Inhibition of dengue virus entry and multiplication into monocytes using RNA interference

Background

Dengue infection ranks as one of the most significant viral diseases of the globe. Currently, there is no specific vaccine or antiviral therapy for prevention or treatment. Monocytes/macrophages are the principal target cells for dengue virus and are responsible for disseminating the virus after its transmission. Dengue virus enters target cells via receptor-mediated endocytosis after the viral envelope protein E attaches to the cell surface receptor. This study aimed to investigate the effect of silencing the CD-14 associated molecule and clathrin-mediated endocytosis using siRNA on dengue virus entry into monocytes.

Methodology/Principal Findings

Gene expression analysis showed a significant down-regulation of the target genes (82.7%, 84.9 and 76.3% for CD-14 associated molecule, CLTC and DNM2 respectively) in transfected monocytes. The effect of silencing of target genes on dengue virus entry into monocytes was investigated by infecting silenced and non-silenced monocytes with DENV-2. Results showed a significant reduction of infected cells (85.2%), intracellular viral RNA load (73.0%), and extracellular viral RNA load (63.0%) in silenced monocytes as compared to non-silenced monocytes.

Conclusions/Significance

Silencing the cell surface receptor and clathrin mediated endocytosis using RNA interference resulted in inhibition of the dengue virus entry and subsequently multiplication of the virus in the monocytes. This might serve as a novel promising therapeutic target to attenuate dengue infection and thus reduce transmission as well as progression to severe dengue hemorrhagic fever.     

Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments

Background

The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells.

Methodology/Principal Findings

Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl β-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island.

Conclusions

Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.     

Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1

Background

Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak.

Results

Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5%) were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19%) dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified.

Conclusion

This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.     

RNA interference mediated inhibition of dengue virus multiplication and entry in HepG2 cells

Background

Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells.

Methodology/Principal Findings

HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78) and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively) in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4%) and extracellular viral RNA load (71.4%) compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7%) in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells.

Conclusions/Significance

Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS. Furthermore, a decrease of viremia in humans can result in the reduction of infected vectors and thus, halt of the transmission cycle.     

Distinct cellular responses differentiating alcohol- and hepatitis C virus-induced liver cirrhosis

Background

Herpes simplex virus (HSV) can utilize multiple pathways to enter host cells. The factors that determine which route is taken are not clear. Chinese hamster ovary (CHO) cells that express glycoprotein D (gD)-binding receptors are model cells that support a pH-dependent, endocytic entry pathway for all HSV strains tested to date. Fusion-from-without (FFWO) is the induction of target cell fusion by addition of intact virions to cell monolayers in the absence of viral protein expression. The receptor requirements for HSV-induced FFWO are not known. We used the syncytial HSV-1 strain ANG path as a tool to evaluate the complex interplay between receptor usage, membrane fusion, and selection of entry pathway.

Results

Inhibitors of endocytosis and endosome acidification blocked ANG path entry into CHO cells expressing nectin-1 receptors, but not CHO-nectin-2 cells. Thus, under these conditions, nectin-2 mediates pH-independent entry at the plasma membrane. In addition, CHO-nectin-2 cells supported pH-dependent, endocytic entry of different strains of HSV-1, including rid1 and HFEM. The kinetics of ANG path entry was rapid (t_1/2 of 5–10 min) regardless of entry route. However, HSV-1 ANG path entry by fusion with the CHO-nectin-2 cell plasma membrane was more efficient and resulted in larger syncytia. ANG path virions added to the surface of CHO-nectin-2 cells, but not receptor-negative CHO cells or CHO-nectin-1 cells, induced rapid FFWO.

Conclusion

HSV-1 ANG path can enter CHO cells by either endocytic or non-endocytic pathways depending on whether nectin-1 or nectin-2 is present. In addition to these cellular receptors, one or more viral determinants is important for the selection of entry pathway. HSV-induced FFWO depends on the presence of an appropriate gD-receptor in the target membrane. Nectin-1 and nectin-2 target ANG path to divergent cellular pathways, and these receptors may have different roles in triggering viral membrane fusion.     

The effect of the histone deacetylase inhibitor M344 on BRCA1 expression in breast and ovarian cancer cells

Background

Chlorotoxin (TM601), a scorpion venom- derived 36-AA peptide, is an experimental drug against recurrent glioma with tumor specificity but unknown route of intracellular distribution. The aim of this study was to evaluate the route of entry and cellular localization of TM601 in glioma cells.

Results

We have found that in human gliomas, lung carcinoma and normal vascular endothelial cells, TM601 localizes near trans-Golgi while in normal human dermal fibroblasts (NHDF) and astrocytes it is dispersed in the cytoplasm. The uptake of TM601 by U373 glioma cells is rapid, concentration and time dependent, not affected by inhibitors such as filipin (caveolae-dependent endocytosis) and amiloride (non-selective macropinocytosis), but significantly affected by chlorpromazine (clathrin-dependent intracellular transport of coated pits) resulting in intracellular build-up of the drug and clathrin near the Golgi. In contrast, TM601 uptake by NHDF cells was significantly affected by amiloride indicating that macropinocytosis is the dominant uptake route of TM601 in these cells.

Conclusions

In conclusion, we found a distinct cellular localization pattern and uptake of TM601 by glioma cells differing from that found in normal cells. Further insight into the cellular processing of TM601 should assist in the development of effective anti-glioma therapeutic modalities.     

Rhesus Rhadinovirus Infection of Rhesus Fibroblasts Occurs through Clathrin-Mediated Endocytosis

Rhesus rhadinovirus (RRV) is a gammaherpesvirus closely related to Kaposi''s sarcoma-associated herpesvirus (KSHV), an oncogenic virus linked to the development of Kaposi''s sarcoma and several other lymphoproliferative diseases, including primary effusion lymphoma and multicentric Castleman''s disease. RRV naturally infects rhesus macaques and induces lymphoproliferative diseases under experimental conditions, making it an excellent model for the study of KSHV. Unlike KSHV, which grows poorly in cell culture, RRV replicates efficiently in rhesus fibroblasts (RFs). In this study, we have characterized the entry pathway of RRV in RFs. Using a luciferase-expressing recombinant RRV (RRV-luciferase), we show that the infectivity of RRV is reduced by inhibitors of endosomal acidification. RRV infectivity is also reduced by inhibitors of clathrin-mediated but not caveola-mediated endocytosis, indicating that RRV enters into RFs via clathrin-mediated endocytosis. Using a red fluorescent protein (RFP)-expressing recombinant RRV (RRV-RFP), we show that RRV particles are colocalized with markers of endocytosis (early endosome antigen 1) and clathrin-mediated endocytosis (clathrin heavy chain) during entry into RFs. RRV particles are also colocalized with transferrin, which enters cells by clathrin-mediated endocytosis, but not with cholera toxin B, which enters cells by caveola-mediated endocytosis. Inhibition of clathrin-mediated endocytosis with a dominant-negative construct of EPS15, an essential component of clathrin-coated pits, blocked the entry of RRV into RFs. Together, these results indicate that RRV entry into RFs is mediated by clathrin-mediated endocytosis.Kaposi''s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is a gammaherpesvirus associated with the development of Kaposi''s sarcoma, a malignancy commonly found in AIDS patients (13). KSHV is also associated with the development of multicentric Castleman''s disease (MCD) and primary effusion lymphoma (PEL), two rare lymphoproliferative diseases. KSHV has a restricted host range, making it difficult to study KSHV and its related malignances directly in an animal model (25). Rhesus rhadinovirus (RRV) is closely related to KSHV. RRV infects its natural host and induces lymphoproliferative diseases resembling MCD and PEL; thus, it has been proposed as an animal model for the study of KSHV (19, 26, 39). Two isolates of RRV (26-95 and 17577) have been independently isolated and sequenced so far (3, 7, 32).To establish a successful infection, a virus needs to enter the target cells and release its genome (20). Thus, defining the entry and trafficking pathway of RRV can help us understand its mechanism of infection and replication in vitro and in vivo. Herpesviruses bind to the cell surface through complex interactions between viral glycoproteins and receptor molecules, leading to either plasma membrane fusion or endocytosis (35). Plasma membrane fusion is a pH-independent event between the viral envelope and the host cell plasma membrane (23). Enveloped viruses also take advantage of cellular endocytosis pathways for their internalization (34). Endocytosis leads to fusion between the membrane of the internalized vesicle and the viral envelope at low pHs and to the release of the viral particle into the cytoplasm. Following membrane fusion, the nucleocapsid traffics to the perinuclear space and delivers the viral genome to the nucleus. Thus, endocytosis offers a convenient and fast transit system enabling the virus to enter and traffic across the plasma membrane and cytoplasm of the infected cell.In mammalian cells, there are several endocytic pathways, including clathrin-mediated endocytosis, caveola-mediated endocytosis, clathrin- and caveola-independent endocytosis, and macropinocytosis (34). These endocytic pathways differ in the nature and size of the cargo. The clathrin-mediated pathway is the most commonly observed uptake pathway for viruses (30). A viral particle is internalized into a clathrin-coated vesicle, which then loses the clathrin-coated subunits before fusing with the early endosome. An activation step occurs in the endosome, leading to the fusion of the viral envelope with the endosomal membrane and the delivery of the viral capsid to the cytosol. The acidic pH in the endosome is thought to play an essential role in triggering the fusion event. Therefore, pH sensitivity is often considered an indication that a virus enters the cell by endocytosis (30).KSHV has been shown to use clathrin-mediated endocytosis to enter human foreskin fibroblasts, activated primary human B cells, and primary human umbilical vein endothelial cells (1, 12, 29); however, the macropinocytic pathway and plasma membrane fusion pathway have also been implicated (17, 28). The mechanism of RRV entry into cells has not been defined. In this study, using two recombinant RRVs expressing luciferase (RRV-luciferase) and red fluorescent protein (RRV-RFP), respectively, we have characterized the entry pathway of RRV in rhesus fibroblasts (RFs), a cell type that RRV can infect efficiently and in which it can replicate. The results show that RRV entry into RFs occurs primarily via clathrin-mediated endocytosis.     

Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses.

Background

In Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.

Results

Convalescent sera from 69 individuals who were known to have had dengue or Japanese encephalitis virus infection were tested by western blotting against dengue, Japanese encephalitis and West Nile virus antigens. We determined that individuals who had been infected with dengue viruses had IgG responses against the premembrane protein of dengue viruses but not Japanese encephalitis, whereas individuals who had been infected with Japanese encephalitis had IgG specific for the premembrane protein of Japanese encephalitis virus but not the dengue viruses. None reacted with the premembrane protein of West Nile virus. Using the Pearson Chi Square test, it was determined that the difference between the two groups was highly significant with a p value of <0.001.

Conclusion

The use of flavivirus premembrane protein in seroepidemiological studies will be useful in determining what flaviviruses have circulated in a community.     

Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry

African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na⁺/H⁺ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.Many animal viruses have evolved to exploit endocytosis to gain entry into host cells after initial attachment of virions to specific cell surface receptors. To date, a number of different routes of endocytosis used by viruses have been characterized, including clathrin-mediated endocytosis, uptake via caveolae/lipid rafts, macropinocytosis, phagocytosis, and other routes that are currently poorly understood.In recent years, viruses have also been used as tools to study cellular endocytosis and membrane trafficking at the molecular level, with there being special interest in the regulation of the diverse routes (31), since examples of viruses using each route can be found (reviewed in references 26, 31, and 38). The clathrin-mediated endocytic route has been the most extensively studied at the molecular level, and it has been shown to be used by diverse mammalian enveloped viruses, such as vesicular stomatitis virus (42), Semliki Forest virus (19), and West Nile virus (11), to infect cells. Influenza virus and HIV-1 also can use this pathway as an alternative route of entry (12, 39). Clathrin is assembled on the inside face of the plasma membrane to form a characteristic coated pit (CCP). During this process, clathrin also interacts with a number of essential molecules, including Eps15, adapter protein AP2, and dynamin GTPase (9). Additionally, clathrin-mediated endocytosis also provides endocytic vesicles as an acidified environment for those viruses that require a low-pH step during the first stages of infection to initiate capsid destabilization and genome uncoating. On the other hand, the lipid raft/caveola-based route is generally used by those acid-independent viruses. Recently, macropinocytosis is generating growing interest, since it has been demonstrated to be induced by some viruses from diverse families, such as vaccinia virus and adenovirus serotype 3 (5, 29), to gain entry into cells.In this study, we have focused on the entry of African swine fever virus (ASFV), a large enveloped DNA virus with a genomic composition similar to that of poxviruses, although the virion structure and morphology resemble those of iridoviruses. At present, it is the sole member of the newly created family Asfarviridae (16, 43). It is the etiologic agent responsible for a highly lethal and hemorrhagic disease affecting domestic swine, which often results in important economic losses in affected countries because of the high rate of mortality associated with this illness and the lack of an effective vaccine.Early studies of the entry of BA71V, a Vero cell-adapted ASFV strain, into host cells showed that this internalization of virus particles is a temperature-, energy-, and low-pH-dependent process, since it does not occur at 4°C or in the presence of inhibitors of cellular respiration or lysosomotropic agents (2, 44). All these features are consistent with a receptor-mediated endocytosis mechanism of entry. However, there are still numerous questions to be answered. One of them is the nature of the cellular receptor(s) that mediates ASFV entry, which remains largely unknown, although a correlation between cell susceptibility to infection and expression of porcine CD163 on the surface of swine monocytes/macrophages has been reported (36). In regard to the viral components involved in this initial step, the p12 and p54 proteins were shown to play a role during attachment to the cell surface and p30 during internalization, as inferred from previous studies with neutralizing antibodies against p30 and p54 (17) and blockage of infection after saturation of virus binding sites with recombinant p12 (6). Early electron microscopy (EM) studies (2, 45) revealed that attachment of ASFV virions to the cell surface often occurs in coated pits; however, their later presence inside coated vesicles is not fully clear. After attachment, virions are detected inside endosomes, where fusion with the viral membrane takes place. The ASFV cycle continues with the transport of viral cores via retrograde transport along microtubules to reach a perinuclear area, known as the viral factory, where replication occurs (4).In recent times, knowledge about different endocytic pathways and their regulatory molecules has notably increased, and the development of molecular tools to study these processes is becoming increasingly precise (38). In the present study, we examined the ASFV infection using a variety of chemical inhibitors and dominant negative molecules to disrupt different endocytic pathways. Our results confirmed a major role for dynamin-dependent and clathrin-mediated endocytosis during the first stages of ASFV infection, with no significant differences in the behavior of the two ASFV strains and the two cell lines analyzed.     

The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu

Background

The integrase (IN) of human immunodeficiency virus type 1 (HIV-1) has been implicated in different steps during viral replication, including nuclear import of the viral pre-integration complex. The exact mechanisms underlying the nuclear import of IN and especially the question of whether it bears a functional nuclear localization signal (NLS) remain controversial.

Results

Here, we studied the nuclear import pathway of IN by using multiple in vivo and in vitro systems. Nuclear import was not observed in an importin α temperature-sensitive yeast mutant, indicating an importin α-mediated process. Direct interaction between the full-length IN and importin α was demonstrated in vivo using bimolecular fluorescence complementation assay (BiFC). Nuclear import studies in yeast cells, with permeabilized mammalian cells, or microinjected cultured mammalian cells strongly suggest that the IN bears a NLS domain located between residues 161 and 173. A peptide bearing this sequence -NLS-IN peptide- inhibited nuclear accumulation of IN in transfected cell-cycle arrested cells. Integration of viral cDNA as well as HIV-1 replication in viral cell-cycle arrested infected cells were blocked by the NLS-IN peptide.

Conclusion

Our present findings support the view that nuclear import of IN occurs via the importin α pathway and is promoted by a specific NLS domain. This import could be blocked by NLS-IN peptide, resulting in inhibition of viral infection, confirming the view that nuclear import of the viral pre-integration complex is mediated by viral IN.     

pH-Dependent entry of chikungunya virus fusion into mosquito cells

Background

Millions of human infections caused by arthropod-borne pathogens are initiated by the feeding of an infected mosquito on a vertebrate. However, interactions between the viruses and the mosquito vector, which facilitates successful infection and transmission of virus to a subsequent vertebrate host, are still not fully understood.

Finding

Here we describe early chikungunya virus (CHIKV) infectious events in cells derived from one of the most important CHIKV vectors, Aedes albopictus. We demonstrated that CHIKV infection of mosquito cells depended on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs chloroquine, ammonium chloride, and monensin, which is consistent with observations in mammalian cells. While all three agents inhibited CHIKV infection in C6/36 cells, ammonium chloride was less toxic to cells than the other agents.

Conclusion

The observation of similar mechanisms for inhibition of CHIKV infection in mosquito and mammalian cell lines suggests that conserved entry pathways are utilized by CHIKV for vertebrate and invertebrate cell types.

     

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

Background

The cellular prion protein (PrP^C) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP^Sc). Although endocytosis appears to be required for this conversion, the mechanism of PrP^C internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved.

Methodology/Principal Findings

We have investigated the mechanism of PrP^C endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP^C internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP^C endocytosis. PrP^C internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP^C co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP^C can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization.

Conclusions/Significance

These findings are of particular interest if we consider that the internalization route/s undertaken by PrP^C can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.     

Polarised Clathrin‐Mediated Endocytosis of EGFR During Chemotactic Invasion

Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.      

Quinic acid derivatives inhibit dengue virus replication in vitro

Background

Dengue is the most prevalent arboviral disease in tropical and sub-tropical areas of the world. The incidence of infection is estimated to be 390 million cases and 25,000 deaths per year. Despite these numbers, neither a specific treatment nor a preventive vaccine is available to protect people living in areas of high risk.

Results

With the aim of seeking a treatment that can mitigate dengue infection, we demonstrated that the quinic acid derivatives known as compound 2 and compound 10 were effective against all four dengue virus serotypes and safe for use in a human hepatoma cell line (Huh7.5). Both compounds were non-virucidal to dengue virus particles and did not interfere with early steps of the dengue virus life cycle, including binding and internalization. Experiments using a replicon system demonstrated that compounds 2 and 10 impaired dengue virus replication in Huh7.5 cells. Additionally, the anti-dengue virus effects of the quinic acid derivatives were preserved in human peripheral blood mononuclear cells.

Conclusions

Taken together, these data suggest that quinic acid derivatives represent a novel chemical class of active compounds that could be used to combat dengue virus infection.

     

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2μ2

Background

Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated.

Methods

We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in μ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out.

Results

We identified μ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2μ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules.

Conclusions

Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2μ2.

General Significance

This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.     

Escherichia coli K1 utilizes host macropinocytic pathways for invasion of brain microvascular endothelial cells

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood‐brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin‐mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin‐mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.      

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na⁺/H⁺ ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via α_v integrin/CD46 in HeLa cells.Human rhinoviruses (HRVs), members of the family Picornaviridae that represent a major cause of the common cold, essentially utilize two different receptor types for host cell attachment. The 12 minor group HRVs, exemplified by HRV2, bind low-density lipoprotein receptor (LDLR), LDLR-related protein (LRP) (20), and very-LDLR (VLDLR) (29) and are internalized via the well-characterized clathrin-dependent endocytic pathway (44); however, these ligands, like others, can switch to different entry portals when the clathrin-dependent pathway is blocked (4). Once the virus arrives in endosomal carrier vesicles or late endosomes, uncoating (i.e., the release of the viral RNA genome) is triggered by the acidic pH (35, 39).The 87 major group HRVs, exemplified by HRV14, bind intercellular adhesion molecule-1 (ICAM-1). Following entry, uncoating is triggered by ICAM-1 itself (3), but the low endosomal pH facilitates this process (37). Based on inhibition of infection by the dominant negative (DN) dynamin-2 mutant dyn^K44A, it was proposed that HRV14 also follows a clathrin-dependent pathway in HeLa-H1 cells (9). However, ICAM-1 lacks a clathrin localization signal and even functions as a viral receptor when its cytoplasmic tail is replaced with a glycosylphosphatidylinositol (GPI) anchor (45). Furthermore, dynamin has also been shown to be involved in pathways other than clathrin-mediated endocytosis (CME), such as caveolae- and lipid raft-dependent entry, as a function of ligand and cell type (reviewed in references 30 and 34). Additionally, dynamin might play a role in formation and closure of circular pinocytic ruffles (31). More recently, a specific entry pathway for ICAM-1 ligands into human umbilical vein endothelial cells was identified and termed “cam-mediated endocytosis”; uptake was found to be triggered upon binding of multivalent ligands, such as immunoconjugates and immunobeads, and to occur independently from clathrin and caveolin. Inhibition by amiloride, actin depolymerization, and protein kinase C inhibitors pointed to macropinocytosis (33). So far, it is not known whether these findings are relevant to the entry pathway of HRVs via ICAM-1 as the uptake kinetics was significantly dependent on particle size. For all these reasons, involvement of clathrin in HRV14 uptake is questionable. Accordingly, we explored entry of HRV14 via ICAM-1 and compared the results with the well-characterized clathrin-dependent entry pathway of HRV2 (44). Employing pharmacological compounds, specific DN inhibitors, immunofluorescence, and electron microscopy, we demonstrate that HRV14 enters rhabdomyosarcoma ICAM-1-expressing (RD-ICAM) cells via a pathway independent of clathrin, caveolin, and flotillin.     

Phylogenetic evidence for the distinction of Saaremaa and Dobrava hantaviruses

Background

Simian virus 40 (SV40) enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi.

Results

We confirmed our earlier finding that SV40 co localizes with the Golgi marker β-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide.

Conclusion

The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with β-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

Clathrin- and caveolin-independent entry of human papillomavirus type 16--involvement of tetraspanin-enriched microdomains (TEMs)

Background

Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway.

Methodology/Principal Findings

Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16.

Conclusions/Significance

Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.