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Figure 14.3 Overview of disease progression in untreated HIV infection. Circulating levels of CD4+ cells are shown during the initial acute viraemic phase, which is usually associated with an acute influenza-like illness, followed by an asymptomatic phase of variable duration at the end of which opportunistic infections become more common as the CD4 T cell count falls (<500 cells/Ml). AIDS typically develops when the CD4 count is <200 cells/Ml. Reprinted with permission from Janeway et al. (2005) .

Box 14.3 Variants of HIV, coreceptor specificity, and disease progression

Variants of HIV have been described specific to particular cell types and coreceptors, which correlate with disease progression (Connor et al. 1997). The terminology can be confusing and related initially to the ability of the virus to infect particular cells in vitro. The main variants of HIV in vivo infect T cells, dendritic cells, and macrophages using the CCR5 coreceptor and are found in newly infected people: they were initially described as macrophage tropic (M-tropic) as in vitro they infected macrophages but not T cells, but are now usually described as R5 based on their coreceptor use. Later in the course of disease, lymphocyte tropic variants are much more common; in vivo they are restricted to infecting CD4+ T cells using the CXCR4 receptor and are known as X4 viruses (previously as T-tropic based on growth in vitro in T cell lines). This phenotypic switch between R5 and X4 is associated with faster disease progression.

amino acids, and causes it to fail to be expressed at the cell surface or be detectable in the cytoplasm (Fig. 14.5). This deletion, which has since been dubbed the CCR5 A32 allele, was confirmed on sequencing genomic DNA of EU2 and EU3. When the investigators looked in more detail at the group of 25 individuals, one further individual was noted to be highly resistant to viral infection and that person was also, like EU2 and EU3, homozygous for CCR5 A32. The polymorphism was found to be relatively common among Europeans with 24 of 122 individuals heterozygous for CCR5 A32, giving a minor allele frequency of 0.098. The resistance to HIV-1 was specific to R5 viruses found in primary infective isolates. These account for more than 95% of incident HIV-1 infections: the cells were, however, fully infectable by X4 strains of HIV-1 that use a different coreceptor, CXCR4.

The role of CCR5 A32 in resistance to HIV-1 infection was independently demonstrated and published by Samson and colleagues (1996) in the journal Nature and Dean et al. (1996) in Science at the same time as Liu et al. (1996) published their findings in the journal Cell. Samson and coworkers had found the CCR5 A32 deletion by sequencing CCR5 in three HIV-1 infected patients in whom disease progression was slow. They postulated that a 10 bp repeat region flanking the deletion may have promoted the recombination event resulting in the A32 deletion. They noted a similar minor allele frequency in Europeans as had Liu and coworkers, at 0.09, but strikingly the deletion was not found in a cohort of individuals from West and Central Africa or from Japan. The allele frequency in a Caucasian cohort of 723 seropositive patients from Belgian and Parisian hospitals was significantly lower than among the general population, at 0.054, but no individuals homozygous for the CCR5 A32 deletion were present among this cohort infected with HIV-1.

Dean et al. had mapped the genetic locus to chromosome 3p21 and then identified the A32 deletion (Dean et al. 1996). They reported that homozygosity for the deletion occurred only among HIV-1 antibody negative individuals and not among HIV-1 infected subjects. Similar results were found by Huang et al. with significant overrepresentation of CCR5 A32 deletion in at-risk but uninfected people (3.6% of 446 Caucasian subjects) and significant underrepresentation among infected people (Huang et al. 1996). In fact among the 2741 HIV-1 infected individuals reported by the three independent cohort studies in 1996, no subjects were found who were homozygous for the CCR5 A32 deletion (Dean et al. 1996; Samson et al. 1996; Huang et al. 1998).

What of those individuals who were heterozygous for the CCR5 A32 deletion, possessing one 'normal' allele and one bearing the deletion? Analysis of lymphocytes heterozygous for CCR5 A32 showed very reduced expression of CCR5 (Wu et al. 1997) with subsequent analysis demonstrating this was due to a gene dosage effect rather than receptor sequestration (Venkatesan et al. 2002). The frequency of individuals heterozygous for the CCR5 A32 deletion was reported to be 35% lower in a cohort

Figure 14.4 HIV virion, CD4 receptor, and CCR5 coreceptor. (A) The HIV virion includes an outer lipoprotein membrane made up of 72 glycoprotein complexes. Individual complexes comprise an external glycoprotein gp120 and a transmembrane protein gp41. The CD4 molecule binds to gp120, 'docking' the HIV-1 virion surface envelope protein. (B) Subsequently, interaction between gp120 and the coreceptor protein CCR5 leads to a change in conformation of the R5 viral gp41 protein which penetrates the cell membrane. The viral envelope then fuses with the host cell membrane allowing entry of the viral core into the cell cytoplasm. (C) The chemokine ligands Mip1 a (macrophage inflammatory protein-1 alpha), Mip1 p, or RANTES (regulated upon activation T cell expressed and secreted), which normally bind to CCR5, block the infection of cells by R5 HIV-1. During the course of HIV-1 infection in most Caucasian populations where HIV clade B is predominant, a change in coreceptor preference occurs from CCR5 to CXCR4 due to a mutational shift in the gene encoding the envelope glycoproteins. For CXCR4, the normal ligand is SDF-1 (stromal cell-derived factor 1) and this protein will inhibit entry of X4 HIV-1. Redrawn by permission from Macmillan Publishers Ltd: Nature Genetics, copyright 2004, and from Dr Robert Doms (University of Pennsylvania).

of Caucasian individuals infected with HIV-1 than in an uninfected cohort (Samson et al. 1996). Protection from infection was also reported among high risk seronegative groups (Marmor et al. 2001). In contrast, other studies found no clear evidence of protection from infection but rather suggested or clearly demonstrated a role in disease progression (Dean et al. 1996; Huang et al. 1996; Zimmerman et al. 1997). Heterozygosity has been associated with a slower rate of progression, being present in some cohorts at twice the frequency in those surviving

Figure 14.5 Structure of the CCR5 coreceptor. (A) The CCR5 chemokine coreceptor showing its seven transmembrane domain G protein-coupled structure. (B) Non-functional CCR5 A32 protein lacks the final three transmembrane segments and the regions involved in G coupling. Redrawn from McNicholl et al. (1997).

Figure 14.5 Structure of the CCR5 coreceptor. (A) The CCR5 chemokine coreceptor showing its seven transmembrane domain G protein-coupled structure. (B) Non-functional CCR5 A32 protein lacks the final three transmembrane segments and the regions involved in G coupling. Redrawn from McNicholl et al. (1997).

greater than 10 years with infection compared to rapid progressors (Dean et al. 1996). There is evidence that the consequences of heterozygosity for the CCR5 A32 deletion depends on the nature of the other allele, with protective or deleterious effects dependent on the particular CCR5 haplotype present on that partner allele (Gonzalez et al. 1999; Mangano et al. 2001; Hladik 2005) (Section 14.2.2). It is also worth noting that the CCR5 A32 deletion, in the homozygous or heterozygous state, explains only a small proportion of cases of highly exposed individuals who are persistently seronegative for HIV-1.

Among populations from Asia or Africa, CCR5 A32 is extremely rare (Su et al. 2000). Resequencing of the coding region of CCR5 in different ethnic groups identified a number of rare nonsynonymous polymorphisms including the CCR5-893 variant, which was restricted to Japanese and Chinese individuals (Ansari-Lari et al. 1997). This polymorphism was associated with substantially reduced coreceptor activity and cell surface expression as it prematurely terminates translation of the CCR5 protein through a frameshift at codon 299, causing the receptor to lack a cytoplasmic tail. The polymorphism is rare and the relationship to HIV-1 infection unclear; in a small case-control study of HIV-1 infected versus uninfected individuals in Japan, no significant effect was found (Shioda et al. 2001).

A variety of other mechanisms have been proposed to explain why highly exposed individuals should be persistently seronegative. These include the cellular and humoral immune response acting at a systemic and mucosal level, production of soluble suppressive factors, and coreceptor mutations (Kulkarni et al. 2003).

14.2.2 Haplotypic structure of the CCR5 locus: evolutionary insights, variation between ethnic groups, and relationship to HIV-1 disease susceptibility

The CCR5 gene and flanking regions is highly polymorphic. Attention was focused initially on the coding gene sequences but it became evident that understanding the extent of variation was important in resolving underlying associations with disease. This is made more difficult by the finding of significant linkage disequilibrium across the locus, including the neighbouring gene CCR2 - a site itself associated with delayed progression to AIDS (Box 14.4). Could there be functional diversity elsewhere in the region of CCR5 responsible for such an observation, perhaps modulating CCR5 gene expression?

The promoter region of CCR5 was analysed among AIDS patients by Martin and colleagues who demonstrated four common allelic variants with at least ten SNPs. Strikingly, one CCR5 promoter haplotype, denoted CCR5 P1, which was present in over 12% of Caucasians, was significantly associated with faster progression to AIDS (Martin et al. 1998). In the same year, 1998, McDermott and coworkers reported a screen of allelic variation in a region of the CCR5 promoter among blood donors which showed a specific SNP was associated with rate of progression to AIDS (McDermott et al. 1998). Further work

Box 14.4 CCR2 polymorphism and disease progression in HIV-1 infection

The chemokine receptor gene CCR2 lies within 10 kb of the CCR5 gene on chromosome 3. On certain cell types, CCR2 acts as a minor coreceptor for HIV-1. Screening for polymorphism in CCR2 identified a G to A single nucleotide polymorphism (SNP) that resulted in a substitution of valine for isoleucine at position 64 (rs1799864, c.190G>A, p.V64I; the rarer allele of the SNP was dubbed CCR2-64I) resulting in a conservative change in the first transmembrane domain of the protein (Smith et al. 1997). Possession of one or two copies of CCR2-64I did not appear to modulate susceptibility to infection with HIV-1 but was associated with a delay in disease progression. The effect appeared independent of CCR5 A32. The disease association was confirmed in an independent study but this study found evidence of linkage between CCR2-64I and a SNP in the CCR5 regulatory region (Kostrikis et al. 1998). The effects of CCR2-64I appeared most significant early in infection (Mulherin et al. 2003) before the emergence of X4 strains (van Rij et al. 1998b). The mechanism whereby CCR2-64I may exert a functional effect remains unclear; it may be serving as a marker for a coinherited variant, for example in CCR5, or be exerting a direct effect as there is some evidence that it may act to downregulate surface expression of CCR5 (Nakayama et al. 2004).

was needed to delineate the haplotypic structure of the locus, understand the regulation of gene expression, and determine the functional significance of noncoding DNA sequence polymorphism. Regulation of CCR5 gene expression was found to be complex, with at least two promoters implicated and multiple alternatively spliced isoforms (Mummidi et al. 1997, 2000).

Sequencing of the CCR5 open reading frame (ORF) and c/s-regulatory region for 60 human alleles defined 27 haplotypes while sequencing of 43 non-human primates (and genotyping of an additional 40) allowed Mummidi and colleagues to define the ancestral haplotype and to resolve an evolutionary framework from which a rooted phylogenetic network could be constructed (Mummidi et al. 2000). This allowed seven distinct clusters of haplotypes to be defined, denoted as haplogroups HHA to HHG (Fig. 14.6). HHA included the ancestral haplotype, while the CCR5 A32 deletion was found in haplogroup HHG. The previously reported allele CCR5 P1 was found to be a mixture of HHE, HHF*1 and HHG*1, illustrating the value of using an evolutionary framework to organize and classify haplotypes.

This classification is important in many ways. It allows the variability between frequencies of haplotypes among human populations to be assessed, and indeed significant variability between populations is found (Fig. 14.7)

(Gonzalez et al. 1999, 2001). The ancestral haplotype HHA was found to be most common in people of African descent with the highest frequency among Mbuti and Biaka pygmies. Haplogroups HHB and HHD were noted to be African-specific, while HHC haplotypes had a higher frequency in Caucasians than African Americans.

Does consideration of haplogroups provide a rational basis for analysis of any association between genetic variation at CCR5 and disease? The picture remains unclear but certain haplogroups or allelic pairs of haplogroups are reported to show significant associations with disease progression. The pairing of HHA and HHF*2 was the most common found in Africans, and was associated with slower disease progression in an African American cohort, while HHF*1 was associated with accelerated progression to AIDS across populations groups (Gonzalez et al. 1999, 2001). It has also been shown that individuals homozygous for HHE have increased risk of acquisition and disease progression (Gonzalez et al. 1999; Mangano et al. 2001). Consideration of haplogroups also facilitates the definition of putative regulatory variants in terms of gene regulation. For example, the HHA haplotype showed the lowest level of transcriptional activity in reporter gene constructs and there was evidence of haplotype-specific protein-DNA binding by transcription factors such as NFkB (Mummidi et al. 2000).

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