Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN

DC-SIGN, a human C-type lectin, is expressed on the surface of dendritic cells (DC), while a closely related human gene, DC-SIGNR or L-SIGN, is found on sinusoidal endothelial cells of liver and lymph node. Both DC-SIGN and DC-SIGNR/L-SIGN can bind ICAM-3 and HIV gp120, and transmit HIV to susceptible cells in trans. Here, we report the cloning of five mouse genes homologous to human DC-SIGN and DC-SIGNR/L-SIGN. Only one gene, named mouse DC-SIGN, is highly expressed in DC, and is not found in a panel of mouse macrophage and lymphocyte cell lines. The other four genes, named mouse SIGNR1 (SIGN-Related gene 1), SIGNR2, SIGNR3 and SIGNR4, are expressed at lower levels in various cells according to RT-PCR and Northern blot analyses on RNA. All the genes of mouse DC-SIGN and SIGNRs map to adjacent regions of chromosome 8 A1.2-1.3. However, like human DC-SIGN, only the mouse DC-SIGN gene is closely juxtaposed to the CD23 gene, while the other four SIGNR genes are located close to each other in a neighboring region. mRNAs of mouse DC-SIGN and three SIGNR genes encode type II transmembrane proteins (DC-SIGN, 238 amino acids; SIGNR1, 325 amino acids; SIGNR3, 237 amino acids; SIGNR4, 208 amino acids), but the SIGNR2 gene only encodes a carbohydrate recognition domain (CRD) without a cytosolic domain and a transmembrane domain (SIGNR2, 178 amino acids). Amino acid sequence similarities between the CRD of human DC-SIGN and the mouse homologues are 67% for DC-SIGN, 69% for SIGNR1, 65% for SIGNR2, 68% for SIGNR3 and 70% for SIGNR4 respectively. However, the membrane proximal neck domains in the mouse genes are much shorter than their counterparts in human DC-SIGN and DC-SIGNR/L-SIGN. This family of mouse C-type lectins is therefore complex, but only one of the new genes, DC-SIGN, is juxtaposed to CD23 and is expressed at high levels in DC.     

Recombinant vesicular stomatitis viruses encoding simian immunodeficiency virus receptors target infected cells and control infection

We have constructed VSV recombinants lacking the viral glycoprotein gene and instead expressing rhesus macaque SIV receptors CD4 and CCR5 with or without the receptor DC-SIGN. The recombinant expressing CD4 and CCR5 specifically infected SIV envelope protein-expressing cells. Incorporation of DC-SIGN into the particles required deletion of the cytoplasmic domain. Inclusion of DC-SIGN in the particles definitely enhanced infection, indicating that the enhancement by coexpression of DC-SIGN with CD4 and CCR5 does not require internalization of the virus into cells. The recombinants also specifically infected, killed, and propagated in CEMx174 cells that were first infected with an SIV expressing EGFP. If cells were superinfected with either of the recombinants after the primary SIV infection, the numbers of SIV-infected cells and titers of infectious SIV in the cultures were significantly reduced. Such antivirals can now be tested in the SIV/non-human primate model for AIDS to determine their therapeutic value in vivo.     

Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural antibodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins

The present study demonstrates that human breast milk and normal human polyclonal immunoglobulins purified from plasma [intravenous immunoglobulins (IVIg)] contain functional natural immunoglobulin A (IgA) and IgG antibodies directed against the carbohydrate recognition domain (CRD) domain of the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule, which is involved in the binding of human immunodeficiency virus (HIV)-1 to dendritic cells (DCs). Antibodies to DC-SIGN CRD were affinity-purified on a matrix to which a synthetic peptide corresponding to the N-terminal CRD domain (amino-acid 342-amino-acid 371) had been coupled. The affinity-purified antibodies bound to the DC-SIGN peptide and to the native DC-SIGN molecule expressed by HeLa DC-SIGN+ cells and immature monocyte-derived dendritic cells (iMDDCs), in a specific and dose-dependent manner. At an optimal dose of 200 microg/ml, natural antibodies to DC-SIGN CRD peptide purified from breast milk and IVIg stained 25 and 20% of HeLa DC-SIGN+ cells and 32 and 12% of iMDDCs, respectively. Anti-DC-SIGN CRD peptide antibodies inhibited the attachment of virus to HeLa DC-SIGN by up to 78% and the attachment to iMDDCs by only 20%. Both breast milk- and IVIg-derived natural antibodies to the CRD peptide inhibited 60% of the transmission in trans of HIV-1(JRCSF), an R5-tropic strain, from iMDDCs to CD4+ T lymphocytes. Taken together, these observations suggest that the attachment of HIV to DCs and transmission in trans to autologous CD4+ T lymphocytes occur through two independent mechanisms. Our data support a role of natural antibodies to DC-SIGN in the modulation of postnatal HIV transmission through breast-feeding and in the natural host defence against HIV-1 in infected individuals.     

Epitope mapping on the dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) pathogen-attachment factor

DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin) is a myeloid pathogen-attachment factor C-type lectin which recognizes mannose- and fucose-containing oligosaccharide ligands on clinically relevant pathogens. Intracellular signaling initiated upon ligand engagement of DC-SIGN interferes with TLR-initiated signals, and modulates the T cell activating and polarizing ability of antigen-presenting cells. The C-terminal carbohydrate-recognition domain (CRD) of DC-SIGN is preceded by a neck domain composed of eight 23-residue repeats which mediate molecule multimerization, and whose polymorphism correlates with altered susceptibility to SARS and HIV infection. Naturally occurring isoforms and chimaeric molecules, in combination with established recognition properties, were used to define seven structural and functional epitopes on DC-SIGN. Three epitopes mapped to the CRD, one of which is multimerization-dependent and only exposed on DC-SIGN monomers. Epitopes within the neck domain were conformation-independent and unaltered upon molecule multimerization, but were differentially affected by neck domain truncations. Although neck-specific antibodies exhibited lower function-blocking ability, they were more efficient at inducing molecule internalization. Moreover, crosslinking of the different epitopes resulted in distinct levels of microclustering on the cell surface. The identification of independent epitopes on the DC-SIGN molecule might facilitate the design of reagents that modulate the T cell activating and polarizing ability of DC-SIGN-expressing cells without preventing its antigen- and pathogen-recognition capacities.     

Productive infection of dendritic cells by simian immunodeficiency virus in macaque intestinal tissues

Dendritic cells (DCs) are potent antigen-presenting cells that likely play multiple roles in human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) pathogenesis. This paper describes the effects of pathogenic SIV infection on the networks of DCs in rhesus macaque (Macaca mulatta) intestinal tissues. Intestinal tissues were obtained from macaques at different stages of disease following infection with the pathogenic SIV/DeltaB670 isolate. The patterns and levels of expression of SIV and DC-associated mRNAs were examined and quantitated directly in intestinal tissue sections. In situ hybridization was performed for SIV, DC-specific ICAM3-grabbing non-integrin (DC-SIGN), DC-specific lysosome-associated membrane glycoprotein (DC-LAMP), DC-specific C-type lectin 1 (DECTIN-1), CC chemokine receptor 6 (CCR6), CCR7, and macrophage inflammatory protein 3alpha (MIP-3alpha/CCL20) mRNAs and quantitative image analysis was performed to measure mRNA expression levels. To identify the cell types productively infected by SIV, simultaneous in situ hybridization and immunohistochemical staining were performed. The DC networks in macaque intestinal tissues were found to be extensive and although they generally remained intact during the course of SIV infection, there were alterations in the expression of markers for immature DCs. One alteration was an increase in the expression in intestinal submucosa of DC-SIGN, a molecule that binds to HIV-1/SIV and increases its infectivity. Concomitant with this increase, it was found that during AIDS, the population of productively infected cells included DCs, based on co-expression of DC-SIGN and DECTIN-1 mRNAs. These data indicate that SIV infection affects subpopulations of macaque intestinal DCs, including productive infection of DC-SIGN+ DCs, the consequences of which are likely to be ongoing viral propagation and decreased immunostimulatory function.     

Rhesus macaque and chimpanzee DC-SIGN act as HIV/SIV gp120 trans-receptors, similar to human DC-SIGN

Dendritic cells (DC) have been implicated in the pathogenesis of both human and simian immunodeficiency viruses (HIV and SIV, respectively). The DC-specific HIV-1 trans-receptor DC-SIGN is thought to be essential for viral dissemination by DC. Abundant expression in lymphoid tissues also implies a function for DC-SIGN in chronic HIV-1 infections, in facilitating persistent infection of T cells. We have therefore isolated the rhesus macaque and chimpanzee homologues of DC-SIGN to investigate their function in a primate model. Both rhesus macaque and chimpanzee DC-SIGN are highly similar to the human homologue. Three monoclonal antibodies against human DC-SIGN, AZN-D1, -D2 and -D3, cross-react with rhesus macaque DC-SIGN, whereas AZN-D2 does not cross-react with chimpanzee DC-SIGN. The primate homologues are abundantly expressed in lymphoid tissues such as lymph nodes, as well as in mucosal tissues involved in sexual transmission of HIV-1, and are functionally similar to human DC-SIGN. They have a high affinity for the immunological ligands of DC-SIGN: ICAM-2 and -3. Moreover, both homologues bind the HIV-1 envelope glycoprotein gp120 and therefore can act as a HIV-1 trans-receptor in the same way as human DC-SIGN. These data demonstrate that primate models are suitable to further dissect the role of DC-SIGN in the transmission and pathogenesis of infection with immunodeficiency viruses.     

Tissue-specific reduction in DC-SIGN expression correlates with progression of pathogenic simian immunodeficiency virus infection

Studies were undertaken to determine whether previously described reductions in splenic DC-SIGN expression in simian acquired immune deficiency syndrome (AIDS) are limited to pathogenic simian immunodeficiency virus (SIV) infection. DC-SIGN expression was evaluated by immunohistochemistry in lymphoid tissues from AIDS-susceptible Asian macaque monkeys as compared with AIDS-resistant sooty mangabey monkeys in the presence and absence of SIV infection. The phenotype of DC-SIGN+ cells in susceptible and resistant species was identical and most consistent with macrophage identity. Significantly lower levels of DC-SIGN expression were identified in spleen, mesenteric lymph node, and bone marrow of macaques with AIDS (P<0.05). Reduced levels of splenic DC-SIGN correlated significantly with CD4T cell depletion in long-term pathogenic infection of macaques (P<0.01), whereas SIV-infected mangabeys retained high levels of DC-SIGN expression in spleen despite persistent infection. Reduced expression of DC-SIGN in spleen specifically characterizes pathogenic forms of SIV infection, correlates with disease progression, and may contribute to SIV pathogenesis.     

Molecular mechanisms that set the stage for DC-T cell engagement

The unsurpassed capacity of dendritic cells (DC) to prime naive T cells is thought to depend on the formation of an immunological synapse. DC-SIGN, a C-type lectin exclusively expressed at the cell surface of DC, functions as an adhesion receptor facilitating T cell binding and priming through recognition of glycosylated ICAM-3 on naive T cells. Yet, DC-SIGN also mediates binding to pathogens such as HIV by recognizing glycosylated gp120. The scope of the present study was to investigate whether DC-SIGN upon recognition of its cellular ligand and pathogenic ligand affects DC synapse formation and activation/mobilization of other adhesion receptors such as LFA-1 to the cell contact site. Using a DC-SIGN deletion mutant, we show that DC-SIGN is a constitutively active receptor that mediates ligand binding independent of signaling through the cytoplasmic domain. Surprisingly, initial binding of gp120 to DC-SIGN did not result in increased adhesion levels of LFA-1 to its ligand ICAM-1 in both immature DC and Raji-DC-SIGN cells. However, ligand binding to DC-SIGN induced recruitment of LFA-1 to the adhesion site. Moreover, we could demonstrate that activation of LFA-1 results in DC-SIGN-LFA-1 co-clustering in the cell membrane. This triggers binding of ligands to LFA-1 that are shared with DC-SIGN, such as ICAM-3, but not of ligands that are not shared with DC-SIGN, such as ICAM-1. Thus, we propose that upon ligand binding DC-SIGN recruits LFA-1 to the contact site, resulting in the formation of DC-SIGN-LFA-1 co-clusters, in which the initial DC-SIGN-mediated interactions with ligand are transient and eventually shift to more stable LFA-1-dependent interactions.     

Contributions of the N- and C-terminal domains of surfactant protein d to the binding,aggregation, and phagocytic uptake of bacteria

Collectins play important roles in host defense against infectious microorganisms. We now demonstrate that the serum collectins mannose-binding lectin (MBL) and conglutinin have less ability to bind to, aggregate, and enhance neutrophil uptake of several strains of gram-negative and gram-positive bacteria than pulmonary surfactant protein D (SP-D). Collectins are composed of four major structural domains (i.e., N-terminal, collagen, and neck and carbohydrate recognition domains). To determine which domains of SP-D are responsible for its greater bacterial binding or aggregating activity, activities of chimeric collectins containing the N-terminal and collagen domains of SP-D coupled to the neck recognition domains and carbohydrate recognition domains (CRD) of MBL or conglutinin (SP-D/Cong(neck+CRD) and SP-D/MBL(neck+CRD)) were tested. The SP-D/Cong(neck+CRD) and SP-D/MBL(neck+CRD) chimeras bound to and aggregated the bacteria more strongly than did wild-type MBL or conglutinin. SP-D/MBL(neck+CRD) also enhanced neutrophil uptake of bacteria more so than MBL. Hence, the SP-D N-terminal and/or collagen domains contribute to the enhanced bacterial binding and aggregating activities of SP-D. In prior studies, SP-D/Cong(neck+CRD) and SP-D/MBL(neck+CRD) had increased ability to bind to influenza virus compared not only with that of conglutinin or MBL but with that of wild-type SP-D as well. In contrast, the chimeras had either reduced or unchanged ability to bind to or aggregate bacteria compared to that of wild-type SP-D. Hence, although replacement of the neck recognition domains and CRDs of SP-D with those of MBL and conglutinin conferred increased viral binding activity, it did not favorably affect bacterial binding activity, suggesting that requirements for optimal collectin binding to influenza virus and bacteria differ.     

Impact of polymorphisms in the DC-SIGNR neck domain on the interaction with pathogens

The lectins DC-SIGN and DC-SIGNR augment infection by human immunodeficiency virus (HIV), Ebolavirus (EBOV) and other pathogens. The neck domain of these proteins drives multimerization, which is believed to be required for efficient recognition of multivalent ligands. The neck domain of DC-SIGN consists of seven sequence repeats with rare variations. In contrast, the DC-SIGNR neck domain is polymorphic and, in addition to the wild type (wt) allele with seven repeat units, allelic forms with five and six sequence repeats are frequently found. A potential association of the DC-SIGNR genotype and risk of HIV-1 infection is currently under debate. Therefore, we investigated if DC-SIGNR alleles with five and six repeat units exhibit defects in pathogen capture. Here, we show that wt DC-SIGNR and patient derived alleles with five and six repeats bind viral glycoproteins, augment viral infection and tetramerize with comparable efficiency. Moreover, coexpression of wt DC-SIGNR and alleles with five repeats did not decrease the interaction with pathogens compared to expression of each allele alone, suggesting that potential formation of hetero-oligomers does not appreciably reduce pathogen binding, at least under conditions of high expression. Thus, our results do not provide evidence for diminished pathogen capture by DC-SIGNR alleles with five and six repeat units. Albeit, we cannot exclude that subtle, but in vivo relevant differences remained undetected, our analysis suggests that indirect mechanisms could account for the association of polymorphisms in the DC-SIGNR neck region with reduced risk of HIV-1 infection.     

树突状细胞特异表面分子DC-SIGN的研究进展

DC-SIGN是一种特异表达于树突状细胞(DC)表面的Ⅱ型跨膜蛋白,在机体生理和病理免疫调节中发挥着重要作用,可以与ICAM-3结合,从而介导DC与T细胞的相互作用,其CRD区可以与HIV-1、HCV、结核杆菌等多种病原体表面糖蛋白结合,从而促进病原体感染。最近的研究表明DC-SIGN与肿瘤免疫、免疫逃避也密切相关。     

DC-SIGN and L-SIGN: the SIGNs for infection

Two closely related trans-membrane C-type lectins dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN or CD209) and liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN also known as DC-SIGNR, CD209L or CLEC4M) directly recognize a wide range of micro-organisms of major impact on public health. Both genes have long been considered to share similar overall structure and ligand-binding characteristics. This review presents more recent biochemical and structural studies, which show that they have distinct ligand-binding properties and different physiological functions. Of importance in both these genes is the presence of an extra-cellular domain consisting of an extended neck region encoded by tandem repeats that support the carbohydrate-recognition domain, which plays a crucial role in influencing the pathogen-binding properties of these receptors. The notable difference between these two genes is in this extra-cellular domain. Whilst the tandem-neck-repeat region remains relatively constant size for DC-SIGN, there is considerable polymorphism for L-SIGN. Homo-oligomerization of the neck region of L-SIGN has been shown to be important for high-affinity ligand binding, and heterozygous expression of the polymorphic variants of L-SIGN in which neck lengths differ could thus affect ligand-binding affinity. Functional studies on the effect of this tandem-neck-repeat region on pathogen-binding, as well as genetic association studies for various infectious diseases and among different populations, are discussed. Worldwide demographic data of the tandem-neck-repeat region showing distinct differences in the neck-region allele and genotype distribution among different ethnic groups are presented. These findings support the neck region as an excellent candidate acting as a functional target for selective pressures exerted by pathogens.     

ICAM-3 influences human immunodeficiency virus type 1 replication in CD4(+) T cells independent of DC-SIGN-mediated transmission

We investigated the role of ICAM-3 in DC-SIGN-mediated human immunodeficiency virus (HIV) infection of CD4(+) T cells. Our results demonstrate that ICAM-3 does not appear to play a role in DC-SIGN-mediated infection of CD4(+) T cells as virus is transmitted equally to ICAM-3(+) or ICAM-3(-) Jurkat T cells. However, HIV-1 replication is enhanced in ICAM-3(-) cells, suggesting that ICAM-3 may limit HIV-1 replication. Similar results were obtained when SIV replication was examined in ICAM-3(+) and ICAM-3(-) CEMx174 cells. Furthermore, while ICAM-3 has been proposed to play a co-stimulatory role in T cell activation, DC-SIGN expression on antigen presenting cells did not enhance antigen-dependent activation of T cells. Together, these data indicate that while ICAM-3 may influence HIV-1 replication, it does so independent of DC-SIGN-mediated virus transmission or activation of CD4(+) T cells.     

Modulation of HIV and SIV neutralization sensitivity by DC-SIGN and mannose-binding lectin

The C-type lectin DC-SIGN binds to oligosaccharides on the human and simian immunodeficiency virus (HIV, SIV) envelope glycoproteins and promotes infection of susceptible cells. Here, we show that DC-SIGN recognizes glycans involved in SIV sensitivity to neutralizing antibodies and that binding to DC-SIGN confers neutralization resistance to an otherwise sensitive SIV variant. Moreover, we provide evidence that mannose-binding lectin (MBL) can interfere with HIV-1 neutralization by the carbohydrate-specific antibody 2G12.     

Species differences in the carbohydrate binding preferences of surfactant protein D

Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each with identical N-terminal tags remote from the ligand-binding surface. Although rat and mouse showed similar affinities for saccharide competitors, both differed markedly from the human protein. The human neck+CRD preferentially recognized N-acetyl-mannosamine, whereas the rat and mouse proteins showed greater affinity for myoinositol, maltose, and glucose. Although human neck+CRDs bound to maltosyl-agarose and fungal mannan, only rat and mouse neck+CRDs showed significant binding to maltosyl-Toyopearl beads, solid-phase maltosyl-albumin neo-glycoprotein, or the Phil82 strain of influenza A virus. Likewise, human SP-D dodecamers and trimeric subunits of full-length rat, but not full-length human SP-D trimers, bound to maltosyl-Toyopearl. Site-directed mutagenesis of the human neck+CRD demonstrated an important role of Asp324-Asp325 in the recognition of N-acetyl-mannosamine, and substitution of the corresponding murine sequence (Asn324-Asn325) conferred a capacity to interact with immobilized maltose. Thus, ligand recognition by human SP-D involves a complex interplay between saccharide presentation, the valency of trimeric subunits, and species-specific residues that flank the primary carbohydrate binding site.     

Inhibition of DC-SIGN-mediated trans infection of T cells by mannose-binding lectin

Some dendritic cells (DC) express a cell-surface lectin called 'dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin' (DC-SIGN). DC-SIGN has been shown to mediate a type of infection called 'trans' infection, where DC bind human immunodeficiency virus (HIV) and efficiently transfer the virus to T cells. We investigated the possibility that mannose-binding lectin (MBL), a soluble lectin that functions as a recognition molecule in innate immunity and that binds to HIV, could block trans infection mediated by DC-SIGN. Binding studies with glycoprotein (gp)120/gp41-positive and -negative virus preparations suggested that DC-SIGN and MBL bind primarily to glycans on gp120/gp41, as opposed to glycans on host-cell-derived proteins, indicating a close overlap in the binding site of the two lectins and supporting the notion that MBL could prevent binding of HIV to DC-SIGN. Preincubation of X4, R5 or dual-tropic HIV strains with MBL prevented DC-SIGN-mediated trans infection of T cells. The mechanism of MBL blocking trans infection of T cells was at least partly caused by blocking of virus binding to DC-SIGN positive cells. This study shows that MBL prevents DC-SIGN-mediated trans infection of T cells in vitro and suggests that in infected persons, MBL may inhibit DC-SIGN-mediated uptake and spread of HIV.     

Development of simian immunodeficiency virus isolation, titration, and neutralization assays which use whole blood from rhesus monkeys and an antigen capture enzyme-linked immunosorbent assay.

Assays that use rhesus macaque whole blood and an antigen capture enzyme-linked immunosorbent assay for the simian immunodeficiency virus (SIV) p27 core protein were developed for the isolation of SIV from the blood of infected animals, the titration of infectivity of SIV inocula, and the quantitation of virus neutralizing antibodies in serum. These assays required small amounts of whole blood, were adaptable to a microtiter format, and used substrates mainly of rhesus macaque origin.     

Method for detection of simian immunodeficiency virus neutralizing antibodies using a noncommercial antigen capture enzyme-linked immunosorbent assay.

A neutralization test (NT) using a noncommercial antigen capture enzyme-linked immunosorbent assay (ELISA) to detect simian immunodeficiency virus (SIV) growth in vitro was developed. The capture antibody was a mixture of purified macaque anti-SIV immunoglobulin G (IgG) and a monoclonal antibody to SIV p27. Captured antigens were detected by using purified macaque anti-SIV IgG conjugated to horseradish peroxidase. The NT reliably and sensitively detected differences when various amounts of SIV were used with positive and negative control macaque sera. Dilutions of sequential sera from a macaque (Macaca nemestrina) that had been experimentally infected with SIV were tested for neutralizing antibody with 300 50% tissue culture infective doses of SIV. In this macaque, neutralizing activity and anti-SIV IgG levels in serum (detected by ELISA) increased with time after SIV inoculation, and high IgG titers were required in serum before neutralization occurred in vitro. This simple NT, which detects the presence of SIV serum neutralizing antibodies at a low cost, will be useful for investigating the role of neutralizing antibodies in the SIV-infected macaque model for AIDS.     

Mechanism of interferon action: identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2 alpha protein kinase.

A molecular cDNA clone of the human RNA-dependent P1/eIF-2 alpha protein kinase was expressed in Escherichia coli. Mutant P1 proteins were examined for RNA binding activity by Northwestern blot analysis using the reovirus s1 mRNA, an activator of the kinase; the adenovirus VAI RNA, an inhibitor of kinase activation; or human immunodeficiency virus (HIV) TAR RNA as probe. Analysis of TrpE-P1 deletion mutant fusion proteins revealed that the 11-kDa N-terminal region of the P1 protein bound reovirus s1 mRNA, adenovirus VAI RNA, and HIV TAR RNA. Neither s1 RNA, VAI RNA, nor TAR RNA was bound by truncated P1 proteins which lacked the N-terminal 98 amino acids. Computer analysis revealed that the human protein P1 sequence corresponding to amino acid residues within the N-terminal RNA binding domain displays high homology (greater than 54% identity; 61 to 94% similarity) with two animal virus proteins which possess RNA binding activity (vaccinia virus E3L; rotavirus VP2) and two proteins of unknown function (murine TIK; rotavirus NS34), but which are likely RNA binding proteins.     

In vitro analysis of an RNA binding site within the N-terminal 30 amino acids of the southern cowpea mosaic virus coat protein

Southern cowpea mosaic virus (SCPMV) is a positive-sense RNA virus with T = 3 icosahedral symmetry. The coat protein (CP) has two domains, the random (R) domain and the shell (S) domain. The R domain is formed by the N-terminal 64 amino acids (aa) and is localized to the interior of the particle where it is expected to interact with the viral RNA. The R domain (aa 1--57) was expressed in Escherichia coli as a recombinant protein (rWTR) containing a nonviral C-terminal extension with two histidine tags. The RNA binding site of the R domain was identified by Northwestern blotting and electrophoretic mobility shift assay (EMSA) using recombinant wild-type and mutant R domain proteins. Deletions within the R domain revealed that the RNA binding site is localized to its N-terminal 30 aa. RNA binding by this element was found to be nonspecific with regard to RNA sequence and was sensitive to high salt concentrations, suggesting that electrostatic interactions are important for RNA binding by the R domain. The RNA binding site includes 11 basic residues, eight of which are located in the arginine-rich region between aa 22 and 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21, did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an alpha-helix in the presence of 50% trifluoroethanol (TFE). The possible role for this structural change in RNA binding by the R domain is discussed.