CFTR is required for cellular entry and internalization of Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular Gram‐negative pathogen affecting over 600 million people worldwide with 92 million new cases occurring globally each year. C. trachomatis enter the cells and replicate to infect different tissues/organs, giving rise to a spectrum of pathological conditions; however, the exact mechanism or receptor(s) for their entry is not well understood. Here we report that CFTR (cystic fibrosis transmembrane conductance regulator), an apical epithelial anion channel, is required for cellular entry and internalization of C. trachomatis. Human epithelial cell lines expressing functional CFTR internalized more C. trachomatis than the cells expressing mutant Δ508 CFTR. The in vitro cellular uptake of C. trachomatis can be blocked by CFTR inhibitors or antibody, and the in vivo cellular uptake of C. trachomatis in CFTR mutant (CFTR^?/?) mice was significantly less compared with that in the wild‐type. Direct interaction between CFTR and C. trachomatis LPS (lipopolysaccharide) is demonstrated by their immune‐co‐localization and co‐immunoprecipitation. Despite an increase in CFTR expression observed upon C. trachomatis LPS challenge, a reduction in its ion channel activity is observed, consistent with the notion that CFTR functions as a receptor for cellular entry and internationization of C. trachomatis, with compromised ion‐channel function. These findings, for the first time, demonstrate that CFTR functions as a cell‐surface receptor for epithelial cell entry, and internalization of C. trachomatis and these findings may lead to the development of new treatment strategies to curtail the spread of chlamydial infections.     

Lipids and flaviviruses,present and future perspectives for the control of dengue,Zika, and West Nile viruses

Flaviviruses are emerging arthropod-borne pathogens that cause life-threatening diseases such as yellow fever, dengue, West Nile encephalitis, tick-borne encephalitis, Kyasanur Forest disease, tick-borne encephalitis, or Zika disease. This viral genus groups > 50 viral species of small enveloped plus strand RNA virus that are phylogenetically closely related to hepatitis C virus. Importantly, the flavivirus life cycle is intimately associated to host cell lipids. Along this line, flaviviruses rearrange intracellular membranes from the endoplasmic-reticulum of the infected cells to develop adequate platforms for viral replication and particle biogenesis. Moreover, flaviviruses dramatically orchestrate a profound reorganization of the host cell lipid metabolism to create a favorable environment for viral multiplication. Consistently, recent work has shown the importance of specific lipid classes in flavivirus infections. For instances, fatty acid synthesis is linked to viral replication, phosphatidylserine and phosphatidylethanolamine are involved on the entry of flaviviruses, sphingolipids (ceramide and sphingomyelin) play a key role on virus assembly and pathogenesis, and cholesterol is essential for innate immunity evasion in flavivirus-infected cells. Here, we revise the current knowledge on the interactions of the flaviviruses with the cellular lipid metabolism to identify potential targets for future antiviral development aimed to combat these relevant health-threatening pathogens.     

Folate receptor-alpha is a cofactor for cellular entry by Marburg and Ebola viruses.

Human infections by Marburg (MBG) and Ebola (EBO) viruses result in lethal hemorrhagic fever. To identify cellular entry factors employed by MBG virus, noninfectible cells transduced with an expression library were challenged with a selectable pseudotype virus packaged by MBG glycoproteins (GP). A cDNA encoding the folate receptor-alpha (FR-alpha) was recovered from cells exhibiting reconstitution of viral entry. A FR-alpha cDNA was recovered in a similar strategy employing EBO pseudotypes. FR-alpha expression in Jurkat cells facilitated MBG or EBO entry, and FR-blocking reagents inhibited infection by MBG or EBO. Finally, FR-alpha bound cells expressing MBG or EBO GP and mediated syncytia formation triggered by MBG GP. Thus, FR-alpha is a significant cofactor for cellular entry for MBG and EBO viruses.     

West Nile virus entry requires cholesterol-rich membrane microdomains and is independent of alphavbeta3 integrin

West Nile virus (WNV) has been the leading cause of viral encephalitis in the United States since 1999. The endocytic processes involved in the internalization of infectious WNV by various cell types are not well characterized, and the involvement of cholesterol-rich membrane microdomains, or lipid rafts, in the life cycle of WNV has not been investigated previously. In this study, we found that the depletion of cellular cholesterol levels by brief treatment with methyl-beta-cyclodextrin resulted in a 100-fold reduction of the titers of infectious WNV released into the culture supernatant, as well as a reduction in the number of WNV genome copies in the cholesterol-depleted cells. The addition of exogenous cholesterol to cholesterol-depleted cells reversed this effect. Cholesterol depletion postinfection did not affect WNV growth, suggesting that the effect occurs at the level of WNV entry. We also showed that while WNV entry did not require alphavbeta3 integrin and focal adhesion kinase, WNV particles failed to be internalized by cholesterol-depleted cells. Finally, we showed the colocalization of the WNV envelope protein and cholera toxin B, which is internalized in a lipid raft-dependent pathway, in microdomain clusters at the plasma membrane. These data suggest that WNV utilizes lipid rafts during initial stages of internalization and that the lipid rafts may contain a factor(s) that may enhance WNV endocytosis.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

Dual prenylation is required for Rab protein localization and function

The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes. In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function. Substitution of different prenyl anchors on Rab GTPases does not lead to correct function. In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4. Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane. We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins. This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.     

Complement activation is required for induction of a protective antibody response against West Nile virus infection

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.     

Rab27a is required for human cytomegalovirus assembly

Human cytomegalovirus (HCMV) completes its final envelopment on intracellular membranes before it is released from the cell. The mechanisms underlying these processes are not understood. Here we studied the role of Rab27a, a regulator of lysosome-related organelle transport, in HCMV production. HCMV infection increased Rab27a expression, and recruitment of Rab27a to membranous strutures at the assembly site. Immuno-gold labelling demonstrated association of Rab27a with viral envelopes. CMV production was reduced after knock-down of Rab27a, and in Rab27a-deficient ashen melanocytes. This study shows a requirement for Rab27a in the CMV life cycle and suggests that CMV and LRO biogenesis share common molecular mechanisms.     

Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus

Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 10(7) PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.     

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.     

The Rab6-binding kinesin, Rab6-KIFL, is required for cytokinesis

The Rab6-binding kinesin, Rab6-KIFL, was identified in a two-hybrid screen for proteins that interact with Rab6, a small GTPase involved in membrane traffic through the Golgi apparatus. We find that Rab6-KIFL accumulates in mitotic cells where it localizes to the midzone of the spindle during anaphase, and to the cleavage furrow and midbody during telophase. Overexpression of Rab6-KIFL causes a cell division defect resulting in cell death. Microinjection of antibodies to Rab6-KIFL results in the cells becoming binucleate after one cell cycle, and time-lapse microscopy reveals that this is due to a defect in cleavage furrow formation and thus cytokinesis. These data show that endogenous Rab6-KIFL functions in cell division during cleavage furrow formation and cytokinesis, in addition to its previously described role in membrane traffic.     

Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus

The replication of flaviviruses requires the correct processing of their polyprotein by the viral NS3 protease (NS3pro). Essential for the activation of NS3pro is a 47-residue region of NS2B. Here we report the crystal structures of a dengue NS2B-NS3pro complex and a West Nile virus NS2B-NS3pro complex with a substrate-based inhibitor. These structures identify key residues for NS3pro substrate recognition and clarify the mechanism of NS3pro activation.     

Matrix metalloproteinase 9 facilitates West Nile virus entry into the brain

West Nile virus (WNV) is the most-common cause of mosquito-borne encephalitis in the United States. Invasion of the brain by WNV is influenced by viral and host factors, and the molecular mechanism underlying disruption of the blood-brain barrier is likely multifactorial. Here we show that matrix metalloproteinase 9 (MMP9) is involved in WNV entry into the brain by enhancing blood-brain barrier permeability. Murine MMP9 expression was induced in the circulation shortly after WNV infection, and the protein levels remained high even when viremia subsided. In the murine brain, MMP9 expression and its enzymatic activity were upregulated and MMP9 was shown to partly localize to the blood vessels. Interestingly, we also found that cerebrospinal fluid from patients suffering from WNV contained increased MMP9 levels. The peripheral viremia and expression of host cytokines were not altered in MMP9(-/-) mice; however, these animals were protected from lethal WNV challenge. The resistance of MMP9(-/-) mice to WNV infection correlated with an intact blood-brain barrier since immunoglobulin G, Evans blue leakage into brain, and type IV collagen degradation were markedly reduced in the MMP9(-/-) mice compared with their levels in controls. Consistent with this, the brain viral loads, selected inflammatory cytokines, and leukocyte infiltrates were significantly reduced in the MMP9(-/-) mice compared to their levels in wild-type mice. These data suggest that MMP9 plays a role in mediating WNV entry into the central nervous system and that strategies to interrupt this process may influence the course of West Nile encephalitis.     

Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5

The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis.     

Rab5 activation by Toll-like receptor 2 is required for Trypanosoma cruzi internalization and replication in macrophages

Trypanosoma cruzi can infect and replicate in macrophages. During invasion, T. cruzi interacts with different macrophage receptors to induce its own phagocytosis. However, the nature of those receptors and the molecular mechanisms involved are poorly understood. In this study, we demonstrate that T. cruzi metacyclic trypomastigotes but not epimastigotes were able to induce Rab5 activation and binding to the early endosomes in peritoneal macrophages. In this process, active Rab5 colocalized with parasites in the phagosome and with the Rab5A effector molecule early endosomal antigen 1. Phagosome formation and T. cruzi internalization were inhibited in Raw 264.7 macrophages expressing a dominant-negative form of Rab5 [(S34N)Rab5]. Using T. cruzi membrane extracts, we verified that the Rab5 activation depends on the interaction between parasite surface molecules and macrophages surface molecule. In addition, during infection of macrophages, phosphatidylinositol 3-kinase (PI3K) pathway was activated. Assays carried out using a selective PI3K inhibitor (LY294002) showed that the PI3K activation is essential for Rab5 activation by T. cruzi infection and for the entrance and intracellular replication of T. cruzi in macrophages. Moreover, using macrophages from knockout mice, we found that activation of Rab5, fusion of early endosomes and phagocytosis induced by T. cruzi infection involved Toll-like receptor (TLR)2 but were independent of TLR4 receptors.     

West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection

The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.