Complement alternative pathway genetic variation and Dengue infection in the Thai population.

Dengue disease is a mosquito-borne infection caused by Dengue virus. Infection may be asymptomatic or variably manifest as mild Dengue fever (DF) to the most severe form, Dengue haemorrhagic fever (DHF). Mechanisms that influence disease severity are not understood. Complement, an integral component of the immune system, is activated during Dengue infection and the degree of activation increases with disease severity. Activation of the complement alternative pathway is influenced by polymorphisms within activation (factor B rs12614/rs641153, C3 rs2230199) and regulatory [complement factor H (CFH) rs800292] proteins, collectively termed a complotype. Here, we tested the hypothesis that the complotype influences disease severity during secondary Dengue infection. In addition to the complotype, we also assessed two other disease-associated CFH polymorphisms (rs1061170, rs3753394) and a structural polymorphism within the CFH protein family. We did not detect any significant association between the examined polymorphisms and Dengue infection severity in the Thai population. However, the minor allele frequencies of the factor B and C3 polymorphisms were less than 10%, so our study was not sufficiently powered to detect an association at these loci. We were also unable to detect a direct interaction between CFH and Dengue NS1 using both recombinant NS1 and DV2-infected culture supernatants. We conclude that the complotype does not influence secondary Dengue infection severity in the Thai population.

Evasion of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells.

Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four-amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4(+) and CD8(+) populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.

Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity.

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5' splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.

TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL.

A subset of the tumour necrosis factor (TNF) receptor family contain a conserved intracellular motif, the death domain. Engagement of these receptors by their respective ligands initiates a signalling cascade that rapidly leads to cell death by apoptosis. We have cloned a new member of this family, TRICK2, the TRAIL (TNF-related apoptosis-inducing ligand) receptor inducer of cell killing 2. TRICK2 is expressed in a number of cell types, and to particularly high levels in lymphocytes and spleen. Two isoforms of the TRICK2 mRNA are generated by alternative pre-mRNA splicing and differ by a 29 amino-acid extension to the extracellular domain. Overexpression of TRICK2 rapidly induced apoptosis in 293T cells; this induction was dependent upon the presence of the death domain of TRICK2. Using a soluble molecule containing the TRICK2 extracellular domain, we demonstrated that TRICK2, like DR4 [1], is a receptor for TRAIL/APO-2L [2,3] and could inhibit TRAIL-induced killing of lymphocyte lines, such as the Jurkat T-cell line. TRAIL is upregulated upon lymphocyte activation, as is the intensively studied ligand for Fas, FasL [4]. TRAIL and its receptors might therefore provide another system for the regulation of lymphocyte selection and proliferation, as well as providing an additional weapon in the armoury of cytotoxic lymphocytes.

T cell life and death signalling via TNF-receptor family members.

An effective immune response requires the rapid and accurate mobilisation of millions of effector cells in an antigen driven fashion. These effector cells must be kept alive long enough to fulfil their function but the majority must then be eliminated, a process known as activation-induced cell death. Recent advances in the field of lymphocyte biology have shed light onto how this balance is maintained and onto the consequences for disease if the homeostatic mechanisms become disturbed.

Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements.

We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. beta-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin mu-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.

Influence of intron length on alternative splicing of CD44.

Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.

Role of SR protein modular domains in alternative splicing specificity in vivo.

The SR proteins constitute a family of nuclear phosphoproteins which are required for constitutive splicing and also influence alternative splicing regulation. They have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain, rich in arginine and serine residues. The functional role of the different domains of SR proteins in constitutive splicing activity has been extensively studied in vitro; however, their contribution to alternative splicing specificity in vivo has not been clearly established. We sought to address how the modular domains of SR proteins contribute to alternative splicing specificity. The activity of a series of chimeric proteins consisting of domain swaps between different SR proteins showed that splice site selection is determined by the nature of the RRMs and that RRM2 of SF2/ASF has a dominant role and can confer specificity to a heterologous protein. In contrast, the identity of the RS domain is not important, as the RS domains are functionally interchangeable. The contribution of the RRMs to alternative splicing specificity in vivo suggests that sequence-specific RNA binding by SR proteins is required for this activity.

The splicing determinants of a regulated exon in the axonal MAP tau reside within the exon and in its upstream intron.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression profile is distinct from that of the other tau regulated exons, implying the utilization of distinct regulatory factors. Previous work had established the use of cryptic splice sites within exon 6 and the influence of flanking exons on the ratio of exon 6 variants. The present work shows that, in addition to the previously identified participants, the ratio of exon 6 isoforms is affected by: (1) suppression of the cryptic sites, (2) deletions of the upstream intron, and (3) the splicing regulators PTB and U2AF, both of which act on the branch point/polypyrimidine tract region. These results strongly suggest that factors binding immediately upstream of exon 6 are involved in regulation of this exon. They also lead to the conclusion that splicing of exon 6 is primarily governed by multiple branch points.

Molecular cloning of htra2-beta-1 and htra2-beta-2, two human homologs of tra-2 generated by alternative splicing.

A yeast two-hybrid screen was performed to find new factors involved in pre-mRNA splicing. Using SC35 as a bait, we isolated a human cDNA bearing high homology to the Drosophila transformer-2 (TRA-2) protein. This cDNA was named htra2-beta1. htra2-beta1 is a nuclear protein that colocalizes with SC35 in a speckled pattern. It interacts with several SR proteins tested in yeast. A second form named htra2-beta2 is generated by alternative splicing. This isoform gives rise to a truncated protein without an SR domain. Both isoforms are evenly distributed throughout adult rat tissue. The ratio of these two isoforms changes after stimulation of primary human T-cell and primary rat spleen cell cultures, indicating that alternative splicing is involved in regulation of htra2-beta activity.

Heparin clearance during continuous veno-venous haemofiltration.

OBJECTIVE: To determine whether premature clotting of haemofiltration circuits could be related to heparin removal across the filter membrane into the ultrafiltrate. DESIGN: Randomised study using either unfractionated (n = 8) or low molecular weight (n = 7) heparin for anticoagulation of the haemofiltration circuit at 1000 and 600 U/h respectively. Samples were drawn at 1 and 2 h from arterial and venous limbs of the haemofilter circuit for measurement of plasma heparin (as anti-Factor Xa activity), antithrombin III and haematocrit. Ultrafiltrate samples were collected at the same time for measurement of anti-Xa activity. SETTING: Intensive care unit. PATIENTS: Patients in acute renal failure requiring haemofiltration. RESULTS: Both unfractionated and low molecular weight heparin plasma levels were within the range required for therapeutic anticoagulation in all but one patient at 2 h. Ultrafiltrate anti-Xa levels were insignificant. Antithrombin III levels in these critically ill patients were subnormal in 11 of the 15 studies. CONCLUSIONS: Despite their small sizes, neither unfractionated nor low molecular weight heparins cross the haemofilter membrane into the ultrafiltrate in any measurable quantity. Both heparins were present in plasma at a level suitable for therapeutic anticoagulation. Subnormal levels of antithrombin III may be an important factor in determining filter longevity.

Resistivity changes in conductive silicone sheets under stretching.

This paper reports a preliminary finding associated with an investigation of how tissues respond to mechanical stress. The stress distribution within the tissue may be the result of normal function, for example, joint forces, or it may result from interventions such as tissue suturing during or after surgery. We sought to combine electrical and mechanical computational models in order to better understand the interaction between the two. For example, if mechanical stress is applied to tissue this may change the cell arrangements within the tissue matrix and hence change the electrical properties. If this interaction could be determined, then it should be possible to use electrical impedance tomography measurements to identify stress patterns in tissues. Measurements of resistivity changes have been made in conductive silicone rubber sheets when subject to a uniaxial stress of up to 10%. Relatively large changes in resistivity are produced (up to 200%). These changes are far larger than those predicted arising from topological changes alone. It is suggested that under stress the conductive islands of carbon within the silicone rubber sheet undergo a reversible disassociation from their neighbours and that the material's electrical properties change under load. If similar stress-resistivity relationships occur within biological materials it may be possible to recover the stress fields within tissues from transfer impedance measurements and thereby predict if actions such as inappropriate suture tension will compromise tissue viability.

A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm.

The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation and this activity is antagonized by members of the hnRNP A/B family of proteins. We show here that whereas some human SR proteins are confined to the nucleus, three of them-SF2/ASF, SRp20, and 9G8-shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, we show that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. In addition, we show that phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that the family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm.

Virus infections: escape, resistance, and counterattack.

Many viruses establish life-long infections in their natural host with few if any clinical manifestations. The relationship between virus and host is a dynamic process in which the virus has evolved the means to coexist by reducing its visibility, while the host immune system attempts to suppress and eliminate infection without damage to itself. This short review describes a variety of strategies that are employed by viruses to evade host immune responses. These include virus-associated escape from T cell recognition, and resistance to apoptosis and counterattack, with special reference to two papers published in this issue of Immunity (Mueller et al., 2001; Raftery et al., 2001).

The identification of a new alternative exon with highly restricted tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor. Comparison of all 10 variable exons between mouse, human, and rat.

The human CD44 cell surface glycoprotein family which has been implicated in lymphocyte homing, tumor metastasis, and extracellular matrix attachment consists of a large number of related isoforms that derive from the differential splicing of a single CD44 gene transcript. Recent mapping of the CD44 locus in man indicated the presence of at least nine alternative exons within the region of the gene encoding the variable membrane proximal extracellular domain. Here we report the identification of a 10th alternative exon (termed V1) in the mouse, human, and rat CD44 genes. We demonstrate tissue-specific patterns of expression for transcripts containing exons V1-V10 in the mouse and a highly restricted usage of exon V1 in transcripts from mouse gastric tissue. Close sequence homology between exons V1-V10 from mouse rat and human points to a specific functional role rather than a purely structural role for the membrane proximal extracellular domain of the CD44 molecule.

Serine-arginine (SR) protein-like factors that antagonize authentic SR proteins and regulate alternative splicing.

We have characterized two RNA-binding proteins, of apparent molecular masses of approximately 40 and 35 kDa, which possess a single N-terminal RNA-recognition motif (RRM) followed by a C-terminal domain rich in serine-arginine dipeptides. Their primary structures resemble the single-RRM serine-arginine (SR) protein, SC35; however their functional effects are quite distinctive. The 40-kDa protein cannot complement SR protein-deficient HeLa cell S100 extract and showed a dominant negative effect in vitro against the authentic SR proteins, SF2/ASF and SC35. Interestingly, the 40- and 35-kDa proteins antagonize SR proteins and activate the most distal alternative 5' splice site of adenovirus E1A pre-mRNA in vivo, an activity that is similar to that characterized previously for the heterogeneous nuclear ribonucleoprotein particles A/B group of proteins. A series of recombinant chimeric proteins consisting of domains from these proteins and SC35 in various combinations showed that the RRM, but not the C-terminal domain rich in serine-arginine dipeptides, has a dominant role in this activity. Because of the similarity to SR proteins we have named these proteins SRrp40 and SRrp35, respectively, for SR-repressor proteins of approximately 40 and approximately 35 kDa. Both factors show tissue- and cell type-specific patterns of expression. We propose that these two proteins are SR protein-like alternative splicing regulators that antagonize authentic SR proteins in the modulation of alternative 5' splice site choice.

Complex regulation of tau exon 10, whose missplicing causes frontotemporal dementia.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 10 of the gene is an alternatively spliced cassette that is adult-specific and that codes for a microtubule binding domain. Recently, mutations that affect splicing of exon 10 have been shown to cause inherited frontotemporal dementia (FTDP). In this study, we establish the endogenous expression patterns of exon 10 in human tissue; by reconstituting naturally occurring FTDP mutants in the homologous context of exon 10, we show that the cis determinants of exon 10 splicing regulation include an exonic silencer within the exon, its 5' splice site, and the relative affinities of its flanking exons to it. By cotransfections in vivo, we demonstrate that several splicing regulators affect the ratio of tau isoforms by inhibiting exon 10 inclusion.

Regulation of insulin gene expression by the IDDM associated, insulin locus haplotype.

A 4.1 kb genomic region, spanning the insulin (INS) gene, confers genetic susceptibility to Type 1 or insulin-dependent diabetes mellitus (IDDM). Ten polymorphisms within this region form two predominant, complementary haplotypes. We have been studying the effects of these polymorphisms on the levels of insulin mRNA. Cloned genomic DNA fragments representing these two separate haplotypes were transiently transfected into a rodent pancreatic beta cell line, HIT-T15. These studies revealed that insulin mRNA levels were consistently higher in the transfectants expressing the diabetic haplotype. Over-expression of insulin mRNA may provide the basic mechanism for the diabetic susceptibility encoded at the INS locus.

CD44 isoform expression mediated by alternative splicing: tissue-specific regulation in mice.

CD44 is a widely distributed cell surface glycoprotein which shows heterogeneity in molecular expression as a result of post-translational modification as well as alternative splicing of CD44 mRNA. Functional studies have indicated that CD44 plays a role as an adhesion molecule and that different CD44-expressing cells differ in their capacities for CD44-dependent ligand binding. These observations have raised the possibility that structural modifications of CD44, including those resulting from alternatively spliced mRNA isoforms, are involved in the functional heterogeneity of CD44. To assess the expression of CD44 isoforms in the mouse, we examined CD44 cDNA by reverse transcription polymerase chain reaction (RT-PCR). Southern blotting of PCR products with a CD44 cDNA probe or with internal oligonucleotides revealed the expression in mouse tumor cell lines and normal tissues of multiple CD44 mRNA products which are larger than that observed in the absence of variable exon expression. Interestingly, different mouse tissues, including lymphoid cells, showed unique patterns of alternative CD44 mRNA in Southern blotting analysis. The use of exon-specific primers allowed detection of multiple alternatively spliced mRNA species involving expression of at least seven variable exons. Cloning and sequencing of these PCR products revealed sequence identity with recently identified genomic CD44 sequences and confirmed that the PCR products correspond to mature mRNA expressing alternatively spliced CD44 exons. Taken together, these findings demonstrate that the mouse expresses multiple variably spliced CD44 isoforms and that expression is regulated in a tissue- and cell-type specific manner.