Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression.

Allelic variants for the HIV-1 co-receptors chemokine receptor 5 (CCR5) and CCR2, as well as the ligand for the co-receptor CXCR4, stromal-derived factor (SDF-1), have been associated with a delay in disease progression. We began this study to test whether polymorphisms in the CCR5 regulatory regions influence the course of HIV-1 disease, as well as to examine the role of the previously identified allelic variants in 1,090 HIV-1 infected individuals. Here we describe the evolutionary relationships between the phenotypically important CCR5 alleles, define precisely the CCR5 regulatory sequences that are linked to the CCR5-delta32 and CCR2-641 polymorphisms, and identify genotypes associated with altered rates of HIV-1 disease progression. The disease-retarding effects of the CCR2-641 allele were found in African Americans but not in Caucasians, and the SDF1-3'A/3'A genotype was associated with an accelerated progression to death. In contrast, the CCR5-delta32 allele and a CCR5 promoter mutation with which it is tightly linked were associated with limited disease-retarding effects. Collectively, these findings draw attention to a complex array of genetic determinants in the HIV-host interplay.

The VP4 and VP7 of bovine rotavirus VMRI are antigenically and genetically closely related to P-type 5, G-type 6 strains.

We have previously reported the isolation of a bovine rotavirus, designated VMRI, with a super-short electropherotype. We have characterized this strain further as it has shown antigenic differences with the prototype G6 strain NCDV-Lincoln. In this communication, we report the antigenic and molecular characterization and the nucleotide sequence of the VP4 and VP7 genes of this strain. Virus neutralization tests indicated 2- to 13-fold differences in the titers between NCDV-Lincoln, B641 and VMRI strains. Northern blot hybridization results indicated a degree of heterogeneity in the VP4 gene of these strains which can be detected under conditions of high stringency. The VP4 and VP7 genes of the VMRI strain were cloned and sequenced and compared with the published sequences of other bovine rotavirus strains. The VP4 gene of VMRI had a high degree of homology with that of UK and B641 strains but differed significantly from that of both NCDV-Lincoln and B223 strains. Sequence analysis of the VP7 gene of VMRI and other strains indicated a high degree of conservation and the amino acid identity between the different strains was 96%. Sequence information regarding these strains and field isolates will assist in the generation of effective vaccination strategies for control of neonatal calf diarrhea.

Survey of porcine rotavirus G and P genotype in Poland and the United States using RT-PCR.

Porcine rotaviruses are a common cause of gastroenteritis. Several serotypes have been detected based on the two surface proteins VP4 (P-types) and VP7 (G-types). However, limited studies have been performed on the relative frequency of rotavirus types in diarrhetic pigs primarily because of the lack of availability of suitable methods. In this study, we describe a reverse transcriptase-polymerase chain reaction (RT-PCR) method for the typing of P and G types of rotavirus. This method allowed to detect G and P types in 96.8 and 87.1% of isolates collected in the United States, respectively and in 54.5 and 38.6% of isolates collected in Poland, respectively. Within the US specimens the G3, G4, G5, G9 and G10 types were detected in combination with P6 and P7 types while among Polish specimens only G3, G4 and G5 types in combination with P6 and P7 types were identified. In both instances the G4 and G5 were the most prevalent types. These studies show that a RT-PCR typing method is suitable for molecular epidemiological studies and that there is more diversity among porcine rotavirus than previously reported.

Human dendritic cell (DC)-based anti-infective therapy: engineering DCs to secrete functional IFN-gamma and IL-12.

An imbalance in the Th1- and Th2-type cytokine responses may allow certain microbes to modify the host response to favor their own persistence. We now show that infection/pulsing of human CD34+ peripheral blood hemopoietic progenitor cell-derived dendritic cells (DCs) with Leishmania donovani promastigotes, Histoplasma capsulatum, and Mycobacterium kansasii impairs the constitutive production of IL-12 from these cells. Thus, strategies aimed at modulating a dysregulated Th1/Th2 response to infection would be of great interest. To both augment the host immune response and deliver potent immunomodulatory cytokines such as IL-12 and IFN-gamma, our goal is to develop a therapeutic strategy using genetically modified, microbial Ag-pulsed DCs. Toward developing such immunotherapies, we used retrovirus-mediated somatic gene transfer techniques to engineer human DCs to secrete biologically active IL-12 and IFN-gamma. DCs pulsed with microbial antigens (e.g., leishmania and histoplasma Ags) were capable of inducing proliferative responses in autologous CD4+ lymphocytes. CD4+ lymphocytes cocultured with IL-12-transduced autologous DCs had enhanced Ag-specific proliferative responses compared with CD4+ lymphocytes cocultured with nontransduced or IFN-gamma- transduced DCs. In this cell culture model system we demonstrate that IL-12 has a negative effect on IL-4 secretion that is independent of its ability to induce IFN-gamma secretion. Taken together, these results indicate that IL-12-transduced DCs may be specifically suited in inducing or down-modulating Ag-specific Th1 or Th2 responses, respectively, and thus may be useful as adjunctive therapy in those intracellular infections in which a dominant Th1 response is critical for the resolution of infection.

The human CC chemokine receptor 5 (CCR5) gene. Multiple transcripts with 5'-end heterogeneity, dual promoter usage, and evidence for polymorphisms within the regulatory regions and noncoding exons.

Human CC chemokine receptor 5 (CCR5), mediates the activation of cells by the chemokines macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and RANTES, and serves as a fusion cofactor for macrophage-tropic strains of human immunodeficiency virus type 1. To understand the molecular mechanisms that regulate human CCR5 gene expression, we initiated studies to determine its genomic and mRNA organization. Previous studies have identified a single CCR5 mRNA isoform whose open reading frame is intronless. We now report the following novel findings. 1) Complex alternative splicing and multiple transcription start sites give rise to several distinct CCR5 transcripts that differ in their 5'-untranslated regions (UTR). 2) The gene is organized into four exons and two introns. Exons 2 and 3 are not interrupted by an intron. Exon 4 and portions of exon 3 are shared by all isoforms. Exon 4 contains the open reading frame, 11 nucleotides of the 5'-UTR and the complete 3'-UTR. 3) The transcripts appear to be initiated from two distinct promoters: an upstream promoter (PU), upstream of exon 1, and a downstream promoter (PD), that includes the "intronic" region between exons 1 and 3. 4) PU and PD lacked the canonical TATA or CAAT motifs, and are AT-rich. 5) PD demonstrated strong constitutive promoter activity, whereas PU was a weak promoter in all three leukocyte cell environments tested (THP-1, Jurkat, and K562). 6) We provide evidence for polymorphisms in the noncoding sequences, including the regulatory regions and 5'-UTRs. The structure of CCR5 was strikingly reminiscent of the overall structure of other chemokine/chemoattractant receptors, underscoring an important evolutionarily conserved function for a prototypical gene structure. This is the first description of functional promoters for any CC chemokine receptor gene, and we speculate that the complex pattern of splicing events and dual promoter usage may function as a versatile mechanism to create diversity and flexibility in the regulation of CCR5 expression.

CC chemokine receptor 5-mediated signaling and HIV-1 Co-receptor activity share common structural determinants. Critical residues in the third extracellular loop support HIV-1 fusion.

There is a close correspondence between the ability of RANTES and macrophage inflammatory proteins 1alpha and 1beta to activate CC chemokine receptor 5 (CCR5) and the ability to inhibit CCR5-dependent membrane fusion mediated by the envelope glycoprotein of human immunodeficiency virus (HIV), type 1. This finding suggests that some of the structural determinants for CC chemokine/CCR5 interactions and CCR5 HIV-1 fusion co-receptor activity may be shared. Recent studies using human CCR5/CCR2B chimeras have suggested that the determinants of CCR5 co-receptor activity are complex and may involve multiple extracellular receptor domains and that viral co-receptor activity is dissociable from ligand-dependent signaling responses. However, conclusive evidence demonstrating an important role for the second and third extracellular regions of human CCR5 is lacking. Furthermore, to determine whether the determinants for CCR5 co-receptor activity overlap with those required for agonist activity, studies that compare the chemokine specificity for inhibition of envelope-mediated cell fusion and the agonist profile of chimeric receptors are necessary. In the present report, using a series of CCR5/CCR2B chimeras we ascribe an important role for the second and third extracellular loop of CCR5 in supporting the co-receptor activity of CCR5. We also provide evidence that the intracytoplasmic tail of CCR5 does not play an important role in supporting HIV-1 entry. The hypothesis that the structural determinants for CC chemokine/CCR5 interactions and CCR5 HIV-1 fusion co-receptor activity may be shared was confirmed by two novel observations: first, the fusion activity supported by two hybrid receptors could be inhibited by both RANTES and monocyte chemoattractant protein-1, chemokines specific to CCR5 and CCR2B, respectively; and second, the chemokine specificity for inhibition of envelope-mediated cell fusion matched the agonist profile of these hybrid receptors. These data shed new light on the structural determinants involved in these distinct activities of CCR5 and may have important implications for the development of CCR5-targeted anti-viral compounds.

Extensive repertoire of membrane-bound and soluble dendritic cell-specific ICAM-3-grabbing nonintegrin 1 (DC-SIGN1) and DC-SIGN2 isoforms. Inter-individual variation in expression of DC-SIGN transcripts.

Expression in dendritic cells (DCs) of DC-SIGN, a type II membrane protein with a C-type lectin ectodomain, is thought to play an important role in establishing the initial contact between DCs and resting T cells. DC-SIGN is also a unique type of human immunodeficiency virus-1 (HIV-1) attachment factor and promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We have identified another gene, designated here as DC-SIGN2, that exhibits high sequence homology with DC-SIGN. Here we demonstrate that alternative splicing of DC-SIGN1 (original version) and DC-SIGN2 pre-mRNA generates a large repertoire of DC-SIGN-like transcripts that are predicted to encode membrane-associated and soluble isoforms. The range of DC-SIGN1 mRNA expression was significantly broader than previously reported and included THP-1 monocytic cells, placenta, and peripheral blood mononuclear cells (PBMCs), and there was cell maturation/activation-induced differences in mRNA expression levels. Immunostaining of term placenta with a DC-SIGN1-specific antiserum showed that DC-SIGN1 is expressed on endothelial cells and CC chemokine receptor 5 (CCR5)-positive macrophage-like cells in the villi. DC-SIGN2 mRNA expression was high in the placenta and not detectable in PBMCs. In DCs, the expression of DC-SIGN2 transcripts was significantly lower than that of DC-SIGN1. Notably, there was significant inter-individual heterogeneity in the repertoire of DC-SIGN1 and DC-SIGN2 transcripts expressed. The genes for DC-SIGN1, DC-SIGN2, and CD23, another Type II lectin, colocalize to an approximately 85 kilobase pair region on chromosome 19p13.3, forming a cluster of related genes that undergo highly complex alternative splicing events. The molecular diversity of DC-SIGN-1 and -2 is reminiscent of that observed for certain other adhesive cell surface proteins involved in cell-cell connectivity. The generation of this large collection of polymorphic cell surface and soluble variants that exhibit inter-individual variation in expression levels has important implications for the pathogenesis of HIV-1 infection, as well as for the molecular code required to establish complex interactions between antigen-presenting cells and T cells, i.e. the immunological synapse.

Concordance between the CC chemokine receptor 5 genetic determinants that alter risks of transmission and disease progression in children exposed perinatally to human immunodeficiency virus.

If CC chemokine receptor 5 (CCR5)-dependent mechanisms at the time of initial virus exposure are important determinants of virus entry and disease outcome, then the polymorphisms in CCR5 that influence risk of transmission and disease progression should be similar; this hypothesis was tested in a cohort of 649 Argentinean children exposed perinatally to human immunodeficiency virus type 1 (HIV-1). Two lines of evidence support this hypothesis. First, CCR5 haplotype pairs associated with enhanced risk of transmission were the chief predictors of a faster disease course. Second, some of the haplotype pairs associated with altered rates of transmission and disease progression in children were similar to those that we previously found influenced outcome in European American adults. This concordance suggests that CCR5 haplotypes may serve as genetic rheostats that influence events occurring shortly after initial virus exposure, dictating not only virus entry but, by extension, also the extent of early viral replication.

Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus.

Polymorphisms in CC chemokine receptor 5 (CCR5), the major coreceptor of human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV), have a major influence on HIV-1 transmission and disease progression. The effects of these polymorphisms may, in part, account for the differential pathogenesis of HIV-1 (immunosuppression) and SIV (natural resistance) in humans and non-human primates, respectively. Thus, understanding the genetic basis underlying species-specific responses to HIV-1 and SIV could reveal new anti-HIV-1 therapeutic strategies for humans. To this end, we compared CCR5 structure/evolution and regulation among humans, apes, Old World Monkeys, and New World Monkeys. The evolution of the CCR5 cis-regulatory region versus the open reading frame as well as among different domains of the open reading frame differed from one another. CCR5 cis-regulatory region sequence variation in humans was substantially higher than anticipated. Based on this variation, CCR5 haplotypes could be organized into seven evolutionarily distinct human haplogroups (HH) that we designated HHA, -B, -C, -D, -E, -F, and -G. HHA haplotypes were defined as ancestral to all other haplotypes by comparison to the CCR5 haplotypes of non-human primates. Different human and non-human primate CCR5 haplotypes were associated with differential transcriptional regulation, and various polymorphisms resulted in modified DNA-nuclear protein interactions, including altered binding of members of the NF-kappaB family of transcription factors. We identified novel CCR5 untranslated mRNA sequences that were conserved in human and non-human primates. In some primates, mutations at exon-intron boundaries caused loss of expression of selected CCR5 mRNA isoforms or production of novel mRNA isoforms. Collectively, these findings suggest that the response to HIV-1 and SIV infection in primates may have been driven, in part, by evolution of the elements controlling CCR5 transcription and translation.

Global survey of genetic variation in CCR5, RANTES, and MIP-1alpha: impact on the epidemiology of the HIV-1 pandemic.

Expression of CC chemokine receptor 5 (CCR5), the major coreceptor for HIV-1 cell entry, and its ligands (e.g., RANTES and MIP-1alpha) is widely regarded as central to the pathogenesis of HIV-1 infection. By surveying nearly 3,000 HIV+ and HIV- individuals from worldwide populations for polymorphisms in the genes encoding RANTES, MIP-1alpha, and CCR5, we show that the evolutionary histories of human populations have had a significant impact on the distribution of variation in these genes, and that this may be responsible, in part, for the heterogeneous nature of the epidemiology of the HIV-1 pandemic. The varied distribution of RANTES haplotypes (AC, GC, and AG) associated with population-specific HIV-1 transmission- and disease-modifying effects is a striking example. Homozygosity for the AC haplotype was associated with an increased risk of acquiring HIV-1 as well as accelerated disease progression in European Americans, but not in African Americans. Yet, the prevalence of the ancestral AC haplotype is high in individuals of African origin, but substantially lower in non-Africans. In a Japanese cohort, AG-containing RANTES haplotype pairs were associated with a delay in disease progression; however, we now show that their contribution to HIV-1 pathogenesis and epidemiology in other parts of the world is negligible because the AG haplotype is infrequent in non-Far East Asians. Thus, the varied distribution of RANTES, MIP-1alpha, and CCR5 haplotype pairs and their population-specific phenotypic effects on HIV-1 susceptibility and disease progression results in a complex pattern of biological determinants of HIV-1 epidemiology. These findings have important implications for the design, assessment, and implementation of effective HIV-1 intervention and prevention strategies.

Race-specific HIV-1 disease-modifying effects associated with CCR5 haplotypes.

Genetic variation in CC chemokine receptor 5 (CCR5), the major HIV-1 coreceptor, has been shown to influence HIV-1 transmission and disease progression. However, it is generally assumed that the same CCR5 genotype (or haplotype) has similar phenotypic effects in different populations. To test this assumption, we used an evolutionary-based classification of CCR5 haplotypes to determine their associated HIV-1 disease-modifying effects in a large well-characterized racially mixed cohort of HIV-1-seropositive individuals. We demonstrate that the spectrum of CCR5 haplotypes associated with disease acceleration or retardation differs between African Americans and Caucasians. Also, we show that there is a strong interactive effect between CCR5 haplotypes with different evolutionary histories. The striking population-specific phenotypic effects associated with CCR5 haplotypes emphasize the importance of understanding the evolutionary context in which disease susceptibility genes are expressed.