct patterns were associated to each virus. Mosquito saliva favored modulation of proteins associated with gene regulation in JEV infected neuroblastoma cells while modulation of proteins associated with protein maturation, signal transduction and ion transporters was found in WNV infected neuroblastoma cells.

Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak.

BACKGROUND: A chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis. METHODS AND FINDINGS: We report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, four of which were located in alphavirus-conserved positions of nsP2 (which contains helicase, protease, and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector. CONCLUSIONS: The unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.

The E glycoprotein plays an essential role in the high pathogenicity of European-Mediterranean IS98 strain of West Nile virus.

West Nile virus (WNV) is the most widespread arbovirus in the world. Several recent outbreaks and epizootics have been reported in Europe and the Mediterranean basin with increased virulence. In contrast to the well-characterized American and Australian strains, little is known about the virulence determinants of the WNV European-Mediterranean strains. To investigate the viral factors involved in the virulence of these strains, we generated chimeras between the highly neuropathogenic Israel 1998 (IS-98-ST1, IS98) strain and the non-pathogenic Malaysian Kunjin virus (KJMP-502). In vivo analyses in a mouse model of WNV pathogenesis shows that chimeric virus where KJMP-502 E glycoprotein was replaced by that of IS98 is neuropathogenic, demonstrating that this protein is a major virulence determinant. Presence of the N-glycosylation site had limited impact on virus virulence and the 5'UTR does not seem to influence pathogenesis. Finally, mice inoculated with KJMP-502 virus were protected against lethal IS98 infection.

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex.

The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

IS-98-ST1 West Nile virus derived from an infectious cDNA clone retains neuroinvasiveness and neurovirulence properties of the original virus.

Infectious clones of West Nile virus (WNV) have previously been generated and used to decipher the role of viral proteins in WNV virulence. The majority of molecular clones obtained to date have been derived from North American, Australian, or African isolates. Here, we describe the construction of an infectious cDNA clone of a Mediterranean WNV strain, IS-98-ST1. We characterized the biological properties of the recovered recombinant virus in cell culture and in mice. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotype of recombinant and parental WNV was indistinguishable as regards viremia, viral load in the brain, and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs. The capacity of different WNV strains to replicate in embryonated chicken eggs closely paralleled their ability to replicate in mice, suggesting that inoculation of embryonated chicken eggs could provide a practical in vivo model for the study of WNV pathogenesis. In conclusion, the IS-98-ST1 infectious clone will allow assessment of the impact of selected mutations and novel genomic changes appearing in emerging European strains pathogenicity and endemic or epidemic potential. This will be invaluable in the context of an increasing number of outbreaks and enhanced severity of infections in the Mediterranean basin and Eastern Europe.

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.

CD8 alpha alpha-mediated intraepithelial lymphocyte snatching of thymic leukemia MHC class Ib molecules in vitro and in vivo.

Thymic leukemia (TL) is a MHC class Ib molecule that interacts with CD8alphaalpha homodimers. CD8alphaalpha is abundantly expressed by intraepithelial T lymphocytes (IELs) located in close proximity to TL-expressing intestinal epithelial cells. In this study, we show that CD8alphaalpha(+) IELs "snatch" TL from the plasma membrane of TL-expressing cells and express TL in its proper orientation on their own cell surface. TL snatching is enhanced by cross-linking of IEL TCRs in a phosphatidylinositol kinase-dependent manner, and results in overall alterations to the IEL cell surface detected by enhanced binding of peanut agglutinin lectin. Induction of bowel inflammation results in the presence of TL on IELs, probably via in vivo snatching, providing the initial evidence for the interaction of CD8alphaalpha IELs with intestinal cells.

The TL MHC class Ib molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes.

Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8alphaalpha homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in beta2m-/- mice but unaffected in TCR-/- mice or CD8alpha chain-/- mice. We demonstrate that the interaction between TL and CD8alphaalpha is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of alpha-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-gamma in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-gamma production suggest that the functional consequences of TL interaction with CD8alphaalpha as well as the more general biological role of TL in mucosal immunity remains to be discovered.