G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4(+)-transformed cells or pharmacological inhibition of G(alpha)i proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Delta32/Delta32). Expression of wild-type (wt) CCR5 in ccr5 Delta32/Delta32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4(+) cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4(+) T lymphocytes or macrophages.     

Frequency analysis of the delta32ccr5 HIV resistance allele in a medieval plague mass grave

The 32 basepair deletion in the gene for the human chemokine receptor CCR5 (delta32ccr5) conferring resistance against HIV-1 infection is present in Caucasian populations. The mutant allele is believed to have originated by a single mutational event in historic times and to have reached its present population frequency of an average 10 % in Europe through selective pressure by a pathogenic agent. Because of their great impact on European populations, the medieval Plague epidemics have been considered as a possible candidate. To test this hypothesis, we studied the delta32ccr5-frequency in 35 individuals from a mass grave containing victims of the 14th century Plague pandemic in Lübeck, Northern Germany, and compared them to the frequency in a control group from the same burial site, dating from the time before the first Plague pandemic. If the delta32ccr5 allele conferred an at least partial resistance against the medieval Plague, its frequency would be expected to be lower in those that died in the pandemic, than it was in the local population before the arrival of the Plague. The CCR5 locus could be typed successfully for 14 Plague victims and for 20 individuals from the medieval control group. We found a delta32ccr5 allelic frequency of 14.2% and 12.5%, respectively. The difference between these figures is not statistically significant. Furthermore, they are comparable to the delta32ccr5 frequency for nowadays Northern Europe. We therefore conclude that the medieval Plague pandemic has not contributed to an increase in the allelic frequency of the mutant delta32ccr5 allele and that, if there has been a positive selection of this allele, it is likely to have occurred before the 14th century and thus before the arrival of the Plague in Europe.     

Reduced cell surface expression of CCR5 in CCR5Delta 32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration

Macrophage tropic (M-tropic) human immunodeficiency virus (HIV) infection of primary human T cells and macrophages requires optimal cell surface expression of the chemokine receptor CCR5 in addition to CD4. Natural mutations of CCR5 that impair surface expression bestow in the homozygous state complete resistance to M-tropic HIV infection. ccr5Delta32 is the major prototype of such mutants. ccr5Delta32 heterozygosity is associated with delayed onset of AIDS and reduced risk of initial transmission, and this correlates with reduced levels of CCR5 and reduced infectability of CD4+ cells. In addition to gene dosage, sequestration of wild type (WT) CCR5 by mutant protein has been proposed as a mechanism to explain reduced surface expression of CCR5 in cells from ccr5Delta32 and CCR5-893(-) heterozygotes. However, here we demonstrate that a molar excess of ccr5Delta32 or related deletion mutants does not significantly impair the cell surface density of co-expressed WT receptor either in human epithelial cells or Jurkat T cells. Further, ligand-dependent signaling and M-tropic HIV usage of WT receptor are also unaffected. Nascent WT receptor does associate with ccr5Delta32 and related mutant proteins and with other unrelated CC and CXC chemokine receptors under transient labeling conditions. However, using confocal microscopy, we demonstrate that in the steady state, WT and truncated CCR5 proteins segregate into nonoverlapping subcellular compartments. These findings together with the observed and known variability in the cell surface density of CCR5 on quiescent PBLs lead us to conclude that reduced CCR5 gene dosage rather than receptor sequestration is the major determinant of reduced CCR5 expression in cells from ccr5Delta32 heterozygotes.     

Effects of education, vaccination and treatment on HIV transmission in homosexuals with genetic heterogeneity

Genetic studies report the existence of a mutant allele Delta32 of CCR5 chemokine receptor gene at high allele frequencies (approximately 10%) in Caucasian populations. The presence of this allele is believed to provide partial or full resistance to HIV. In this study, we look at the impact of education, temporarily effective vaccines and therapies on the dynamics of HIV in homosexually active populations. In our model, it is assumed that some individuals possess one or two mutant alleles (like Delta32 of CCR5) that prevent the successful invasion or replication of HIV. Our model therefore differentiates by genetic and epidemiological status and naturally ignores the reproduction process. Furthermore, HIV infected individuals are classified as rapid, normal or slow progressors. In this complex setting, the basic reproductive number R0 is derived in various situations. The separate or combined effects of therapies, education, vaccines, and genetic resistance are analyzed. Our results support the conclusions of Hsu Schmitz that some integrated intervention strategies are far superior to those based on a single approach. However, treatment programs may have effects which counteract each other, as may genetic resistance.     

Mutational analysis of the CCR5 and CXCR4 genes (HIV-1 co-receptors) in resistance to HIV-1 infection and AIDS development among intravenous drug users

We analysed a group of Spanish intravenous drug users and controls to determine the role of mutations at the chemokine receptor-5/HIV-1 cofactor (CCR5), previously implicated in resistance to HIV-1 infection, and CXCR4 genes in susceptibility to HIV-1 infection. The complete coding sequence of both genes was amplified by the polymerase chain reaction from genomic DNA of 50 seropositive slow progressors and 10 long-term non-progressors, and analysed by the single-strand conformation polymorphism technique in a search for mutations. No mutation in CXCR4 was found, and Δccr5 was the only mutation identified at the CCR5 gene. We genotyped (Δccr5 allele) 150 HIV-1+ intravenous drug users and 250 healthy controls from the same population (Asturias, Northern Spain). Patients were divided into rapid progressors, presenting an event indicating progression to the acquired immunodeficiency syndrome (AIDS) in the 2 years after infection (100 patients), and slow progressors, remaining asymptomatic for 2–10 years (50 patients). The frequencies of the Δccr5 allele were 0.105 and 0.040 in controls and HIV-1+ patients, respectively. Eighteen per cent of the controls (45/250) and 8% (12/150) of the patients carried the Δccr-5 allele (P=0.013). The frequency of Δccr5 carriers among rapid and slow disease progressors was 3 and 15%, respectively. A highly significant difference was found between rapid progressors and controls (P=0.0014). No patient (0/150) was Δccr5 homozygous compared with 1% among controls. Thus, the Δccr5 allele (the only CCR5 mutation found in our HIV-1 patients) was rare among seropositive intravenous drug users, suggesting that the absence of this mutation confers an advantage to the virus when infecting cells in vivo. In addition, patients carrying the Δccr5 allele tend to show a slow progression towards HIV-1-related disease, remaining asymptomatic for longer periods of time. Received: 15 October 1997 / Accepted: 12 January 1998     

HIV neutralizing IgA in exposed seronegative subjects recognise an epitope within the gp41 coiled-coil pocket

Human immunodeficiency virus (HIV)-specific IgA can be detected in cervical secretions, saliva, and sera of HIV-infected and HIV-uninfected individuals with a known exposure to the virus. IgA from HIV-uninfected exposed seronegative individuals (ESN) neutralize in vitro primary strains of HIV-1. We analyzed the epitopes of HIV recognized by serum HIV-specific IgA of ESN individuals to identify the antigenic correlates of HIV neutralization in exposed-uninfected subjects, and to verify whether different epitopes would be recognized by HIV-specific IgA of ESN and of HIV-infected patients. Results confirmed that HIV-neutralizing IgA are detected in sera of ESN and showed that neutralization of primary HIV strains is mediated by the recognition of different epitopes in HIV-infected patients and ESN. Thus, whereas IgA of HIV+ individuals recognize epitopes expressed both within gp120 and gp41, IgA of ESN exclusively bind to gp41-expressed epitopes. Epitope mapping revealed that the epitope recognized by serum IgA of ESN on gp41 is restricted to aa 581-584 (LQAR) and corresponds to coiled coil pocket in the alpha helic region. In contrast, the epitope seen by IgA of HIV-infected patients on gp41 is identified by two regions; the first is contained within the cystein loop (aa 589-618), the second correspond to C terminal region in the extra membrane region of gp 41 (aa 642-673). Thus, we have identified and characterized the epitopes that mediate neutralization of HIV in individuals in whom infection does not occur despite multiple exposures to the virus. These results have important implications for the development of a new therapy against HIV infection.     

CC chemokine receptor 5 cell-surface expression in relation to CC chemokine receptor 5 genotype and the clinical course of HIV-1 infection.

CCR5 cell-surface expression was studied in relation to CCR5 genotype and clinical course of HIV-1 infection. HIV-1 infected CCR5+/+ individuals had higher percentages of CCR5-expressing CD4+ T cells as compared with HIV-1-infected CCR532/+ individuals. For both genotypic groups, the percentages of CCR5-expressing cells were higher than for the uninfected counterparts (CCR5+/+, HIV+ 28% and HIV- 15% (p < 0.0001); CCR532/+, HIV+ 21% and HIV- 10% (p = 0.001), respectively). In HIV-1-infected individuals, high percentages of CCR5-expressing cells were associated with low CD4+ T cell numbers (p = 0.001), high viral RNA load in serum (p = 0.046), and low T cell function (p = 0.054). As compared with nonprogressors with similar CD4+ T cell numbers, individuals who did progress to AIDS had a higher percentage of CCR5-expressing CD4+ T cells (32% vs 21% (p = 0.002). Longitudinal analysis of CCR5+/+ individuals revealed slight, although not statistically significant, increases in CCR5-expressing CD4+ T cells and CD4+ T cell subsets characterized by the expression of CD45 isoforms, during the course of HIV-1 infection. Preseroconversion, the percentage of CCR5-expressing CD4+ T cells was higher in individuals who subsequently developed AIDS (28%) than in those who did not show disease progression within a similar time frame (20%; p = 0.059). Our data indicate that CCR5 expression increases with progression of disease, possibly as a consequence of continuous immune activation associated with HIV-1 infection. In turn, CCR5 expression may influence the clinical course of infection.     

First report of a healthy Indian heterozygous for Δ32 mutant of HIV-1 co-receptor-CCR5 gene

The β-chemokine receptor, CCR5, is a major co-receptor for macrophage tropic non-syncytia-inducing isolates of HIV-1. Recently a 32 bp homozygous deletion in the coding region of CCR5 has been reported in a very small percentage (<1%) of Caucasian individuals who remain uninfected, despite multiple exposure to the wild-type virus. This mutant allele in the heterozygous form (CCR5/32 ccr5) was readily detected in a normal unrelated Caucasian population of European heritage with varying frequencies (13–20%). However, when a large number of the non-Caucasian population (261 Africans and 423 Asians) were screened for the presence of this deleted allele, not a single case of either homozygous or heterozygous mutant for Δ32 allele of CCR5 was detected. We screened 100 normal individuals and found a single heterozygous case with an identical 32 bp deletion in CCR5 gene reported earlier, the rest possessed wild-type alleles. This deleted gene was inherited in Mendelian fashion among the family members of this individual. Thus, the frequency of this deleted allele in India among unrelated normal individuals is likely to be very low (<1%). We observed a moderate transdominant effect of this mutant allele in a fusion assay. Finally, we show a significant inhibition of fusion of cell membranes when the 176-bp region of CCR5 was used as an antisense.     

Frequency of the CCR5-delta32 mutation in the Atlantic island populations of Madeira, the Azores, Cabo Verde, and São Tomé e Príncipe

There is evidence that the CCR5-delta32 mutation confers protection against HIV-1 infection to homozygous individuals. It is believed that this mutation spread through Europe with the Vikings and that it has been subjected to positive selection, leading to a high frequency in Europe (approximately 10%). We carried out the present study to determine the 32-bp deletion allele and genotype frequencies of the CCR5 gene (CCR5-delta32) in the Atlantic island populations of Madeira, the Azores, Cabo Verde, and S?o Tomé e Principe. These Atlantic archipelagos were all colonized by the Portuguese in the 15th and 16th centuries, but the latter two received most of their settlers from the West African coast. The frequency of the CCR5-delta32 mutation varies between 0% in S?o Tomé e Príncipe and 16.5% in the Azores. The Azores Islands have one of the highest frequencies of homozygotes found in Europe (4.8%). There are significant differences (P < 0.05) between some of these populations, for example, between S?o Tomé e Príncipe and Cabo Verde, and even within populations (e.g., Portugal, Madeira, and the Azores).     

Role for CCR5Delta32 protein in resistance to R5, R5X4, and X4 human immunodeficiency virus type 1 in primary CD4+ cells

CCR5Delta32 is a loss-of-function mutation that abolishes cell surface expression of the human immunodeficiency virus (HIV) coreceptor CCR5 and provides genetic resistance to HIV infection and disease progression. Since CXCR4 and other HIV coreceptors also exist, we hypothesized that CCR5Delta32-mediated resistance may be due not only to the loss of CCR5 function but also to a gain-of-function mechanism, specifically the active inhibition of alternative coreceptors by the mutant CCR5Delta32 protein. Here we demonstrate that efficient expression of the CCR5Delta32 protein in primary CD4(+) cells by use of a recombinant adenovirus (Ad5/Delta32) was able to down-regulate surface expression of both wild-type CCR5 and CXCR4 and to confer broad resistance to R5, R5X4, and X4 HIV type 1 (HIV-1). This may be important clinically, since we found that CD4(+) cells purified from peripheral blood mononuclear cells of individuals who were homozygous for CCR5Delta32, which expressed the mutant protein endogenously, consistently expressed lower levels of CXCR4 and showed less susceptibility to X4 HIV-1 isolates than cells from individuals lacking the mutation. Moreover, CD4(+) cells from individuals who were homozygous for CCR5Delta32 expressed the mutant protein in five of five HIV-exposed, uninfected donors tested but not in either of two HIV-infected donors tested. The mechanism of inhibition may involve direct scavenging, since we were able to observe a direct interaction of CCR5 and CXCR4 with CCR5Delta32, both by genetic criteria using the yeast two-hybrid system and by biochemical criteria using the coimmunoprecipitation of heterodimers. Thus, these results suggest that at least two distinct mechanisms may account for genetic resistance to HIV conferred by CCR5Delta32: the loss of wild-type CCR5 surface expression and the generation of CCR5Delta32 protein, which functions as a scavenger of both CCR5 and CXCR4.     

RNA-binding protein Khd1 and Ccr4 deadenylase play overlapping roles in the cell wall integrity pathway in Saccharomyces cerevisiae

The Saccharomyces cerevisiae RNA-binding protein Khd1/Hek2 associates with hundreds of potential mRNA targets preferentially, including the mRNAs encoding proteins localized to the cell wall and plasma membrane. We have previously revealed that Khd1 positively regulates expression of MTL1 mRNA encoding a membrane sensor in the cell wall integrity (CWI) pathway. However, a khd1Δ mutation has no detectable phenotype on cell wall synthesis. Here we show that the khd1Δ mutation causes a severe cell lysis when combined with the deletion of the CCR4 gene encoding a cytoplasmic deadenylase. We identified the ROM2 mRNA, encoding a guanine nucleotide exchange factor (GEF) for Rho1, as a target for Khd1 and Ccr4. The ROM2 mRNA level was decreased in the khd1Δ ccr4Δ mutant, and ROM2 overexpression suppressed the cell lysis of the khd1Δ ccr4Δ mutant. We also found that Ccr4 negatively regulates expression of the LRG1 mRNA encoding a GTPase-activating protein (GAP) for Rho1. The LRG1 mRNA level was increased in the ccr4Δ and khd1Δ ccr4Δ mutants, and deletion of LRG1 suppressed the cell lysis of the khd1Δ ccr4Δ mutant. Our results presented here suggest that Khd1 and Ccr4 modulate a signal from Rho1 in the CWI pathway by regulating the expression of RhoGEF and RhoGAP.     

CCR5-reactive antibodies in seronegative partners of HIV-seropositive individuals down-modulate surface CCR5 in vivo and neutralize the infectivity of R5 strains of HIV-1 In vitro

Exposure to HIV does not necessarily result in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1 beta (Mip1 beta) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1 beta binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89-102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1beta-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1beta in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.     

Homozygosity for Pc 1 Duarte-like protein in primates and other animals.

Pc 1 Duarte is a mutant brain protein present in 32% of the normal human population with 2.6% being homozygous. Preliminary studies suggest an increased frequency of this mutation in individuals with affective disease. To determine if this mutant was present in primates, 32 fetal (wild)-born baboons were examined. All 32 were homozygous for a Pc 1 Duarte-like protein.     

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.     

Polymorphisms of diacylglycerol acyltransferase 2 gene and their relationship with growth traits in goats

Diacylglycerol acyltransferase (DGAT) plays a critical role in the synthesis of triacylglycerol. In this study, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variations of DGAT-2 gene in 299 goats from three breeds (Boer goat, Chinese Xuhuai white goat and Chinese Haimen goat). Three fragments of DGAT-2 gene were investigated, only exon 3 of DGAT-2 gene showed polymorphism. The alignment between nucleotide sequences of NM_205793.2 in GenBank and the sequencing results of three PCR products with different patterns revealed that there was one mutation (A????G) in exon 3 of DGAT-2 gene, which resulted in amino acid change (Lys????Arg) and constructed two genotypes (AA, AB). The frequencies of allele A and genotype AA were dominant in all three breeds. And there was no significant difference for genotypic and allelic frequencies among the three breeds. The genotype distributions were in good agreement with Hardy?CWeinberg equilibrium (P?>?0.05) in each breed. Significant statistical differences were only found in withers heights (P?<?0.05) in Xuhuai goat between genotypes. The results indicated that individuals with genotype AA were significantly higher than those of individuals with genotype AB in withers height (P?<?0.05). No polymorphism was detected in the intron 3, exon 8 and 3?? flanking region. So we suggested that DGAT-2 gene had the close relationship with growth traits in goats. And this mutation could be used as a perfect molecular marker for marker-assisted selection (MAS) in animal genetics and breeding.     

Plant growth promotion and Penicillium citrinum

Background

Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months.

Results

Half of the patients were homosexual men. Only 4/36 (11%) patients were HBV DNA negative. As expected for a Brazilian group, genotypes A (24/32 positive individuals, 75%), D (3/32, 9.3%) and F (1/32, 3%) were present. One sample was from genotype C, which is a genotype rarely found in Brazil. Three samples were from genotype G, which had not been previously detected in Brazil. Lamivudine resistance mutations were identified in 20/32 (62%) HBV DNA positive samples. Mean HBV loads of patients with and without lamivudine resistance mutations were not very different (2.7 × 10⁷ and 6.9 × 10⁷ copies/mL, respectively). Fifteen patients showed the L180M/M204V lamivudine resistant double mutation. The triple mutant rt173V/180M/204V, which acts as a vaccine escape mutant, was found in two individuals. The three isolates of genotype G were entirely sequenced. All three showed the double mutation L180M/M204V and displayed a large genetic divergence when compared with other full-length genotype G isolates.

Conclusion

A high (55%) proportion of patients submitted to long term lamivudine therapy displayed resistant mutations, with elevated viral load. The potential of transmission of such HBV mutants should be monitored. The identification of genotypes C and G, rarely detected in South America, seems to indicate a genotype distribution different to that observed in non treated patients. Disparities in routes of transmission (genotype G seems to be linked to homosexual behavior) and in pathogenic properties (genotype C is very aggressive) among HBV genotypes may explain the presence of rare genotypes in the present work.     

Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases

HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.     

CCR5Delta32 protein expression and stability are critical for resistance to human immunodeficiency virus type 1 in vivo

Human immunodeficiency virus type 1 (HIV-1) infection of individuals carrying the two alleles of the CCR5Delta32 mutation (CCR5(-/-)) has rarely been reported, but how the virus overcomes the CCR5Delta32 protective effect in these cases has not been delineated. We have investigated this in 6 infected (HIV(+)) and 25 HIV(-) CCR5(-/-) individuals. CD4(+) T lymphocytes isolated from HIV(-) CCR5(-/-) peripheral blood mononuclear cells (PBMCs) showed lower levels of CXCR4 expression that correlated with lower X4 Env-mediated fusion. Endogenous CCR5Delta32 protein was detected in all HIV(-) CCR5(-/-) PBMC samples (n = 25) but not in four of six unrelated HIV(+) CCR5(-/-) PBMC samples. Low levels were detected in another two HIV(+) CCR5(-/-) PBMC samples. The expression of adenovirus 5 (Ad5)-encoded CCR5Delta32 protein restored the protective effect in PBMCs from three HIV(+) CCR5(-/-) individuals but failed to restore the protective effect in PBMCs isolated from another three HIV(+) CCR5(-/-) individuals. In the latter samples, pulse-chase analyses demonstrated the disappearance of endogenous Ad5-encoded CCR5Delta32 protein and the accumulation of Ad5-encoded CCR5 during the chase periods. PBMCs isolated from CCR5(-/-) individuals showed resistance to primary X4 but were readily infected by a lab-adapted X4 strain. Low levels of Ad5-encoded CCR5Delta32 protein conferred resistance to primary X4 but not to lab-adapted X4 virus. These data provide strong support for the hypothesis that the CCR5Delta32 protein actively confers resistance to HIV-1 in vivo and suggest that the loss or reduction of CCR5Delta32 protein expression may account for HIV-1 infection of CCR5(-/-) individuals. The results also suggest that other cellular or virally induced factors may be involved in the stability of CCR5Delta32 protein.