ABSTRACT Diverse intracellular pathogens rely on eukaryotic cell surface disulﬁde reductases to invade host cells. Pharmacologic inhibition of these enzymes is cyto- toxic, making it impractical for treatment. Identifying and mechanistically dissecting microbial proteins that co-opt surface reductases could reveal novel targets for dis- rupting this common infection strategy. Anaplasma phagocytophilum invades neutro- phils by an incompletely deﬁned mechanism to cause the potentially fatal disease granulocytic anaplasmosis. The bacterium’s adhesin, Asp14, contributes to invasion by virtue of its C terminus engaging an unknown receptor. Yeast-two hybrid analysis identiﬁed protein disulﬁde isomerase (PDI) as an Asp14 binding partner. Coimmuno- precipitation conﬁrmed the interaction and validated it to be Asp14 C terminus de- pendent. PDI knockdown and antibody-mediated inhibition of PDI reductase activity impaired A. phagocytophilum infection of but not binding to host cells. Infection during PDI inhibition was rescued when the bacterial but not host cell surface disul- ﬁde bonds were chemically reduced with tris(2-carboxyethyl)phosphine-HCl (TCEP). TCEP also restored bacterial infectivity in the presence of an Asp14 C terminus blocking antibody that otherwise inhibits infection. A. phagocytophilum failed to pro- ductively infect myeloid-speciﬁc-PDI conditional-knockout mice, marking the ﬁrst demonstration of in vivo microbial dependency on PDI for infection. Mutational anal- yses identiﬁed the Asp14 C-terminal residues that are critical for binding PDI. Thus, Asp14 binds and brings PDI proximal to A. phagocytophilum surface disulﬁde bonds that it reduces, which enables cellular and in vivo infection.
16 Read more
including meningitis, myocarditis, gastroenteritis, polio- myelitis, common cold and diabetes . The enterovirus genome is a positive single stranded RNA molecule of approximately 7.500 nucleotides starting with a 5'untranslated region (5'UTR) followed by an open read- ing frame encoding a polyprotein of about 2.200 amino acids and a 3'UTR ending with a poly A tail . Several cel- lular receptors have been identified as attachment mole- cules for Picornaviridae, including the poliovirus receptor (PVR) , various types of integrins [7-10], intracellular adhesion molecule 1 (ICAM-1) [11,12], decay-accelerat- ing factor (DAF or CD55) [13,14] and coxsackie- and ade- novirus receptor (CAR) [15,16]. Group B coxsackieviruses (CVB) with its six serotypes, CVB1-6, may enter the sus- ceptible cell by attachment to CAR, a 46-kDa transmem- brane protein that also serves as a receptor for many adenoviruses . In addition, some strains of CVB1, 3 and 5 can interact with an additional receptor, DAF, a 70- kDa regulatory protein consisting of four short consensus repeats (SCRs) . CVBs can attach to DAF, but are usu- ally unable to enter the cell in the absence of CAR [19,20] unless the DAF receptors are cross-linked by specific anti- DAF monoclonal antibodies (MAbs) . Thus, binding to DAF is a characteristic feature of many enteroviruses including enterovirus 70 and echovirus 7 [13,14,21-25]. Interactions between a virus and the host cell surface are generally studied using purified radiolabeled virions that are allowed to attach to cultured cells.
FIV-34TF10 is a particularly useful molecular clone for study of OrfA function, in that this Petaluma-derived isolate has a premature stop codon in the OrfA open reading frame (28, 35). There is an overall sequence identity level of 91% between this virus and FIV-PPR, with 71% and 85% homology at the amino acid level for the orfA and env genes. FIV-34TF10 is CD134 independent, and the viral entry assay in this study also showed that 34TF10 Env pseudotyped virons were more efficient for CrFK cell-based entry than for entry into GFox cells. However, it is important to emphasize that 34TF10 SU still binds CD134 strongly, even though it is apparently already in a conformation suitable for binding to CXCR4, thus facili- tating infection. The current study indicated that FIV-34TF10 can readily infect CD134-negative CrFK cells and generate high RT activity levels in the culture but does not launch efficient infection on primary T cells, feline T-cell lines, CD134-positive GFox cells, or CrFK cells expressing domain 1 of feline CD134 on the background of human CD134 (fD1- CrFK; data not shown) (5). In contrast, an OrfA-repaired 34TF10 strain, OrfArep, can produce robust infection in CD134-positive 104-C1 cells and GFox cells. This phenome- non implies that there is some direct or indirect interaction between FIV OrfA and CD134 and that this interplay is es- sential for productive viral infection mediated by CD134. This supposition is borne out by the FACS analysis and Western blot data in this study, which clearly show that OrfA causes FIG. 6. ␤ -Gal assay for measurement of the entry of FIV-PPR or
Previous studies have indicated that SAG1, the major surface molecule of the protozoan parasite Toxoplasma gondii, is an important attachment ligand for the host cell. However, the research data that supports this claim comes largely from studies investigating tachyzoite binding, and not SAG1 binding per se. In this study we successfully developed an in vitro attachment assay to directly evaluate the mechanism of SAG1-host cell binding. Competition experiments were then performed using SAG1 that had been pre-treated with the neoglycoprotein BSA-glucosamide or with antibody. Soluble BSA-glucosamide blocked SAG1 attachment to MDBK cells in a dose-dependent manner, implying that SAG1 binding is mediated, in part, via attachment to host cell surface glucosamine. Interestingly, pre-incubation of SAG1 in polyclonal sera from chronically infected mice failed to block binding. This challenges the assumption that anti-SAG1 antibodies block parasite attachment through the masking of SAG1 host cell binding domains. Taken together, this evidence presents new strategies for understanding SAG1-mediated attachment.
Cargo uptake via caveolin-1-mediated endocytosis is normally triggered through ligand-receptor interactions (45, 61–69; see also reference 70 for a review). However, support for ligand-independent caveolin-1 function at plasma membrane caveolae also exists (71). Could ligand receptor-mediated signaling also play a role in the recruitment of caveolin-1 and other caveolar proteins to invaginations? From our experiments involving isolated L. monocytogenes membrane protrusions, the results implied that contact of the structures with the host cell surface alone is sufﬁcient to trigger recruitment of caveolin-1, cavin-2, and EHD2. Coupled with our recent ﬁnding of the host plasma membrane receptor CD147 at L. monocytogenes membrane invaginations (23), the potential for the presence of a ligand(s) on the surface of protrusions, interacting with a corresponding receptor(s) on the cell forming the invaginations, presents a compelling area of further investigation. Furthermore, the visually apparent elevated levels of these caveolar proteins surrounding the structures also point to the possibility that preassembled caveolar vesicles could incorporate their proteins into the plasma membrane as an endocytic unit at sites of membrane invaginations (72, 73) rather than being recruited separately. Unlike those aforementioned pro- teins, dynamin-2 remained absent at contact sites of the isolated membrane protru- sions and the underlying cell. During cell-to-cell-spreading experiments, dynamin-2 showed an unusual localization at the invagination, being restricted to the region of the bacterium. This recruitment contrasts with its classical function at the neck of endocytic pits, where assemblies of dynamin-2 collars catalyze, via GTP, membrane scission of the budding vesicle (19, 74–76). One potential explanation may pertain to the L. monocy- togenes internalin family of proteins which are involved in several stages of its infection cycle. Cell surface internalin A (InlA) and InlB control initial bacterial invasion into cells (77, 78; see also reference 79 for a review), whereas secreted internalin C (InlC) and InlP have been shown to promote bacterial cell-to-cell and cell-to-basement membrane transfer, respectively (80, 81). Consequently, future investigations into the L. monocy- togenes infection cycle should focus on evaluating whether or not these bacterial components are also involved in the caveolin-mediated engulfment of membrane protrusions.
22 Read more
Microsporidia possess a unique, highly specialized invasion mechanism that involves the spore wall (SW), polar tube, and infectious sporoplasm. The spore (Sp) wall consists of proteins and chitin that protect the organism from harsh environmental conditions, thereby permitting transmission of the organism via water or food (12). The spore wall contains two layers: an electron-dense outer exospore layer and an electron-lucent inner endospore layer. Chitin is the main component of the endospore layer and is important for maintaining spore structure. Several spore wall proteins play crucial roles in spore adherence, signaling, and host cell interactions during the infection (13–17). The polar tube is a highly specialized invasion organelle of the microsporidia that coils around the sporoplasm inside the spore before germination (18, 19). Five polar tube proteins (polar tube protein 1 [PTP1] through PTP5) have been identiﬁed, among which PTP1 and PTP4 interact with host cells via mannose binding receptors and transferrin receptor 1 (TfR1), respectively, thereby enabling the polar tube to bind to the host cell surface and create an invasion synapse in which the sporoplasm can penetrate into the host cell (20–25). The infectious sporoplasm enters the host cell and undergoes development from meronts (proliferative forms) to sporonts, sporoblasts (Sb), and, ﬁnally, mature spores (6, 12, 26). Following entrance of the sporoplasm into the host cell, microsporidia (depending on the species) can reside directly in the host cell cytosol without additional barriers or can be found in a parasitophorous vacuole during various stages of the replicative cycle. Members of the genus Encephalitozoon spend their entire host cell cycle inside a PV (27–29). Ultrastructural analysis showed that the plasma membrane of meronts was closely associated with the PV membrane, and it has been suggested that they interact directly with the PV membrane (30–32). Meronts (Me) then detach from the PV membrane at a later stage of development, and maturing spores move to the center of the vacuole (32).
24 Read more
α-amylase (malS), and the genetically linked and corre- lated transport functions for maltodextrins (mdxG), as well as two 6-phospho-beta-glucosidases (pbg5, and pbg4). Among these 6-phospho-beta-glucosidase encod- ing genes, the pgb4 gene appeared to be induced in all cps cluster deletion strains, a feature shared by only two additional genes that appeared to be consistently down- regulated in all mutants, and encode a prephenate dehydrogenase (tyrA), and a sodium-coupled N-acetyl- neuramidate transporter (lp_3563). In addition, expres- sion of a FAD/FMN-containing dehydrogenase encoding gene (lp_0291) appeared to be differentially affected in the 4 cps deletion strains, i.e., downregulated upon dele- tion of cps cluster 1 or 2, but upregulated when cps clus- ter 3 or 4 is deleted. Importantly, compensatory activation of one of the alternative cps clusters was not observed in any of the cps cluster deletion mutants. These results show that especially deletion of cps cluster 1 or 2 elicited pleiotropic transcriptome consequences, which appeared to be centered on genes with functions associated with metabolism and transport of various amino acids, but also included genes encoding several other functions. The observation that deletion of the capacity to produce CPS affected the expression of genes encoding specific transport and metabolism functions, may suggest that the presence of polysac- charides in the cell envelope plays a role in the access that the bacteria have to nutrients from the environ- ment. Polysaccharides may function as macromole- cules that sequester nutrients and thereby facilitate their import [28,29], or alternatively they may form a capsular structure that surrounds the cell and retards or inhibits diffusion or transport of nutrients towards the membrane surface and thereby reduces trans-membrane transport efficiencies.
10 Read more
exchange of molecules between neighboring cells (Cheval and Faulkner 2018). To date, whether F. graminearum modulates its host plasmodesmata for successful penetra- tion and/or cell-to-cell movement remains to be dissected. Furthermore, there has not been a consensus about the biotrophic lifestyle of F. graminearum during the initial stages of host colonization (Jansen et al. 2005; Trail 2009; Brown et al. 2010; Kazan et al. 2012). Brown et al. (2010) found no indication of necrotrophy at those stages after infection as the advancing F. grami- nearum hyphae remained in the intercellular space of the wheat rachis cells before subsequent intracellular growth, which leads to cell death and necrosis. How- ever, another group found that as soon as the pathogen enters the cytosol of the epicarp cells in both barley and wheat, plant cell death is induced, suggesting the absence of biotrophic lifestyle of the fungus at that stage (Jansen et al. 2005). In the present study, we first demonstrated by live cell imaging that the first- and later-invaded wheat coleoptile cells were still alive after colonization by F. graminearum (Fig. 4). To further confirm this, we stained these cells with FM4–64 and found that the biotrophic invasive hyphae of the fungus were separately sealed within a plant membrane as they colonized the living plant cells (Fig. 5). In addition, we demonstrate that the invasion pegs of the hyphae ex- perience extreme constriction as they pierce the wheat cell walls (Figs. 3 and 4), which is a typical feature of biotrophic growth. Our results are consistent with an extended biotrophic invasion strategy reported in rice blast disease (Kankanala et al. 2007). This is also in line with a previous study which affirmed that F. grami- nearum initiates infection of coleoptiles using covert penetration strategies (Zhang et al. 2012).
12 Read more
timally benefit from the host milieu and withstand stress condi- tions it may encounter. In return, infection alters the structure and integrity of the ciliated bronchial epithelium, a condition that is coupled to immune modulators, leading to the onset of disease. Here, we present a valuable data resource that informs the regu- lation of the majority of NTHi genomic determinants under con- ditions that mimic the first stages of bacterial infection. However, one limitation of our study is that the dynamics of the events may affect the transcriptome evaluation. Indeed, during each time point, bacteria exist in different cell cycle states and cellular local- izations, while eukaryotic cells are exposed to a different number of bacteria, resulting in a mixed population of transcripts that is averaged by the analysis. Nevertheless, the proposed model allows a plausible assessment of gene modulation during NTHi infection of human mucosae. Importantly, we observed that a significant portion of the highly upregulated genes were annotated as having an unknown function or no clear cellular localization of their products. Therefore, our attempt to reproduce a temporal infec- tion model could be taken as a unique opportunity to disclose new factors that may act as important players in NTHi pathogenesis. Using this approach to monitor the expression dynamics of viru- lence factors in real time represents a novel tool to complement classical screening procedures used for NTHi vaccine discovery, with a potential application to other bacterial pathogens.
14 Read more
It is now well established that during their intracellular life cycle, a fraction of M. tuberculosis bacteria escape the phagosome (20 –22; see reference 23 for review). Thus, increasing numbers of bacteria can be found in the cytosol over time until a threshold is reached and the host cell undergoes necrosis, allowing for exit of M. tu- berculosis (21, 24). The process of host cell necrosis induction involves potentially multiple bacterial effectors and host cell signaling pathways that are only beginning to be understood (3, 8–10). Recently, our group described a hypervirulent M. tuberculosis mutant resulting from the deletion of the gene Rv3167c, a putative member of the tetracycline repressor (TetR)-like family of transcriptional regulators (TFR). Infection of macrophages with the M. tuberculosis Rv3167c deletion (ΔRv3167c mutant) strain resulted in a signiﬁcant increase in phagosomal escape, autophagy, and host cell necrosis compared to wild-type M. tuberculosis-infected cells (25). However, we did not identify the Rv3167c-regulated genes, which are responsible for the observed pheno- types. In the present study, we further investigated the virulence regulation by Rv3167c and uncovered a novel molecular mechanism for the action of PDIM on the host cell.
12 Read more
The target virus we selected is measles virus (MV). Measles virus (MV) is a member of the morbillivirus subgroup of paramyxoviruses, containing glycosylated envelope proteins hemagglutinin (H) and fusion (F) that are embedded on the phospholipid bilayer envelope. 182 Live attenuated MV has been shown to possess promising oncolytic activity against many tumor cells, 183 which enables the possibility of virotherapy for cancer treatment. 184 For the sake of virotherapy, it is urgent to develop labeling strategies to site- specifically modify the virus surface with functional handles such as a folate and folate receptor-specific antibody, which can achieve targeting to tumor cells while avoiding normal cells. 185 The surface modification of enveloped virus in literature has focused on both surface proteins and on the phospholipid envelope. The covalent linkage of functionalities to surface proteins that is achieved by chemical modification 186 , genetic engineering 187 and metabolic incorporation of azido sugars, 188,189 likely affects the normal properties of viruses including their interaction with host cells. Since the virus envelope is derived from a host cell membrane, 190 the metabolic incorporation of phospholipids that carry functional groups into the host cell membrane has enabled the subsequent modification of virus envelopes during virus replication and assembly 191 .
183 Read more
significant resemblance to bacterial virulence factors or their virulence domains in particular with the help of the virulence factors database . We identified a few L. donovani proteins possibly having an Internalin-A (Inl-A) like domain similar to Listeria monocytogenes Inl-A, which is a surface LRR protein that helps mediate host cell inva- sion by interacting with E-cadherin on the cell membrane . We devised a strategy referred to as forward and re- verse search analysis comprising of profile versus sequence (Hidden Markov Model (HMM)  based) and sequence versus sequence (BLASTp ) comparisons to confirm the existence of such Inl-A-like remote orthologs in L. donovani. With the help of this search strategy we iden- tified remote but significant similarities between certain L. donovani proteins (L. donovani Inl-A-like proteins) and LRR containing Inl-A protein from Listeria mono- cytogenes. We next asked questions like, whether these remote orthologs shared a significant evolutionary rela- tionship with Inl-A, or what is the extent of similarity between such L. donovani Inl-A-like proteins and their bacterial counterparts? Moreover, it would also be in- teresting to study whether these L. donovani Inl-A-like proteins share similar subversion mechanism in host cells as L. monocytogenes Inl-A? We addressed the first question by studying the orthologs of these proteins and by comparing the signature leucine rich repeat (LRR) motifs between the two sets of proteins. Whereas the second question was assessed by homology modeling of L. donovani Inl-A-like proteins and their subsequent docking studies with the protein interaction partner (human E-cadherin [hEC1]) of their bacterial ortholog (Inl-A). Based on these analyses we suggest the existence of a new group of virulence factors capable of employing a yet to be known mode of host invasion mechanism in L. donovani.
17 Read more
Immunologic characterization of cell lines. Radioimmunoprecipitations (RIPs) were performed as previously described (32). To perform immunoblots, whole-cell lysates were prepared by incubating cell pellets in 0.5 ml of lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Nonidet P-40) for 15 min on ice. Nuclei and cell debris were removed by centrifugation, and protein concentrations were determined with the BCA Protein Assay Reagent (Pierce, Rockford, Ill.). Pro- tein (100 mg) from the lysates was subjected to sodium dodecyl sulfate-polyac- rylamide gel electrophoresis (SDS-PAGE) and electroblotted to 0.45-mm nitro- cellulose membranes (Schleicher and Schuell, Keene, N.H.). The membranes were blocked in 5% dried milk in phosphate-buffered saline (PBS) for 1 h, followed by overnight incubation with primary antibodies at a concentration of 10 mg/ml in blocking buffer. The blots were washed three times in PBS plus 0.1% Tween-20, incubated with horseradish peroxidase-conjugated secondary antibod- * Corresponding author. Mailing address: Laboratory of Microbial
10 Read more
The construction of hybrid genomes consisting essentially of the JFH1 replicase and the core-to-NS2 region of other HCV isolates from divergent subtypes or even genotypes, as well as the construction of a highly cell culture-adapted variant of the H77 isolate, substantially extended the scope of this cell cul- ture system (18, 24, 35), although particle yields are very low with the latter. Clearly, viral determinants contribute to the efficiency of virus assembly and release (18, 24), but it is likely that cellular proteins also modulate the efficiency of HCV production. This is illustrated by the differential outcomes of passaging of naive Huh-7 cells or a subclone of Huh-7.5 cells, designated Huh-7.5.1, that had been transfected with JFH1 RNA: Wakita and colleagues reported that JFH1 copy num- bers in transfected naive Huh-7 cells slowly decreased upon continuous passaging, and even 13 days posttransfection, only 50 to 60% of the cells were HCV antigen positive (33); in contrast, Zhong and colleagues observed a robust increase of JFH1 RNA at later passages, overall about 50-fold-higher virus titers, and eventually the spread of JFH1 to almost 100% of cells (see reference 38; also discussed in reference 3). These data suggest that Huh-7 cells differ substantially with respect to their permissiveness for HCV.
11 Read more
Binding assays. Amphotropic 4070A virus was obtained by centrifugation of 1.5 liters of medium from a culture of infected mouse BALB/c 3T3 fibroblasts (11). The gp70 was then released from the virions by freeze-thawing and was recovered in the supernatants after sedimentation of the residual viral particles as described elsewhere (11, 35). The amphotropic gp70 preparations (aliquots were adjusted to 4 mg of total protein per ml and were frozen at 2808C) maintained their receptor-binding activities through repetitive freeze-thaw cy- cles. For analyses, gp70 preparations were incubated with cell cultures for 2 h at 378C in amounts specified for each experiment. After removal of unbound gp70, the cells were rinsed with fresh medium and were then incubated for 1 h at 378C with a 1:200 dilution of a goat antiserum made to Friend ecotropic viral gp70 that cross-reacts strongly with amphotropic gp70 (10, 19). After the cells were rinsed with fresh medium, the adsorbed gp70-antibody complexes were detected by three methods. In one method, cells were incubated with fluorescein-labeled rabbit anti-goat immunoglobulin G (1:200 dilution in culture medium; Organon Teknika, Durham, N.C.) for 1 h at 378C and then rinsed three times for 5 min each with fresh medium. The cells were then fixed with ice-cold methanol, mounted in 10% glycerol in phosphate-buffered saline, and examined by immu- nofluorescence microscopy. A second detection method involved incubation with 0.4 m Ci of [ 125 I]protein A (DuPont NEN, Wilmington, Del.) per ml for 1 h,
IBDV infection begins with virus attachment to the putative cellular receptors on the surface of host cells. So far, three putative IBDV receptors on host cells have been identiﬁed, including IgM in chicken B lymphocytes (11), integrin in mouse NIH 3T3 cells (12), and HSP90 (heat shock protein 90) in chicken ﬁbroblasts (13). While integrins are cell surface-expressed membrane proteins, HSP90, a highly conserved molecular chap- erone, can be present inside the cell, expressed on the cell surface (13, 14), and secreted into the extracellular space (15). Beside its role as a cell attachment receptor for IBDV, which confers susceptibility to nonpermissive cells in IBDV infection (13), surface HSP90 was recently shown to initiate the autophagy cascade by interacting with VP2 of IBDV (14). The capsid protein VP2 is the primary immunogen of IBDV and is displayed as trimeric clusters of outer-protruding structures on IBDV capsids (16). This protein contains three distinct domains, namely, the base (B), shell (S), and projection (P) domains (17, 18). The P domain contains a conserved putative ␣ 4 ␤ 1 integrin binding motif of Ile-Asp-Ala (IDA), which mediates the interaction of IBDV with integrin recep- tors on the surface of permissive cells (19). After receptor binding, IBDV actively modulates and engages cytoskeletons and is then internalized to enter target cells via a mechanism of adsorptive or receptor-mediated endocytosis that is independent of clathrin (20). Endosomal penetration by IBDV is mediated by a pep46-dependent pore-forming mechanism. pep46, a membrane permeabilization protein present in the virus capsid, is generated from the self-cleavage of precursor VP2 during VP2 matura- tion. This protein interacts with the lipid bilayer to induce pores with a diameter of ⬍ 10 nm in endosomal membranes, resulting in the release of viral particles from the endosome into the cytoplasm (21). However, the signaling events that occur after IBDV particles attach to cellular receptors and the cellular factors that regulate these virus entry events have not yet been clear.
16 Read more
The accumulation of DVGs can have consequences on viral replication by directly competing with the full-length genomes and modulating host innate immune re- sponses (reviewed in references 27 to 30, 49, and 51 to 53). The immune-modulatory effect of DVGs has been attributed to efﬁcient activation of the IFN induction cascade and antiviral immunity, a mechanism especially well known for the copy-back DVGs found in many negative-sense RNA viruses, such as SeV (reviewed in references 28 to 30 and 36). Consistently with previous reports, we detected enrichment of highly expressed genes involved in the IFN response in one subpopulation of cells with signiﬁcantly attenuated viral transcription. In this cluster of cells, we also observed that DVG transcripts accumulated to a high level. The observation was further validated in our DI or WT infection assay, in which we detected a signiﬁcantly higher expression of some ISGs in DI-infected cells independently of the level of viral transcription and of IFNs at the late stage of the infection. The observed strong innate immune response in DI-infected cells may be explained by reduced expression of the NS1 protein, a major IFN antagonist, compared to that in WT-infected cells. However, the observation of DI-mediated antiviral protection against lethal homologous WT virus infection in type I IFN receptor knockout mice suggests the involvement of innate immune factors other than type I IFN (54). Alternatively, the IFN mRNA dose-independent ISG expression in DI-infected cells may suggest a potential novel mechanism of DVG immunostimulation that is independent of IFN induction. Expression of a subset of ISGs can be induced in the absence of IFN signaling (55, 56). It is likely to be at least partially attributed to similar and overlapping consensus DNA-binding motifs for the interferon regulatory factor (IRF) family, most notably IRF3 and IRF7, which are key regulators of type I IFN
19 Read more
In prokaryotes, BspA and Pmp proteins aid attachment to host tissue. In T. vaginalis, this might be the case for those that carry a TMD and were found to be part of the pathogen’s surface proteome; it seems natural to assume that they are anchored into the plasma membrane of the eukaryotic parasite. It is evidently more complicated than that. The BspA and Pmp HA-fusion constructs localize to intracellular compartments and not the plasma mem- brane (Fig. 5a, Additional file 2: Figure S2, Additional files 4, 5, 6: Figures S4–S6). The defined localization around the entire nucleus is typical for the endoplasmic reticu- lum of the parasite that embraces the nucleus in several layers and is largely absent from the remaining cytosol, while the two adjacent rings at the apical end, and in close proximity to the nucleus, are typical for the Golgi appara- tus of trichomonads [76, 77]. However, a sole localization of the analyzed BspA and Pmp proteins to compartments of the endomembrane system appears unlikely, as it is somewhat incompatible with the detection of some as part of a surface proteome analysis , the presence of the BspA protein TVAG_240680 in exosomes , and the increasing adhesion to host tissue we observed.
15 Read more
139 Bhana, N.; Ormrod, D.; Perry, C. M.; Figgitt, D. P., Zidovudine: a review of its use in the management of vertically-acquired pediatric HIV infection. Paediatr Drugs. 2002, 4 (8), 515-53. 45. (a) Bai, J.; Rossi, J.; Akkina, R., Multivalent anti-CCR ribozymes for stem cell-based HIV type 1 gene therapy. AIDS Res. Hum. Retroviruses 2001, 17 (5), 385-99; (b) McCaffrey, A. P.; Meuse, L.; Karimi, M.; Contag, C. H.; Kay, M. A., A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 2003, 38 (2), 503-8; (c) Deas, T. S.; Binduga-Gajewska, I.; Tilgner, M.; Ren, P.; Stein, D. A.; Moulton, H. M.; Iversen, P. L.; Kauffman, E. B.; Kramer, L. D.; Shi, P. Y., Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication. J. Virol. 2005, 79 (8), 4599-609.
151 Read more
targets for chemotherapeutic attack and the advent of a number of specific antiviral agents, it has become increasingly clear that a selective chemotherapy of virus infections can be achieved and that virus reproduction can be suppressed without deleterious effects on the host. The viral replication cycle can be roughly divided into 10 steps: virus- cell adsorption (binding, attachment), virus-cell fusion (entry, penetration), uncoating (decapsidation), early transcription and early translation, replication of the viral genome, late transcription, late translation, virus assembly, and release. All these steps could be envisaged as targets for chemotherapeutic intervention 17 . Some of synthetic drugs and