Rh type B glycoprotein is a new member of the Rh superfamily and a putative ammonia transporter in mammals

Ammonium transporters play a key functional role in nitrogen uptake and assimilation in microorganisms and plants; however, little is known about their structural counterpart in mammals. Here, we report the molecular cloning and biochemical characterization of Rh type B glycoproteins, human RhBG and mouse Rhbg, two new members of the Rh family with distinct tissue specificities. The RhBG orthologues possess a conserved 12-transmembrane topology and most resemble bacterial and archaeal ammonium transporters. Human RHBG resides at chromosome 1q21.3, which harbors candidate genes for medullary cystic kidney disease, whereas mouse Rhbg is syntenic on chromosome 3. Northern blot and in situ hybridization revealed that RHBG and Rhbg are predominantly expressed in liver, kidney, and skin, the specialized organs involving ammonia genesis, excretion, or secretion. Confocal microscopy showed that RhBG is located in the plasma membrane and in some intracellular granules. Western blots of membrane proteins from stable HEK293 cells and from mouse kidney and liver confirmed this distribution. N-Glycanase digestion showed that RhBG/Rhbg has a carbohydrate moiety probably attached at the NHS motif on exoloop 1. Phylogenetic clustering, tissue-specific expression, and plasma membrane location suggest that RhBG homologous proteins are the long sought major ammonium transporters in mammalians.     

Characterization of putative proteins encoded by variable ORFs in white spot syndrome virus genome

Three transketolase genes have been identified in the human genome to date: transketolase (TKT), transketolase-like 1 (TKTL1) and transketolase-like 2 (TKTL2). Altered TKT functionality is strongly implicated in the development of diabetes and various cancers, thus offering possible therapeutic utility. It will be of great value to know whether TKTL1 and TKTL2 are, similarly, potential therapeutic targets. However, it remains unclear whether TKTL1 and TKTL2 are functional transketolases. Homology modelling of TKTL1 and TKTL2 using TKT as template, revealed that both TKTL1 and TKTL2 could assume a folded structure like TKT. TKTL1/2 presented a cleft of suitable dimensions between the homodimer surfaces that could accommodate the co-factor-substrate. An appropriate cavity and a hydrophobic nodule were also present in TKTL1/2, into which the diphosphate group fitted, and that was implicated in aminopyrimidine and thiazole ring binding in TKT, respectively. The presence of several identical residues at structurally equivalent positions in TKTL1/2 and TKT identified a network of interactions between the protein and co-factor-substrate, suggesting the functional fidelity of TKTL1/2 as transketolases. Our data support the hypothesis that TKTL1 and TKTL2 are functional transketolases and represent novel therapeutic targets for diabetes and cancer.     

Fenneropenaeus indicus is protected from white spot disease by oral administration of inactivated white spot syndrome virus

Fenneropenaeus indicus could be protected from white spot disease (WSD) caused by white spot syndrome virus (WSSV) using a formalin-inactivated viral preparation (IVP) derived from WSSV-infected shrimp tissue. The lowest test quantity of lyophilized IVP coated onto feed at 0.025 g(-1) (dry weight) and administered at a rate of 0.035 g feed g(-1) body weight d(-1) for 7 consecutive days was sufficient to provide protection from WSD for a short period (10 d after cessation of IVP administration). Shrimp that survived challenges on the 5th and 10th days after cessation of IVP administration survived repeated challenges although they were sometimes positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. These results suggest that F. indicus can be protected from WSD by simple oral administration of IVP.     

The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily

The malaria parasite's chloroquine resistance transporter (CRT) is an integral membrane protein localized to the parasite's acidic digestive vacuole. The function of CRT is not known and the protein was originally described as a transporter simply because it possesses 10 transmembrane domains. In wild-type (chloroquine-sensitive) parasites, chloroquine accumulates to high concentrations within the digestive vacuole and it is through interactions in this compartment that it exerts its antimalarial effect. Mutations in CRT can cause a decreased intravacuolar concentration of chloroquine and thereby confer chloroquine resistance. However, the mechanism by which they do so is not understood. In this paper we present the results of a detailed bioinformatic analysis that reveals that CRT is a member of a previously undefined family of proteins, falling within the drug/metabolite transporter superfamily. Comparisons between CRT and other members of the superfamily provide insight into the possible role of the protein and into the significance of the mutations associated with the chloroquine resistance phenotype. The protein is predicted to function as a dimer and to be oriented with its termini in the parasite cytosol. The key chloroquine-resistance-conferring mutation (K76T) is localized in a region of the protein implicated in substrate selectivity. The mutation is predicted to alter the selectivity of the protein such that it is able to transport the cationic (protonated) form of chloroquine down its steep concentration gradient, out of the acidic vacuole, and therefore away from its site of action.     

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays.     

The cell surface receptor G6b, a member of the immunoglobulin superfamily, binds heparin

The G6b gene, located in the human Major Histocompatibility Complex, encodes a receptor of the immunoglobulin (Ig) superfamily. In this study, we show using a variety of techniques that the extracellular domain of the G6b protein, containing a single Ig-like domain, binds to heparin with high affinity. In an ELISA assay, this binding was displaceable with soluble heparin with an IC50 value of approximately 0.5 microg/ml. Other sulfated glycans showed weaker or no competition. The observed interaction between G6b and heparin is strongly salt dependent suggesting a mainly electrostatic interaction. Heparin might modulate the interaction of G6b with its as yet unidentified protein ligand.     

A model for apoptotic interaction between white spot syndrome virus and shrimp

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.     

ORF390 of white spot syndrome virus genome is identified as a novel anti-apoptosis gene

Apoptosis serves as an important defense strategy employed by host cells against viral invasion. Many viruses contain the anti-apoptotic genes to block the defense-by-death response of host cells. In this study, we tried to identify the putative anti-apoptotic genes in white spot syndrome virus (WSSV) genome. We confirmed that actinomycin D could induce apoptosis of shrimp primary cells. However, the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPVDelta35k/pol+ lacks a functional P35 gene undergoing apoptosis and its infection could induce Sf9 cell apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPVDeltaP35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPVDeltaP35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anti-apoptotic gene. The ORF displays two putative caspase9 cleavage sites LLVETDGPS, VKLEHDGSK, and a caspase3 cleavage site EEDEVDGVP. The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells did not show obvious characteristics of apoptosis when infected with AcMNPVDeltaP35k/pol+. And the transient expression of ORF390 allowed AcMNPVDeltaP35k/pol+ replication in Sf9 cells and resulted in the formation of polyhedra successfully. The results indicate that function of ORF390 in WSSV is a kind of apoptotic suppressor like P35 in AcMNPV.     

对虾白斑杆状病毒DNA聚合酶调控序列的克隆

为了揭示对虾白斑杆状病毒的致病机理,将该病毒基因组中推测的DNA聚合酶上游调控序列克隆进荧光素酶报告基因载体中,以便寻找一个能表达该病毒基因的细胞系统.     

Ultrastructure of white spot syndrome virus development in primary lymphoid organ cell cultures

Primary cell cultures from the lymphoid organ of Penaeus monodon were used to investigate in vitro propagation and morphogenesis of white spot syndrome virus (WSSV). Double-strength Leibovitz's L15 supplemented with 20% fetal bovine serum, pH 7.5, with a final osmolarity of 530 +/- 5 mOsm kg-1 was identified as the most suitable culture medium. In this medium, the lymphoid cells remained viable for more than 1 wk. Migrating cells were inoculated with WSSV, and the consequent cytopathic effects documented by light and electron microscopy. WSSV appears capable of following 2 alternative assembly sequences, one similar to the morphogenesis of the Oryctes rhinocerus virus and another which is more typical of baculoviral assembly. Possible relationships between WSSV, Oryctes virus, and baculoviruses are discussed.     

A putative thiamine transport protein is a receptor for feline leukemia virus subgroup A

Feline leukemia virus (FeLV) is a horizontally transmitted virus that causes a variety of proliferative and immunosuppressive diseases in cats. There are four subgroups of FeLV, A, B, C, and T, each of which has a distinct receptor requirement. The receptors for all but the FeLV-A subgroup have been defined previously. Here, we report the identification of the cellular receptor for FeLV-A, which is the most transmissible form of FeLV. The receptor cDNA was isolated using a gene transfer approach, which involved introducing sequences from a feline cell line permissive to FeLV-A into a murine cell line that was not permissive. The feline cDNA identified by this method was approximately 3.5 kb, and included an open reading frame predicted to encode a protein of 490 amino acids. This feline cDNA conferred susceptibility to FeLV-A when reintroduced into nonpermissive cells, but it did not render these cells permissive to any other FeLV subgroup. Moreover, these cells specifically bound FeLV-A-pseudotyped virus particles, indicating that the cDNA encodes a binding receptor for FeLV-A. The feline cDNA shares approximately 93% amino acid sequence identity with the human thiamine transport protein 1 (THTR1). The human THTR1 receptor was also functional as a receptor for FeLV-A, albeit with reduced efficiency compared to the feline orthologue. On the basis of these data, which strongly suggest the feline protein is the orthologue of human THTR1, we have named the feline receptor feTHTR1. Identification of this receptor will allow more detailed studies of the early events in FeLV transmission and may provide insights into FeLV pathogenesis.     

Proteomic analyses of the shrimp white spot syndrome virus

White spot syndrome virus (WSSV), a unique member within the virus family Nimaviridae, is the most notorious aquatic virus infecting shrimp and other crustaceans and has caused enormous economic losses in the shrimp farming industry worldwide. Therefore, a comprehensive understanding of WSSV morphogenesis, structural proteins, and replication is essential for developing prevention measures of this serious parasite. The viral genome is approximately 300kb and contains more than 180 open reading frames (ORF). However, most of proteins encoded by these ORF have not been characterized. Due to the importance of WSSV structural proteins in the composition of the virion structure, infection process and interaction with host cells, knowledge of structural proteins is essential to understanding WSSV entry and infection as well as for exploring effective prevention measures. This review article summarizes mainly current investigations on WSSV structural proteins including the relative quantities, localization, function and protein-protein interactions. Traditional proteomic studies of 1D or 2D gel electrophoresis separations and mass spectrometry (MS) followed by database searches have identified a total of 39 structural proteins. Shotgun proteomics and iTRAQ were initiated to identify more structural proteins. To date, it is estimated that WSSV is assembled by at least 59 structural proteins, among them 35 are defined as the envelope fraction (including tegument proteins) and 9 as nucleocapsid proteins. Furthermore, the interaction within several major structural proteins has also been investigated. This identitification and characterization of WSSV protein components should help in the understanding of the viral assembly process and elucidate the roles of several major structural proteins.     

Polychaete worms--a vector for white spot syndrome virus (WSSV)

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.     

Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus

The envelope protein from vesicular stomatitis virus (VSV) has become an important tool for gene transfer and gene therapy. It is widely used mainly because of its ability to mediate virus entry into all cell types tested to date. Consistent with the broad tropism of the virus, the receptor for VSV is thought to be a ubiquitous membrane lipid, phosphatidylserine (PS). However, the evidence for this hypothesis is indirect and incomplete. Here, we have examined the potential interaction of VSV and PS at the plasma membrane in more detail. Measurements of cell surface levels of PS show a wide range across cell types from different organisms. We demonstrate that there is no correlation between the cell surface PS levels and VSV infection or binding. We also demonstrate that an excess of annexin V, which binds specifically and tightly to PS, does not inhibit infection or binding by VSV. While the addition of PS to cells does allow increased virus entry, we show that this effect is not specific to the VSV envelope. We conclude that PS is not the cell surface receptor for VSV, although it may be involved in a postbinding step of virus entry.     

Crassostrea gigas oysters as a shrimp farm bioindicator of white spot syndrome virus

This study explored whether Crassostrea gigas oysters can be used as a bioindicator of white spot syndrome virus (WSSV) in shrimp farm water canals. Bioassays showed that C. gigas can accumulate WSSV in their gills and digestive glands but do not become infected, either by exposure to seawater containing WSSV or by cohabitation with infected shrimp. The use of a WSSV nested PCR to screen oysters placed in water canals at the entry of a shrimp farm allowed WSSV to be detected 16 d prior to the disease occurring. The finding that C. gigas can concentrate small amounts of WSSV present in seawater without being harmed makes it an ideal sentinel species at shrimp farms.     

Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus

Aims:  White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) for quantitative detection of WSSV.
Materials and Methods:  A set of six specially designed primers that recognize eight distinct sequences of the target. The whole process can be completed in 1 h under isothermal conditions at 63°C. Detection and quantification can be achieved by real-time monitoring in an inexpensive turbidimeter based on threshold time required for turbidity in the LAMP reaction. A standard curve was constructed by plotting viral titre against the threshold time ( T _t) using plasmid standards with high correlation coefficient ( R ² = 0·988).
Conclusions:  Sensitivity analysis using 10-fold dilutions (equivalent to 35 ng  μ l⁻¹ to 35 ag  μ l⁻¹) of plasmid standards revealed this method is capable of detecting upto 100 copies of template DNA. Cross-reactivity analysis with DNA/cDNA of IHHNV, TSV, YHV-infected and healthy shrimp showed this method is highly specific for quantitative detection of WSSV.
Significance and Impact of the Study:  WSSV real-time LAMP assay appears to be precise, accurate and a valuable tool for the detection and quantification of WSSV in large field samples and epidemiological studies.     

A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.