Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism.Malaria, caused by the parasitic protist P. falciparum and just one member in some other Apicomplexan species.Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups. Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite. A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif. The FIKK family seems restricted to Apicomplexa, with 20 members in The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells. Modulation of protein phosphorylation through the antagonistic effects of protein kinases and protein phosphatases is a major regulatory mechanism of most cellular processes. Dysregulation of protein phosphorylation in human cells plays a major role in many diseases such as cancers and neurodegenerative disorders . This haPlasmodium falciparum, the species responsible for the lethal form of human malaria, begins with the bite of an infected Anopheles mosquito, which delivers sporozoites into the bloodstream. These cells establish an infection inside hepatocytes, where they undergo an intense multiplication generating several thousand merozoites, a process called exo-erythrocytic schizogony. The merozoites invade erythrocytes, where they also undergo schizogony, the process that is responsible for malaria pathogenesis. Some merozoites, however, arrest the cell cycle and differentiate into male or female gametocytes, which are infective to the mosquito. Once ingested by the insect, the gametocytes develop into gametes  and fuse into a zygote. Further development in the mosquito involves a process of sporogony, producing sporozoites that accumulate in the salivary glands and are now ready to infect a new human host .Malaria parasites have a complex life cycle. Infection of human beings by Plasmodium falciparum [Plasmodium biology and in the definition of potential novel drug targets.The observation that many parasitic ePKs display profound structural and functional divergences from their counterparts in their vertebrate hosts -7 suggeslciparum , now perPrior to the genomic era, the initial classification system of Hanks and Quinn  distribu• the cyclic-nucleotide- and calcium/phospholipid-dependent kinases (the AGC group);• the CMGC group, constituted of the cyclin-dependent- (CDK), mitogen-activated- (MAPK), glycogen-synthase- (GSK) and CDK-like kinases;• the calmodulin-dependent kinases (CaMK), and• the tyrosine kinases (TyrK).ePKs that did not clearly fit into any of these groups were placed into the OPK ("other protein kinases") group. The primary structure of all enzymes in these groups conform to the model described by Hanks, in which the catalytic domain is subdivided into eleven subdomains, which can be aligned across all groups. In addition to the "typical" ePKs, several enzymes possessing protein kinase activity, but which are unrelated (or only distantly related) to ePKs at the primary structure level, have been identified and termed "atypical protein kinases" (aPKs). These include phosphatidyl-inositol 3' kinase (PI3K), DNA-dependent protein kinase, and members of pyruvate dehydrogenase kinase family.S. cerevisiae contains 115 ePKs [D. melanogaster, C. elegans and H. sapiens comprise 239, 454 and 510–520 ePK-coding genes, respectively [Exhaustive analyses of the kinome of some model organisms have now been published. The kinome of 115 ePKs , and theectively . On the • the casein kinase 1 (CK1) group;• the STE group, which includes many enzymes functioning in MAPK pathways, although the MAPKs themselves belong to the CMGC group ;• the tyrosine kinase-like (TKL) group, which, as its name indicates, includes enzymes that are related to those in the TyrK group, although they are serine-threonine protein kinases.Arabidopsis thaliana has recently been published [Drosophila have 4 and 10 members of the casein kinase 1 (CK1) group respectively, whereas the C. elegans genome encodes 85 CK1-related genes.Furthermore, a description of the 369 non-receptor serine/threonine protein kinases of the plant ublished . Comparaublished . DespiteA. thaliana, all eukaryotes whose kinome has been characterised, from yeast to man, belong to the Opisthokonta phylogenetic group. As depicted in Fig. P. falciparum kinome confirms this prediction.With the exception of the plant P. falciparum kinome .Only one malarial kinase, the previously described PfCK1 [PF11_0377] , clearlyP. falciparum genome (see below). Two additional sequences [PFC0385c and PF11_0464] form a separate cluster attached to the base of the AGC branch. There appears to be no clear member of the PKC subfamily.Five malarial kinases cluster within this group, three of which have been characterized: the cAMP-dependent PfPKA [PFI1685w] , the cGMThe main branch of the tree that contains the human CamKs also contains 13 PfePKs, which underlines the importance of calcium signalling in the parasite . A tightPlasmodium kinome. Interestingly, in other eukaryotic systems a majority of CMGC kinases are involved in the control of cell proliferation and development, and their relative abundance in the P. falciparum kinome may reflect the variety of successive proliferative and non-proliferative stages which constitute the life cycle of malaria parasites. Six enzymes are related to the cyclin-dependent kinase family, 5 of which were identified previously , the laiewed in -33 and Piewed in , clusteriewed in  and Pfcriewed in ,37, formiewed in . The comFive malarial enzymes appear in the vicinity of the TyrK-like group, including two [MAL6P1.191 and PFB0520w] that display maximal homology to MAPKKK-related or MLK (mixed-lineage kinases) enzymes upon BLASTP analysis. PFB0520w clusters with the TGFβ receptor (TGFβ1). The malarial sequence is much more similar to TGFβ receptors than to mammalian Raf, and furthermore, in common with TGFβ receptors, the malarial enzyme has a predicted transmembrane sequence N-terminal to the kinase domain. Mammalian TGFβ receptors assemble as heterodimers, and it remains to be seen whether the malarial enzyme forms a homodimer or has the capacity to coassemble with a mammalian subunit.in vitro and in silico attempts at identifying MAPKK malarial homologues [P. falciparum NIMA-related enzymes (see below) possesses an activation site that closely mimics that of MEK1/2. This enzyme, Pfnek-1 [PFL1370w], is able to specifically phosphorylate Pfmap-2  in vitro, and to act in synergy with Pfmap-2 towards the phosphorylation of exogenous substrates [No malarial protein kinase clusters with the STE7/11/20 group, which is consistent with the lack of success of earlier mologues ,40 and pbstrates . This substrates .Plasmodium genes form a cluster of NIMA-related sequences that includes the NIMA-related kinase Nek1. Of these five, four are recognised by BLASTP analysis as being related to the NIMA/Nek family [Five k family , includik family . The fifP. falciparum exported protein kinase" (PfEST, MAL7P1.91) [Several protein kinases appear not to cluster clearly with any defined group, or to constitute small "satellite" clusters. Examples of such "orphan" kinases are (i) the cluster formed by PfKIN [PF140516], an enzyme previously described as related to the SNF1 family , with twL7P1.91) , which fSo far, four PfePKs have been described as appearing as "composite" enzymes displaying features from more than one established ePK family. As mentioned above, PfPK6 [PF130206] and Pfcrk-4 [PFC0755c] both display relatedness to CDKs and MAPKs, and this is confirmed by their position on the tree. The MAPKK-like activation site of Pfnek-1 [PFL1370w] has been discussed above. The fourth example is that of PfPK7 [PFB0605w], an enzyme whose C-terminal region carries a sequence which is conserved in MAPKKs but whose N-terminal region is more closely related to that of PKAs . This seP. falciparum genome, we carried out a more thorough analysis in which the 18 malarial kinases belonging to this group were compared with comprehensive sets of related kinases from the yeast and human genomes  previously reported as belonging to the ERK family clustered, as expected, with the MAP kinases. However, in contrast to previous suggestions brought forward before the full complement of mammalian ERKs had been characterised [P. falciparum therefore provides a first clear example that in vivo regulation of a kinase related to ERK8/7 does not require a typical MEK, since no member of the latter family is present in the parasite's genome (see above). It is perhaps unsurprising that P. falciparum lacks MAP kinases of the ERK1/ERK2, p38 of JNK subfamilies, given the absence of MAPKKs and STE-like MAPKKKs in the genome. In summary, our data indicate that although the malaria parasite uses MAPK homologues, these are not part of three-component modules – to our knowledge, P. falciparum is the first eukaryote demonstrated to lack such modules.In this analysis, both cterised ,45, theycterised . In the cterised ,48. It hP. falciparum kinases cluster with the cell division kinase group that includes the human cell cycle CDKs. PfPK5 [MAL13P1.279] appears orthologous to yeast cdc28 and to human CDK1-3. PfPK5 displays similar levels (60% identity) of overall homology to both mammalian CDK1 and CDK5; however, in our analysis this enzyme clearly clusters with the former, lending support to the idea that this enzyme is a functional homologue of the major cell cycle control kinases, as previously suggested [P. falciparum protein with maximal homology to cyclin H [in vitro by human cyclin H (a CDK7 activator) and p25 (a highly specific CDK5 activator), among other cyclin-related proteins [Three uggested ,50. The uggested  that thiuggested  as a putcyclin H . Howeverproteins . Extremeproteins .P. falciparum genome is smaller than that in the yeast or human genomes, and may represent a minimum complement of such kinases that are necessary for the completion of a eukaryotic cell cycle. Alternatively, some cell cycle control functions assured by CDKs in human cells may be taken over, in Plasmodium, by some of the CMGC kinases with no clear relatedness to established families.The positions of the clusters containing (i) PfPK6 [PF130206]  and PfcrSchizosaccharomyces pombe, but not Saccharomyces cerevisiae) [P. falciparum kinases (PFC0105w and PFl4_0408) cluster with SPRK. Both these kinases have an insertion between domains VIb and VII that is a distinctive feature of SRPKs. Previously described PfLAMMER [PF14_0431] [A number of CMGC group kinases interact with factors involved in mRNA splicing. PF11_0156 clearly is an orthologue of human PRP4, a kinase that is associated with mRNA splicing and histone deacetylation and that is conserved in most eukaryotic genomes (including evisiae) . Human Sevisiae) ,59. Two 14_0431]  associat14_0431]  and the 14_0431] .P. falciparum kinome. As detected on the general tree  is half the size of the P. falciparum genome (24.8 megabases), encodes approximately twice as many enzymes of this family.That only 65 typical ePKs were identified in this search is somewhat surprising, as P. falciparum genome showed that they share most of the residues that are conserved in the ePK catalytic domain. Indeed, with the exception of the Glycine triad in subdomain I, all residues which are crucial for phosphotransfer or structural stability of protein kinases, and therefore well conserved throughout the family, are present in all members of this family  [P. falciparum gene [In preliminary analyses, 21 sequences identified in the HMM search appeared to form a tight cluster that is relatively distantly related to the more typical ePK groups discussed above. Based on an amino acid motif corresponding to subdomain II of the ePK catalytic domain, and which is well conserved in members of this novel family, we called this group "FIKK". In addition to the ePK catalytic domain-like region, the FIKK sequences all have a highly variable N-terminal extension, and in some cases the catalytic domain itself is interrupted by large insertions . An alignment of the FIKK kinase-like domain with the 65 typical ePKs in the ciparum) , and it rum gene , remainsApicomplexan species: 20 sequences in the P. falciparum genome, one in P. berghei (Pb75h08p1c-3-1074-4583), one in P. yoelii (chrPy1 00951-1-3319-5523), one in P. knowlesi (Pk2145b11q1c-4-8079-3688) and one in P. vivax (Pv402596-4-9942-5746). In contrast, no FIKK family member was found in the (yet incompletely sequenced) genomes of P. chabaudi or P. reichenowi. Searches of the NRprot database, which contained sequences representing all eukaryotic and prokaryotic phyla, yielded only the Plasmodium sequences mentioned above . In agreement with the motif searches, BLAST analysis of the NRprot database with PF10_0160 finds 20 Plasmodium falciparum and one yoelii sequences among the top hits (E < 10-37). Weaker hits (E > 10-5) are mostly MAPKs from a variety of organisms. Further investigations using Apicomplexan genome project databases (Sanger and TIGR) allowed us to identify one such sequence in Toxoplasma gondii and one in Cryptosporidium parvum. Taken together, these data strongly suggest that the FIKK group is specific to Apicomplexa, and has undergone a dramatic expansion in P. falciparum. Interestingly, of the 20 FIKK sequences in the P. falciparum genome, 7 are located on chromosome 9, where they are arranged in a contiguous subtelomeric tandem array, a common location for genes involved in antigenic variation such as those of the var/PfEMP1 [P. falciparum being a recent event. Obviously, a definite conclusion about the species distribution of this gene family will have to await the completion of additional genome sequencing projects, especially with respect to other Plasmodium species and other Apicomplexan genera.In addition to the residues conserved in typical ePKs, several amino-acid motifs are fully conserved in all members of the FIKK family Fig.  and 5. Tr/PfEMP1  or Rifinr/PfEMP1  familiesP. falciparum antigens in the context of immunological studies: the R45 trophozoite antigen (PFD1175w) [P. falciparum genome has been discovered independently in the context of research into the R45 antigen .Although no experimental evidence is available that associates PK activity with any of the FIKK sequences, the fact that all residues required for phosphotransfer and ePK folding are present strongly suggests that these proteins are indeed protein kinases. Some FIKKs have a predicted signal peptide (PFD1165w, PFE0045c, MAL13P1.109, PFI0095c, PFI0105c, PFI0110c) and/or transmembrane helix (PFD1165w, PFD1175w, PF10_0160, PFI0110c, PFI0125c, PFI0100c has two) at the N-terminus. Otherwise, aside from their similarity to the kinase domain, no recognised Pfam domains are found in these proteins. Two of the FIKK sequences have been identified as FD1175w)  and the Table Like in many other plasmodial proteins, large extensions rich in charged and/or polar residues, and in some cases repeated amino acid motifs, are found adjacent to the catalytic domain of several PfPKs. Several enzymes also carry such sequences as insertions within the catalytic domain. The function of these elements is as yet undetermined, although there is evidence in some cases [e.g. Pfmap-1, ] that exMalaria parasites possess two organelles with extra-chromosomal DNA: the apicoplast and the mitochondrion. The apicoplast is a four-membrane organelle carrying a circular 35 kb DNA whose structure is very similar to that of plastid genomes. It is specific to the Apicomplexa (hence its name), and thought to originate from secondary endosymbiosis . As is tProteins devoid of kinase activity but which are known to associate with, and regulate the activity of, ePKs have been identified in PlasmoDB. These include four previously characterised cyclins [PF14_0605, PF13_0022, PFL1330c and PFE0920c] which have been demonstrated to associate with histone H1 kinase activities in parasite extracts ,55, a PKHomo sapiens as queries. GeneDB was also used to look for relevant Pfam domains (ABC1, FAT, FATC, Bromodomain, RIO). Two members of the RIO kinase family were found: PFL1490w (RIOK1-like) and PFD0975w (RIOK2-like). Enzymes of this family are involved in rRNA processing in S. cerevisiae [P. falciparum genes (e.g. PFD0685c and PF14_0326) display regions with low-level similarity to the histidine kinase domain (scores between 4 and 5 with Pfam entries PF00512 and PF06580), but the significance of this observation remains to be established.BLASTP searches of PlasmoDB were performed using atypical protein kinases (aPKs) from revisiae . We alsoL. major, T. brucei and T. cruzi genomes, where ABC1 and RIO kinases were found, as were PIKK (with the FAT and FATC domains), PDHK and Alpha kinase family members (Parsons and Ward, unpublished).No significant hits were obtained with A6 kinases, Alpha kinase, pyruvate dehydrogenase kinase, aminoglycoside phosphotransferases or DNA-dependent kinases. In contrast, we identified a malarial phosphatidyl-inositol-3 kinase homologue [PFE0765w], in agreement with experimental studies  and the P. falciparum transcriptome during development are available on the PlasmoDB database. We compiled these data to obtain a general picture of PfePK gene expression during erythrocytic development . A full alignment of the kinase domains is shown in P. falciparum sequences. Only gap-free conserved regions of the alignment were used for the construction of the tree (164 amino acid positions). Bootstrap values supporting the branches of the tree are rather low; this is to be expected given the diversity of the protein kinase family.The set of predicted peptides of the nome 3D7  was downnome 3D7 . A Hiddenome 3D7  of the pnome 3D7 . In addinome 3D7  of 'prot Artemis . Eight pin silico and in vitro analyses of the FIKK family. JP generated the HMM-derived alignments, constructed the ePK phylogenetic trees and contributed significantly to their description in the text. CD coordinated the study and wrote the larger part of the manuscript. All authors read and approved the manuscript.PW performed most of the database searches for ePK-related sequences and constructed the FIKK phylogenetic tree; LE contributed to the searches for aPKs, compiled expression data and performed the P. falciparum ePKs used for constructing the tree in Fig. 2. Please see the Methods section for details on how the alignment was generated.Alignment of the 65 "typical" Click here for filepartial sequence of the cDNA for the gene PF14_0733/PF14_0734.Click here for fileAlignement of the 20 FIKK sequences used to construct the tree in Fig. 6.Click here for file


BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied.The recombinant antigen W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera.PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses.Wb-BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.This study demonstrates that homologs of the  Brugia malayi and Brugia timori is endemic in several Asian countries and infects approximately 13 million people. In May 2000, The Global Program for Elimination of Lymphatic Filariasis (GPELF ) was officially formed with the goal of eliminating the disease as a public health problem by the year 2020. To this end, sensitive and specific field-applicable diagnostic tools are required for mapping the distribution of the disease and monitoring the various phases of the program. Many areas endemic for LF are remote and have poor access to well-equipped laboratories, thus a rapid and field-applicable diagnostic test is important to ensure that it can be easily be performed by field workers and reliable, reproducible, results can be obtained. For bancroftian filariasis caused by Wuchereria bancrofti, the ICT antigen card test (Binax Inc., USA ) is widely used for this purpose. This test is based on the detection of a circulating adult worm antigen of W. bancrofti. Although this antigen is also present in Brugia [B. malayi infection is not available. Therefore, despite its inconvenience and insensitivity, routine diagnosis of brugian filariasis is made by light microscopy of night blood.Lymphatic filariasis (LF) caused by Although PCR assays are highly sensitive, these mainly detect individuals with circulating microfilariae (mf); and they are both time consuming and labour-intensive requiring well-equipped laboratory facilities.Detection of anti-filarial IgG4 antibody provides a good alternative diagnostic tool for brugian filariasis, as this antibody subclass has been shown to be elevated in active infection and decline post-treatment -9. RecomBmR1 recombinant antigen, expressed by gene pPROEXHT/Bm17DIII (GenBank accession no. AF225296), has been shown by us to be a highly specific and sensitive antigen for IgG4 assays to detect exposure to both B. malayi and B. timori infections. The antigen has been used in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats, and evaluation in various laboratories and field trials has revealed a sensitivity of 93%–100% in detecting microfilariaemic individuals [BmR1 antigen is highly specific (99%–100%) with respect to reactivity with sera from non-filarial infections [W. bancrofti, followed by Loa loa, while only one sample of nine patients with Dirofilaria infection was found to be reactive. No cross-reactivity was exhibited in patients infected with O. volvulus or Mansonella [The ividuals . Furtherividuals -15. The fections ,12. The nsonella ,16.BmR1 antigen more closely. The varying degree of BmR1 recognition in other filarial infection raises the question of whether the homologous antigen is also present in W. bancrofti, L. loa and O. volvulus. In the present study we have shown that BmR1 antigen is highly conserved (99–100% amino acid identity), and that almost identical antigens are present in the other human filarial parasites of public health importance. Interestingly, however, the ability of the hosts to mount IgG4 response to BmR1 homologs was found to be highly variable in some infections. In addition, the antibody responses of other IgG subclasses to BmR1 and its homolog were also investigated.Due to its diagnostic significance, it is therefore important to characterize the W. bancrofti microfilaria (mf), adult male and adult female cDNA libraries were obtained from the Filarial Genome Project Resource Centre (Smith College, Northampton, Massachusetts, USA genome@smith.edu). Genomic DNA of W. bancrofti mf were prepared from samples provided by Dr. B Ravindran, Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Bhubaneswar, India . The samples were comprised of two from individuals whose serum/blood samples were negative by the Brugia Rapid test and two from individuals who were positive by the Brugia Rapid test. L. loa L3 and O. volvulus mf cDNA libraries were kindly provided by Dr. P Fischer, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany .Bm17DIII gene sequence the following PCR primers were used: RNF (24 mer) 5' ATT ACT GAT TAG TAT TTT ATC GTT 3' and RNR (24 mer) 5'ATG ATA AAA ATG AAT GAG AAA TAT 3'. λ phage plaques were amplified and the DNA was extracted using a λ DNA extraction kit (Qiagen, Germany ). PCR was then performed in a thermocycler (Perkin Elmer, USA ) at the following conditions: 94°C, 5 mins.; 55°C, 5 mins.; 35 cycles for 94°C, 45 sec; 55°C, 45 sec & 72°C, 90 sec; 72°C, 10 mins.To amplify the entire W. bancrofti mf was prepared using Genispin Tissue DNA Kit . PCR amplifications using the above primers were then performed using the following thermocycler conditions: 94°C, 5 mins; 35 cycles for 94°C, 1 min; 55°C, 1 min & 72°C, 1 min; 72°C, 10 mins.Genomic DNA from W. bancrofti, O. volvulus and L. loa, the PCR products were cloned into TOPO-TA vector (Invitrogen, USA ), then transformed into E. coli TOP10 host (Invitrogen, USA ). The recombinant plasmids were then amplified, purified using QIAprep® Spin Miniprep Kit (Qiagen, Germany ), and subsequently sent for sequencing (ACGT Inc, USA ). The results of the DNA sequences were analyzed using vector NTI software (Invitrogen, USA ).For sequence analysis of the gene homologs in Bm17DIII gene homologs in O. volvulus and L. loa were subcloned into a bacterial expression vector, pPROEX-HT which contain 6-His tag (Life Technologies, USA ), then transformed into E. coli TOP 10 host cells.The ). The suspension was sonicated at 200 W for 10 minutes, followed by centrifugation at 12 000 g for 30 minutes. The resulting supernatant was purified using Ni-NTA resin (Qiagen, Germany ) and buffers containing imidazole. The protein-containing fractions were then pooled.The recombinant bacteria were cultured in Terrific broth and placed in a shaker incubator at 37°C until the optical density reached 0.5. The culture was then induced with 1 mM IPTG  for 3 hrs at 30°C. The bacterial pellet was reconstituted with lysis buffer containing 50 mM Tris HCl (pH 8.5), 5 mM 2-mercaptoethanol and a cocktail of protease inhibitors (Roche Diagnostics, Germany ) were coated with 100 μl of either BmR1 (20 μg/ml) or the homologous recombinant antigens (5, 10 or 20 μg/ml) in NaHCO3 buffer (pH 9.6). After a blocking step, serum samples (1:20 or 1:50 or 1:100) were incubated for 2 h, followed by 0.5 h incubation with the secondary antibody HRP conjugated to monoclonal anti-human IgG1 (1:6000), IgG2 (1:1000, 1:2000), IgG3 (1:1000, 1:2000) or IgG4 (1:4500) (CLB Sanquin Blood Supply Foundation, Netherlands ). Subsequently ABTS substrate (Roche Diagnostics, ) was added for 30 minutes before the optical densities (OD) were read at 410 nm with an ELISA spectrophotometer (Dynatech (now DYNEX Technologies), USA ).The methodology employed was as previously reported . BrieflyO. volvulus, L. loa, W. bancrofti, B. malayi and other parasitic infections. In addition serum samples from endemic normals  and non-endemic normals  were also tested. The O. volvulus sera were from microfilaremic from western Uganda [L. loa sera were from microfilaremic individuals from the clinical department of the Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. W. bancrofti sera samples were from India; while sera from B. malayi infections, endemic normals, non-endemic normals and other parasitic infections were from Malaysia. Infections with other parasites comprised of patients from Malaysia:Serum samples were from existing serum banks, collected according to the ethical requirements of each institution. The samples were as follows: n Uganda . L. loa lumbricoides, Trichuris trichiura, hookworm, Strongyloides stercoralis)• whose stool specimens were positive for parasite ova/larva • with BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (LI-BmR1). PCR of W. bancrofti cDNA libraries and W. bancrofti genomic DNA  produced a single band of 618 bp. PCR products were eluted from band 618 and cloned into TOPO vector. A total of 12 recombinant clones from six TOPO reactions  were sequenced. A total of 31 DNA sequencing reactions were analyzed and all obtained sequences were identical. Comparison of the obtained nucleotide sequence showed that it was identical to cDNA sequence of BmR1, irrespective whether the template DNA came from cDNA libraries, or microfilaria originated from individuals positive or negative for Brugia Rapid.In order to explain the pattern of antigen recognition in patients with other filarial infection we identified the homologs of the BmR1 and Ll-BmR1, a total of 5 and 3 recombinant clones were sequenced, respectively . The Ov-BmR1 and Ll-BmR1 were 100% identical to each other, and only two base pairs were different from BmR1 and Wb-BmR1 i.e. at 97 bp and 483 bp. When the amino acid sequences were compared only one amino acid difference was observed: the uncharged polar isoleucine at position 33 was substituted by a neutral threonine  using a panel of 201 sera samples from individuals with various parasitic infections and 61 healthy controls  indicated that the exchange of one amino acid had no influence on the reactivity of IgG4 antibodies. The IgG4-ELISA results showed all recombinant antigens were identical in reactivity with the various categories of sera  readings of ≥0.300 were considered to be positive . The comBmR1 and its homologs (Ov-BmR1 and Ll-BmR1) with serum antibodies of the other three IgG subclasses (IgG1, IgG2 and IgG3) using samples from O. volvulus and L. loa infected individuals showed positive reactions with only IgG1. Most IgG1 positive samples had an OD >1.000. Similarly, the reactivities of anti-filarial IgG1, IgG2 and IgG3 antibody subclasses with BmR1 on serum samples from active and chronic cases of W. bancrofti and B. malayi showed positive reactions only with IgG1. It is also noted that sera from non-endemic normals and soil-transmitted infections also showed similar reactivities i.e. IgG1 positive and IgG2- & IgG3-negative , has been consistently shown to be a sensitive and specific antigen for the immunodiagnosis of brugian filariasis in studies employing either ELISA or immunochromatographic rapid test (Brugia Rapid) formats [Bm17DIII cds exhibited 94% homology with the reported EST sequence derived from B. malayi microfilaria cDNA (GenBank AW244981). Southern blot hybridization assays performed on cDNA libraries of L3, L4, mf, adult male and adult female B. malayi showed that it is present in all of the above stages . Bands of the correct molecular weight were observed in a Western blot of B. malayi mf, adult male and adult female soluble antigens probed with monopurified antibody to BmR1 . formats ,11-13,15BmR1 to sera of W. bancrofti-infected patients. Reactivity to sera from Chennai, India was 54.5% (12/22); from Indonesia was 70% (14/20) and from the Cook Islands was 90% (9/10) [W. bancrofti is identical to the cDNA of BmR1 – irrespective of the source of the parasites – whether the mf were isolated from the individuals whose sera showed positive or negative reactivity with the Brugia Rapid test. Thus the observed differences in the reactivity of BmR1 antigen with W. bancrofti sera collected from different geographical regions does not appear to be due to genotypic variability between different isolates of mf. Further studies are currently underway to determine if the variability in the expression of the gene could account for the variability in the Brugia Rapid results with serum samples collected from W. bancrofti infected individuals.Multicenter evaluations performed with Brugia Rapid showed variable reactivity of as 54.5% /22; from% (9/10) . The widW. bancrofti genomic DNA samples to obtain an amplicon with a size greater than 618 bp . However, only one prominent band of 618 bp was obtained . PCR on W. bancrofti genomic DNA to amplify the intron sequence (using primers based on the Bm17DIII intron) produced a sequence that shared ~75% homology to the intron of Bm17DIII. This is believed to be an amplification on another part of W. bancrofti genome, since PCR using a pair of internal primers that flank the possible intron site produced a PCR product of ~300 bp (a size that is expected if there was no intron). Conversely, amplification of B. malayi genomic material produced two kinds of amplicons: 618 bp and 1010 bp. The latter was comprised of an intron (393 bp) and two flanking exons (237 bp and 381 bp), the sequences of which were consistent with B. malayi data at TIGR website . Thus at Universiti Sains Malaysia, genomic DNA of Wb17DIII was found to be intronless, whereas genomic DNA of Bm17DIII was shown to have two variants (i.e one with and one without an intron). These results, though seemingly controversial, were a result of exhaustive efforts with appropriate PCR controls. Data from other laboratories will hopefully confirm these results.PCR experiments were performed on the BmR1 IgG4 was detected in 84.6% (44/52) of L. loa sera but generally not detected in O. volvulus serum samples [BmR1 and Ll-BmR1 were identical to each other and 99.7% similar to BmR1 (and to Wb-BmR1) on the nucleotide level  did not alter their antigenicity. It is interesting to note that although IgG4 has been shown to be elevated in onchocerciasis with assays using other recombinant antigens [BmR1 or Ov-BmR1 in O. volvulus was generally negative. One possible explanation for this is that adult worms mostly express Ov-BmR1 and the immune response to O. volvulus is predominantly due to mf [BmR1 and Ov-BmR1. It is possible that the uptake of antigen from lymphatic filariae by antigen presenting cells is significantly different compared to O. volvulus .Anti- samples ,16. Ov-B4.6% 44/5 of L. loantigens ,19, the ue to mf . This maBmR1, Ov-BmR1 and Ll-BmR1 recombinant antigens were also used to determine if IgG1, IgG2 or IgG3 antibodies in O. volvulus and L. loa serum samples were reactive with the recombinant proteins. In addition, the three IgG subclasses were also tested with BmR1 on assays using sera collected from patients with B. malayi and W. bancrofti infections. In all cases only anti-filarial IgG1 was reactive, while anti-filarial IgG2 and IgG3 assays were consistently negative. It is important to note that IgG1 antibodies to BmR1 and its homologs are unspecific and without any diagnostic value. The BmR1 antigen obviously contains widespread epitopes that are recognized by IgG1 antibodies.The BmR1 and its homologs in W. bancrofti, O. volvulus and L. loa induce IgG antibody responses restricted to IgG1 and IgG 4 subclasses only. Unlike the anti-filarial IgG4 response in B. malayi infection, the IgG4 response to BmR1 in W. bancrofti and L. loa was not consistently detected in all infected individuals, indicating that this recombinant antigen will not be of much use in the diagnosis of these two filarial infections. Although IgG1 response to BmR1 was observed in all the filarial infections tested, it lacks specificity since it was also positive when tested with serum samples from normal individuals and with those infected with other parasites.Thus based on the current study, BmR1 antigen in other filarial parasites. However, they do not seem to induce consistent antibody responses in all infected subjects. Thus the immunogenicity of BmR1 in brugian filariasis appears to be clearly different from that of bancroftian filariasis, onchocerciasis and loiasis.The study demonstrates the presence of identical and almost identical homologs of the diagnostic The author(s) declare that they have no competing interests.RN – was the principle researcher, designed the study, supervised the experiments and result analysis, wrote the first draft of the manuscript.RAAA – performed the experiments and participated in the analysis of the data.BR – provided parasite materials, collected patients' sera, edited the paper.


II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export.Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing ~33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes.Herein, we further characterized the role of p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.We are the first to demonstrate that p30 Althougprovirus . The welprovirus . Tax is provirus . Emerginprovirus ,8. Theresubjects , as wellsubjects . Antibodsubjects ,12 and cI and p13II/p30II in the establishment of infection and maintenance of viral loads in a rabbit model of infection [II contains a highly conserved bipartite nuclear localization signal (NLS) and localizes within the nucleus of cells [II contains serine- and threonine-rich regions with distant homology to transcription factors Oct-1 and -2, Pit-1, and POU-M1 [II also co-localizes with p300 in the nucleus and physically interacts with CREB binding protein (CBP)/p300 and differentially modulates cAMP responsive element (CRE) and TRE mediated transcription [II and HTLV-2 p28II (homologous protein encoded in the HTLV-2 pX ORF II region), in modulating the export of tax/rex RNA from the nucleus [II appears to be a multi-functional protein with transcriptional and post-transcriptional roles in regulating viral gene expression. Based on these reports, we hypothesized that p30II functions as a regulator of cellular and viral gene expression to promote HTLV-1 replication.Using molecular clones of HTLV-1 with selective mutations of ORF I and II, we have tested the requirement of p12nfection -16. ORF of cells -19. In ad POU-M1 . Previoucription ,21. Rece nucleus ,23. TherII as a regulator of gene expression and identified several novel and important alterations in gene expression profiles, unique to cell cycle regulation, apoptosis and T cell signaling/activation. In addition, using semi-quantitative RT-PCR, we have confirmed the expression of multiple genes modulated by p30II in Jurkat T cells and primary CD4+ T lymphocytes. We then tested the influence of p30II in T cell signaling using reporter assays representing critical T lymphocyte transcription factors. This is the first report that demonstrates the role of p30II as an activator of key transcription factors involved in T cell signaling/activation. Together, our data suggests that HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes to promote lymphocyte activation to enhance clonal expansion of infected cells and maintain proviral loads in vivo.Gene arrays have primarily been employed to study the changes in gene expression profile of HTLV-1-immortalized and transformed cell lines or in cells from ATL patients and attempts to test the influence of individual HTLV-1 viral proteins on cellular gene expression have been limited to Tax ,24-27. HII in Jurkat T lymphocytes was established using recombinant lentiviruses   using methods as previously reported [II and verified for cluster formation [Stable expression of HTLV-1 p30es) Fig. . RT-PCR lls Fig. . p30II preported . Differeormation . Qualityormation ,30.II into those upregulated or downregulated in expression. We further grouped genes deregulated by p30II based on their functions, such as apoptosis, cell cycle, cell adhesion, transcription/translation factors and T cell activation or cell signaling. In all the categories, p30II was an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes (Table 1, see We then categorized genes deregulated by HTLV-1 p30II expressing cells (Table 1), p30II would be predicted to modulate apoptosis. These include Bcl-2 related/interacting genes such as anti-apoptotic Bcl-2-related protein A1, anti-apoptotic MCL1, cell-death regulator Harakiri, apoptotic protector BNIP1 (downregulated) and pro-apoptotic BIK (upregulated). In addition, p30II expression correlated with downregulation of genes associated with Fas mediated apoptosis pathway such as tumor suppressing subtransferable candidate 3 and TNF receptor superfamily member 25. p30II expression was also associated with decreased expression of caspases (2 and 4) and increased expression of genes associated with the DNA fragmentation pathway (CIDE-B and CIDE-3). In addition, p30II expression correlated with decreased expression of many other apoptosis related genes including CD28, Lck, cyclin B1, Cullin 5, Adenosine A2a receptor, TAF4B and NCK-associated protein 1.Based on changes in gene expression in p30II expressing Jurkat T lymphocytes. These include checkpoint suppressor 1, cytosolic branched-chain amino acid transaminase 1, histone deacetylase 6, cyclin B1, WEE1 kinase, CDC14A, Lck, JAK2, GAS7, BZAP45, Cullin, Rab6 GTPase activating protein (downregulated) and TERF1, AKAP8, DDX11, MSH2 and JUN-D (upregulated). Another gene down regulated by p30II expression was MDM2, which is over expressed in certain types of leukemia [Multiple genes involved in cell cycle regulation were altered in p30leukemia  and capaleukemia .II expression was associated with altered expression of several genes involved in cell-to-cell adhesion. These include decrease in integrin (integrin β8) immunoglobulin (MADCAM1), a counter-receptor for P-selectin (SELPLG), cadherin (desmocollin 3), protocadherin (PC-LKC) liprin (PPF1BP1), CD84/Ly-9, CD58, CD43/sialophorin and glycosyl-phosphatidyl-inositol phospholipase D1. Expression of p30II correlated with increase in integrin receptor α1 subunit and KIT ligand.p30II expressing Jurkat T lymphocytes. These included decreased expression of TATA-binding protein associated factor 4 (TAF4), two co-repressors (Enolase-1 and Chromosome 19 ORF2 protein), a novel specific coactivator for mammalian TEFs, namely TONDU [II expression correlated with down regulation of zinc finger proteins (zinc finger protein 36), a group of transcription regulators proposed to be candidates in malignant disorders [II was also associated with downregulation of many genes with positive transcriptional effects . Reduced expression of HDAC6, a histone deacetylase and nuclear receptor coactivator 3 (CBP interacting protein) with histone acetyltransferase and pCAF/CBP recruiting abilities [II contains multiple highly conserved lysines, which could play a role in acetylation [II was also associated with decrease in GAS 7, which has sequence homology to Oct and POU family of transcription factors [II expression in Jurkat T lymphocytes was associated with an increase in expression of eukaryotic translation elongation factor 1α (EEF1A2), a putative oncogene [A number of genes encoding transcriptional control factors or regulators of transcription were repressed in p30ly TONDU , homeo bisorders  and coilbilities  are parttylation ,21. Exproncogene , and enhII expression. Expression of p30II was associated with decreased expression of CD28, a co-stimulatory molecule with a distinct role in T lymphocyte activation [II had enhanced Vav-2 and CD72 gene expression. Additionally, p30II expression correlated with decrease in the level of CHP, an endogenous calcineurin inhibitor, which would be predicted to promote NFAT expression by p30II (see below). Moreover, p30II expression was associated with increased expression of Jun-D and c-Fos, suggesting activation of AP-1 mediated transcription. p30II expression was associated with decreased expression of protein kinase D (PKD), which negatively modulates JNK signaling pathway [II, there were no detectable levels of I kappa B kinase gamma (IKKγ), which is important for NF-κB signaling in response to both T cell activation signals and Tax [II expression was associated with increased Hematopoetic Progenitor Kinase-1 (HPK-1), a known NF-κB activator [II expression was also associated with decreased Ras GRP2, a guanyl nucleotide exchange factor that increases Ras-GTP, suggesting a decrease in the level of activated Ras (Ras-GTP). Seminquantitative RT-PCR analysis in Jurkat T lymphocytes and primary CD4+ T lymphocytes correlated directly with the gene array and confirmed the altered expression of each of three selected genes involved in these T cell activation/signaling pathway  use of triplicate samples and appropriate controls (b) use of multiple software for data analysis (c) minimization of nonspecific hybridization and background signals by using Affymetrix chip [Our study represents a comprehensive analysis of gene expression patterns influenced by a retrovirus accessory protein in T lymphocytes. Overall, this study confirmed that p30rix chip  (d) use rix chip . Our stuet al [et al [et al, 2001 [II on cellular gene expression profile of a larger number of genes. Gene expression data from cells in which exogenously expressed proteins, which may also be tagged for identification, may not represent what would occur during the natural infection. However, these patterns provide important clues for functional alterations which may occur during the viral protein expression. The "natural" or in vivo amount of expression of regulatory and accessory gene products encoded from the HTLV-1 pX gene region has not been clearly defined. Recent studies using RT-PCR analysis of cell lines suggests that pX ORF 1 and 2 mRNA is expressed at significantly lower amounts compared to tax/rex mRNA, full length genomic, or singly spliced envelope mRNA [Others have used gene array approaches to study HTLV-1-related changes in gene expression. Harhaj et al  studied l [et al  to studyl [et al . Using Nal, 2001  comparedal, 2001 . In thisope mRNA .in vitro and in leukemia cells in vivo [II in T cell activation and to test the comparative role of p30II expression in context to other regulators of transcription such as Tax.Expression from the IL-2 promoter requires binding of several transcription factors, including NFAT, AP-1 and NF-κB. NFAT is vital to proliferation of peripheral lymphocytes for HTLV-1 infection  while AP in vivo . NF-κB i in vivo  and due I stimulates NFAT mediated transcription, when stimulated with PMA, indicating that p12I acts synergistically with Ras/MAPK pathway to promote NFAT activation and thus may facilitate host cell activation and establishment of persistent HTLV-1 infection [II enhanced NFAT driven transcription significantly when stimulated with ionomycin or CD3, and therefore likely uses a different mechanism than p12I. To modulate NFAT driven transcription and subsequent T cell activation/signaling, it is possible that these two accessory proteins act synergistically. AP-1 is able to interact with transcriptional coactivator CBP/p300, as well as viral CREs and mediate HTLV-1 gene expression [II interacts with CBP/p300 at the KIX domain of CBP, influences CRE and TRE mediated transcription [II modestly activates the transcriptional activity of NFAT, NF-κB and AP-1, at least in part, by its interaction with CBP/p300. In parallel, the HIV-1 accessory protein Vpr causes a modest increase in NF-κB, NFAT and AP-1 mediated transcription in a cell-cycle dependent fashion by causing G2 arrest [II expression was associated with decrease in cyclin B1 and WEE1 kinase levels, suggesting that p30II expression likely cause G2 arrest and may thus modulate transcriptional activity of NFAT, NF-κB and AP-1, in a cell-cycle dependent manner. An important caveat of our data is the use of Jurkat T cells, which while representing human T cells, are IL-2 independent and transformed. Thus, differences in responsive genes expected from non-transformed T cells for the transcription factors screened in our study may be due to our cell line model.We have previously reported that another HTLV-1 accessory protein p12nfection . Our datpression ,51,52. Icription  and disrcription . NF-κB acription  are also2 arrest . SimilarII also deregulates multiple genes resulting in possible pro-apoptotic and anti-apoptotic effects. Since apoptosis is a well-known mechanism of cellular defense against viral infection, a possible role of p30II in lymphocyte apoptosis might correlate with the requirement of p30II in maintaining proviral loads in vivo [HTLV-1 mediated interference with normal T-cell apoptosis is thought to be a mechanism of tumorigenicity , but spe in vivo . Previou in vivo ,55.II appears to regulate viral gene expression and modulate immune response. We have previously reported that, p30II activated HTLV-1 LTR at lower concentrations and repressed at higher concentrations [II expression was associated with downregulation of lck (p56), which suppresses the HTLV-1 promoter [II expression was associated with altered expression of cellular genes involved in immune modulation such as CD46, CD43, CD58, IFNγ and CD72.p30trations . Interes of lck p, which spromoter . AdditioII in gene expression [II functions in HTLV-1 replication or leukemogenesis. Our data confirmed that p30II while a negative regulator of cellular genes, also influences T cell signaling, apoptosis and the cell cycle. Many of the effects of p30II appear to overlap or counteract the influence of other HTLV-1 regulatory proteins like Tax or other accessory proteins such as p12I. It is possible that these proteins act coordinately or synergistically. We postulate that, by modulating the expression of various HTLV-1 proteins, the virus employs selective use of these viral proteins during different stages of the infection. However, since information on the expression profile of HTLV-1 proteins during stages of the infection is limited, additional studies are required to explore this possibility. Such future studies might provide new directions in the development of therapeutic interventions against HTLV-1 disorders, which are associated with immune-mediated mechanisms.Overall, this study supports our earlier reports on the repressive role of HTLV-1 p30pression ,21,23 anIIHA-IRES-GFP was created by cloning the p30II sequence from ACH [IIHA expression were confirmed by fluorescence activated cell sorting (FACS) analysis (Beckman Coulter, Miami, FL) and western blot respectively. GFP expression from each of the plasmids was confirmed by flow cytometry (Beckman Coulter) and the p30IIHA expression from pWPT-p30IIHA-IRES-GFP plasmid was confirmed by western blot using mouse monoclonal anti-hemagglutinin antibody (1:1000) (Covance, Princeton, NJ) as described previously [IIHA was created by cloning p30II sequence from ACH with HA1 tag, into pME-18S (G. Franchini, NIH). Other plasmids used include previously reported pRSV-βGal [The plasmid pWPT-IRES-GFP was generated by cloning the internal ribosome entry site (IRES) sequence from pHR'CMV/Tax1/eGFP   or pWPT-IRES-GFP (control) as described previously [60]. Briefly, 293T cells (5 × 106) were seeded in a 10-cm dish and transfected the following day with 2 μg of pHCMV-G, 10 μg of pCMVΔR8.2 and 10 μg of pWPT-p30IIHA-IRES-GFP or pWPT-IRES-GFP using the calcium phosphate method. Supernatant from 10 to 20 dishes was collected at 24, 48 and 72 h post transfection, cleared of cellular debris by centrifugation at 1000 rpm for 10 min at room temperature and then filtered through a 0.2 μm filter. The resulting supernatant was then centrifuged at 6,500 g for 16 h at 4°C. The viral pellet was suspended in cDMEM (DMEM containing 10% FBS and 10% streptomycin and penicillin) overnight at 4°C and the concentrated virus was aliquoted and stored at -80°C. To determine the virus titer, serial dilutions of the virus stock were used to spin infect 293T cells and 48 h post infection, eGFP expression and p30II expression was measured by flow cytometry and RT-PCT respectively. Briefly, on the day before infection, 293T cells (1 × 105) were seeded in a 6-well plate. The medium was removed the following day and the cells were then incubated with the diluted virus containing 8 μg/ml polybrene (Sigma, St. Louis, MO). Cells were then spin-infected by centrifugation at 2700 rpm for 1 h at 30°C, supplied with fresh medium and cultured for 48 h. Then cells were treated with trypsin (Invitrogen, Carlsbad, CA), pelleted and resuspended in D-PBS (Invitrogen) for fluorescence activity cell sorting (FACS) analysis on an ELITE ESP flow cytometer (Beckman Coulter). One × 106 cells were used to perform western blot to detect the expression of p30II HA. Jurkat T lymphocytes (clone E6.1, American Type Culture Collection) were transduced with recombinant virus at multiplicity of infection of 4 in the presence of 8 μg/ml polybrene (Sigma) and spin-infected at 2700 rpm for 1 h at 25°C. Primary CD4+ T cells were extracted using dynabead CD4 positive isolation kit  according to manufacturer's instructions. Primary CD4+ T cells were stimulated with Phytohemagglutinin (PHA) for 48 h, transduced with recombinant virus at multiplicity of infection of 20 in the presence of 8 μg/ml polybrene (Sigma) and spin-infected at 2700 rpm for 1 h at 25°C. At 10 days post-transduction, GFP expression of controls and samples were verified to be above 90% by FACS analysis and the presence of p30II mRNA expression in samples (and absence in controls) was verified by RT-PCR . Equal amounts of proteins were mixed with Laemmli buffer (62.5 mM Tris [pH 6.8], 2% SDS, 10% glycerol, 0.2% bromophenol blue, 100 mM dithiothreitol). After boiling for 5 min, samples were electrophoresed through 12% polyacrylamide gels. The fractionated proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotechnology) at 100 V for 1 h at 4°C. Membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween for 16 hours, then incubated with mouse anti-HA monoclonal Ab (1:1,000) (clone 16B-12) (Covance Research Products, Princeton, NJ), for overnight at 4°C, and developed by using horseradish peroxidase-labeled secondary Ab (1:1,000) and enhanced chemiluminescence reagent .Cells were lysed in buffer containing phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Cell lysates were prepared by centrifugation at 14,000 rpm (Beckman) for 20 min at 4°C. Protein concentrations were determined by BCA assay (micro-BCA Protein AssayIn vitro transcription was performed on the cDNA to produce biotin-labeled cRNA with ENZO RNA Transcript labeling kit (Affymetrix), according to the manufacturer's instructions. Complimentary RNA (cRNA) cleanup was performed using Genechip Sample Cleanup module (Affymetrix). The quality of total RNA and biotin-labeled cRNA of all the samples and controls were checked by calculating the ratio of absorbance at 260 nm and 280 nm (between 1.9 to 2.1) using a spectrophotometer (Genequant) and agarose gel electrophoresis. The labeled cRNA was fragmented to 50–200 nucleotides, and hybridized to U133A arrays (Affymetrix) using GeneChip® Hybridization Oven (Affymetrix). Arrays were washed and stained using GeneChip® Fluidics Station 400 (Affymetrix) and scanned by GeneArray Scanner (Affymetrix).According to the instructions of manufacturers, total cellular RNA was isolated from transduced Jurkat T lymphocytes using RNAqueous (Ambion, Austin, TX). To test the concentration and purity of the RNA samples, absorbance at 260 nm and 280 nm were measured and the 260/280 ratio was calculated using a spectrophotometer (Genequant, Amersham Pharmacia, Piscataway, NJ). The 260/280 ratio of all the RNA samples were between the range of 1.9–2.1. The probe preparation for GeneChip was performed according to the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Briefly, cDNA was synthesized using genechip T7-Oligo (dT) promoter primer kit (Affymetrix) and superscript double stranded cDNA synthesis kit (Invitrogen), according to the manufacturers instructions. cDNA cleanup was done using Genechip Sample Cleanup module (Affymetrix). ® (Microarray suite 5) to identify probes with at least 1.5 fold difference in expression between control and p30II and verified for cluster formation by dCHIP software [Quality control criteria evaluations done as part of the basic analysis include (1) The ratios of 3' signal to 5' signal of two housekeeping genes, beta-actin and GAPDH were between 0 and 3. (2) The hybridization controls BioB, BioC, BioD, and Cre were all present and in a linear relationship of intensity. (3) The scale factors between arrays did not vary by 3 fold. (4) The background intensity was not significantly higher than expected. (5) The percent of gene present was monitored and found to be not less than the standard 30%. To determine the quantitative RNA level, the average differences representing the perfectly matched minus the mismatched for each gene-specific probe set was calculated. Differential gene expression and comparative analysis was done using Data Mining Toolsoftware . The bioII were as follows: TAG CAA ACC GTC AAG CAC AG (forward) and CGA ACA TAG TCC CCC AGA GA (reverse). The PCR primers for CHP were as follows: CCC ACA GTC AAA TCA CTC GCC (forward) and ATG GTC CTG TCT GCG ATG CTG (reverse). The PCR primers for JUN-D were as follows: CTC TCA GTG CTT CTT ACT ATT AAG CAG (forward) and TTA TCT AGG AAT TGT CAA AGA GAA GATT (reverse). The PCR primers for NFATc were as follows: TTG GGA GAG ACA TGT CCC AGA TT (forward) and TCA TTT CCC CAA AGC TCA AAC A (reverse). The results were expressed as a graph. Statistical analysis was performed using Student's t test, P < 0.05.One μg of RNA was converted to cDNA (Reverse Transcription system, Promega, Madison, WI) as described by the manufacturer. cDNA from 100 ng of total RNA was amplified with AmpliTaq DNA polymerase (Perkin Elmer, Boston, MA), PCR products were separated by agarose gel electrophoresis, normalized to GAPDH and quantified using alpha imager spot densitometry (Alpha Innotech, San Leandro, CA). DNA contamination was tested by performing a control with no reverse transcriptase. The PCR primers for p30II-transfected Jurkat T lymphocytes was performed as described previously [7 cells in cRPMI  and 10% streptomycin and penicillin) at 350 V and 975 μF using Bio-Rad Gene Pulser II (Bio-Rad, Laboratories, Hercules, CA) with 30 μg of pME-p30 or pME empty plasmid, 10 μg of reporter plasmid (NFAT-Luc, AP-1 Luc or NF-κB Luc), and 1 μg of pRSV-Gal plasmid or 1 ug pWPT-IRES-GFP plasmid. The transfected cells were seeded in six-well plates at a density of 5 × 105/ml and were either left untreated or stimulated with 20 ng/ml of phorbol myristate acetate (PMA) (Sigma) or with 2 μM ionomycin (Sigma), or both at 6 h post-transfection, followed by incubation for 18 h prior to lysis for analysis of luciferase activity. Stimulations with anti-CD3 and/or anti-CD28 antibodies (each at 3 μg/ml) (BD Pharmingen, San Diego, CA) were carried out 18 h post-transfection. Following 8 h of stimulation, to measure luciferase activity, the cells were lysed with Cell Culture Lysis Reagent (Promega), and the cell lysates were tested for luciferase activity according to the manufacturer's protocol. Transfection efficiency was normalized by staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside  (Sigma) and counting β-Gal expressing cells. Transfection efficiency was also normalized by counting GFP positive cells under the fluorescence microscope. Results were expressed as mean of optimized luciferase activity  in arbitrary light units (ALU) with standard error (SE) from a minimum of triplicate experiments. Statistical analysis was performed using Student's t test, P < 0.05.Analysis of AP-1, NF-κB, and NFAT transcriptional activity in pME- and pME-p30eviously . BrieflyArbitrary light units, ALUPhorbol myristate acetate, PMAFetal bovine serum, FBSHuman T-lymphotropic virus type 1, HTLV-1Adult T cell leukemia/lymphoma, ATLThe author(s) declare that they have no competing interests.Bindhu Michael, Amrithraj M. Nair, Hajime Hiraragi, Lei Shen, Gerold Feuer, Kathleen Boris-Lawrie and Michael D. Lairmore have all met the definition of author as outlined by the Retrovirology journal. Each has made substantive intellectual contributions to a published study. Bindhu Michael, Amrithraj Nair, Hajime Hiraragi Gerold Feuer, Kathleen Boris-Lawrie and Michael D. Lairmore have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. Lei Shen performed drafting the article or revising it critically for important intellectual content in particular sections related to biostatistical analysis. Each author has given final approval of the version to be published. Each author have participated sufficiently in the work to take public responsibility for appropriate portions of the content.II.Listing of genes modulated by HTLV-1 p30Click here for file


The human immunodeficiency virus type I (HIV-1) transactivator protein Tat is an unusual transcriptional activator that is thought to act solely by promoting RNA polymerase II processivity. Here we study the mechanism of Tat action by analyzing transcription complex (TC) assembly in vivo using chromatin immunoprecipitation assays. We find, unexpectedly, that like typical activators Tat dramatically stimulates TC assembly. Surprisingly, however, the TC formed on the HIV-1 long terminal repeat is atypical and containsTATA-box-binding protein (TBP) but not TBP-associated factors (TAFs). Tat function involves direct interaction with the cellular cofactor positive transcription elongation factor b (P-TEFb). Artificial tethering of P-TEFb subunits to HIV-1 promoter DNA or nascent RNA indicates that P-TEFb is responsible for directing assembly of a TC containing TBP but not TAFs. On the basis of this finding, we identify P-TEFb-dependent cellular promoters that also recruit TBP in the absence of TAFs. Thus, in mammalian cells transcription of protein-coding genes involves alternative TCs that differ by the presence or absence of TAFs. The transcriptional activator, HIV-1 Tat, not only acts by promoting RNA polymerase processivity, but it is able to promote transcription complex assembly in the absence ofTATA- box-binding protein-associated factors In eukaryotes, gene regulation is largely controlled at the transcriptional level. Factors involved in the accurate transcription of eukaryotic structural genes by RNA polymerase II (class II genes) can be classified into two groups. First, general (or basic) transcription factors (GTFs) are necessary and can be sufficient for accurate transcription initiation in vitro (for review, see ). Such fTranscriptional activity is greatly stimulated by the second class of factors, promoter-specific activator proteins (activators). In general, cellular activators are sequence-specific DNA-binding proteins whose recognition sites are usually present in sequences upstream of the core promoter (reviewed in ,6). In aThe Tat protein of human immunodeficiency virus type I (HIV-1) is a potent activator of the viral long terminal repeat (LTR) and is required for viral replication (reviewed in ,12,13). The chromatin immunoprecipitation (ChIP) assay has provided a powerful approach to study in vivo the mechanism by which activators stimulate transcription and to delineate the composition of transcription complexes (TCs). In yeast, ChIP analysis has been used to show that activators function, at least in part, by stimulating TC assembly. These studies have also revealed that at certain promoters TBP is recruited in the absence of TAFs ,17, indiTo analyze transcription stimulation by Tat and other activators we performed ChIP experiments in transiently transfected mammalian tissue culture cells. Plasmids expressing a reporter construct, containing a core promoter and various combinations of activator-binding sites, were transiently transfected into HeLa or 293T cells. In some experiments, a second plasmid expressing an activator was co-transfected. Reporter activity or primer extension was used to quantitate transcription levels. TC assembly was analyzed by a ChIP assay using antibodies directed against components of four representative GTFs involved in distinct stages of TC assembly: TFIID , TFIIB, mediator (CDK8 and hMed6), and RNA polymerase II (RBP1). In most experiments the analysis was performed using two sets of primers: one set encompassed the core promoter, transcription start site, and immediate downstream region, and a second set was located far downstream of the transcription start site, within the open reading frame (ORF).In yeast, it has been shown that activators stimulate TC assembly, which is evident at the earliest step of this process, interaction of TBP with theTATA box ,19. To cThe artificial activator Gal4-VP16 contains an unusually potent acidic activation domain and can stimulate transcription in many species and cell types including mammalian tissue culture cells . Figure cis-acting enhancer element that is often located at a distance from the transcription start site . The encription . Figure Finally, we analyzed transcription directed by the Ad E1a protein. Efficient transcription from Ad early promoters requires the viral E1a protein as well as the participation of cellular transcription factors that also bind to the promoter (reviewed in ). For exIn summary, the results of We next investigated Tat-mediated transcription activation and TC assembly on the HIV-1 LTR promoter. We note that recruitment of Tat was not observed in the ChIP assay, which detects proteins bound either directly or indirectly to DNA (reviewed in ). UnlikeTo determine whether the lack of TAF recruitment was a general feature of Tat-directed transcription, we analyzed a chronically HIV-1-infected cell line, 8E5/LAV, which harbors an integrated provirus that is constitutively transcribed . Figure We also analyzed a second chronically HIV-1-infected cell line, U1, which has been used as a model to study viral latency. U1 cells harbor an integrated viral genome, but, unlike 8E5/LAV cells, the constitutive level of viral expression is extremely low. Viral expression can be induced by phorbol esters, which stimulate transcription through upstream NF-κB-binding sites in the HIV-1 LTR, leading to Tat synthesis and a subsequent substantial Tat-mediated transcriptional increase ,28. ThusTo investigate further the composition of the TC formed on the HIV-1 LTR, we obtained a panel of antibodies directed against nine additional human TAFs (TAF2, TAF4, TAF6, TAF7, TAF8, TAF9, TAF11, TAF12, and TAF13), as well as antibodies against four subunits of the STAGA complex (PAF65β, hSpt3, hGCN5, and TRRAP) and the TBP-interacting protein Mot1. The results A show thThe results of the ChIP experiments strongly suggested that transcription activation by Tat did not require TAFs. To confirm this supposition, we examined the ability of Tat to stimulate transcription following TAF inactivation. First, we examined the ability of Tat to activate transcription in ts13 cells, which harbor a temperature-sensitive mutation in TAF1 ,30. FiguThe results described above indicate that on the HIV-1 LTR transcription activation involves assembly of an atypical TC that contains TBP but not TAFs. However, these experiments do not distinguish whether the critical determinant for recruitment of TBP and not TFIID is Tat or the promoter, the HIV-1 LTR. To address this issue we performed a series of artificial tethering experiments.Previous studies have shown that Tat can activate transcription when directed to the HIV-1 LTR through a heterologous DNA-binding domain ,32,33. WAs discussed above, Tat interacts directly with CycT1, a subunit of the transcription elongation factor P-TEFb. Thus, Tat is an adaptor whose function is to recruit P-TEFb. We therefore considered that P-TEFb was ultimately responsible for the selective recruitment of TBP in the absence of TAFs. To address this possibility, we analyzed transcription activation by the two P-TEFb subunits, CycT1 and CDK9. Consistent with previous results ,35, bothIn the experiments shown in P-TEFb is also a cofactor for several cellular activators, the best studied of which is CIITA, a transcription factor involved in the expression of major histocompatibility complex (MHC) class II genes (reviewed in ). In facHLA-DM and HLA-DR, and, as a negative control, GAPDH. RT-PCR analysis confirmed, as expected, that both HLA-DM and HLA-DR were expressed in 293T cells (data not shown). The associated ChIP experiment , We have shown that transcription from the HIV-1 LTR involves TBP but not TFIID. This result was particularly unexpected because in mammalian cells TBP is bound to TAFs extremely tightly and numerous biochemical studies have failed to find free TBP ,3,4. Nonβ-globin, E4CAT, G6(−83)HIV LTRΔTARCAT, and GAPDH). Third, when TC assembly was directed by Tat or P-TEFb the ChIP assay detected all GTFs analyzed, including TBP, but not TAFs on six different promoters  or lacking (pB/E) the SV40 enhancer were provided by Walter Schaffner; pBS contains a 196-bp SV40 enhancer–containing fragment inserted into a plasmid derived from pB6 [β-globin gene. The E4CAT construct, pE4CAT, was previously described [To generate the G4E1bCAT construct, pDSG39, four Gal4-binding sites were inserted upstream of the E1bTATA box and the CAT ORF in the vector pSP72 (Promega, Madison, Wisconsin, United States). The from pB6 , which hescribed . The pFRescribed . The , E1a , α-CDK8 goat polyclonal (sc-1521), α-CDK9 goat polyclonal (sc-7331), α-CycT1 goat polyclonal (sc-8127), α-Med6 goat polyclonal (sc-9434), α-GCN5 goat polyclonal (sc-6303); α-TRRAP rabbit polyclonal (sc-11411), α-TAF1 mouse monoclonal (sc-735), α-TAF5 mouse monoclonal (sc-743), α-Sp1 rabbit polyclonal (sc-59X), and α-alpha tubulin mouse monoclonal (sc-5268) antibodies were obtained from Santa Cruz Biotechnology . The α-RNA polymerase II mouse monoclonal antibody (8WG16) was obtained from BAbCO . The α-Tat rabbit antiserum was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program . The α-14C chloramphenicol followed by thin layer chromatography. Signals were visualized by autoradiography and quantitated using National Institutes of Health Image 1.62 software.Transcription was measured either by RT-PCR analysis from total RNA isolated from transfected cells or by chloramphenicol acetyl transferase (CAT) assay  using 142 transfection methods [24H39NaO4, 1 mM EDTA, and 10 mM Tris-HCl [pH 8.0]), and twice with TE (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA). Chromatin was eluted with freshly prepared elution buffer (1% SDS and 0.1 M CHNaO3). After reversal of the cross-links, the samples were treated with proteinase K for 1 h at 45 °C, extracted by phenol/chloroform, and ethanol precipitated. The pellet was resuspended in TE, and PCR was performed. Autoradiograms were scanned and quantitated by the National Institutes of Health ImageJ program. IP DNA was quantitated and presented as the ratio of IP to input. Primer sequences are available upon request.HeLa and 293T cells were transfected with 10 μg of effector plasmids and 5 μg of reporter plasmid using standard CaCl methods . Forty-eFor TAF1 inactivation, ts13 cells  were inihttp://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the genes and gene products discussed in this paper are CDK8 (1024), CDK9 (1025), CIITA (4261), CycT1 (904), GAPDH (2597), hGCN5 (2648), HLA-DM (3109), HLA-DR (3122), hSpt3 (8464), MED6 (10001), Mot1 (9044), PAF65β (27097), RBP1 (5430), TAF1 (6872), TAF11 (6882), TAF12 (6883), TAF13 (6884), TAF2 (6873), TAF4 (6874), TAF5 (6877), TAF6 (6878), TAF7 (6979), TAF8 (129685), TAF9 (6880), Tat (155871), TBP (6908), TFIIB (2959), and TRRAP (8295).The LocusLink (