HIV-1 Coreceptor Activity of CCR5 and Its Inhibition by Chemokines: Independence from G Protein Signaling and Importance of Coreceptor Downmodulation

AbstractHIV-1 infection requires the presence of specific chemokine receptors on CD4+ target cells to enable the fusion reactions involved in virus entry. CCR5 is a major fusion coreceptor for macrophage-tropic HIV-1 isolates. HIV-1 entry and fusion are mediated by the viral envelope glycoprotein (Env) and are inhibited by CCR5 ligands, but the mechanisms are unknown. Here, we test the role of G protein signaling and CCR5 surface downmodulation by two separate approaches: direct inactivation of CCR5 signaling by mutagenesis and inactivation of Gi-type G proteins with pertussis toxin. A CCR5 mutant lacking the last 45 amino acids of the cytoplasmic C-terminus (CCR5306) was created that was expressed on transfected cells at levels comparable to cells expressing CCR5 and displayed normal chemokine binding affinity. CCR5 ligands induced calcium flux and receptor downmodulation in cells expressing CCR5, but not in cells expressing CCR5306. Nevertheless, CCR5 or CCR5306, when coexpressed with CD4, supported comparable HIV-1 Env-mediated cell fusion. Consistent with this, treatment of CCR5-expressing cells with pertussis toxin completely blocked ligand-induced transient calcium flux, but did not affect Env-mediated cell fusion or HIV-1 infection. Also, pertussis toxin did not block chemokine inhibition of Env-mediated cell fusion or HIV-1 infection. However, chemokines inhibited Env-mediated cell fusion less efficiently for CCR5306than for CCR5. We conclude that the C-terminal domain of CCR5 is critical for G protein signaling and receptor downmodulation from the surface, but that neither function is required for CCR5 fusion coreceptor activity. The contrasting phenotypes of CCR5 and CCR5306suggest that coreceptor downmodulation and direct blockage of Env interaction sites both contribute to chemokine inhibition of HIV-1 infection

Counterpoint: Cord blood stem cell therapy for acquired immune deficiency syndrome

Comment on Cord blood stem cell therapy for acquired immune deficiency syndrome. [Stem Cells Dev. 2009

Measles virus gene expression

Note:A recombinant cDNA library corresponding to measles virus (MV)-specific mRNAs has been constructed. Cloned cDNAs were identified representing (a) the monocistronic mRNA encoding the virus hemagglutinin(HA) and (b) the functionally bicistronic P/C mRNA whose translation leads to synthesis of two virus proteins (P and C) by initiation of two different overlapping protein coding reading frames at alternative AUG codons. The entire nucleotide sequence of the virus mRNA encoding the HA protein was determined from cloned cDNAs. The amino acid sequence predicts that the only hydrophobic region of sufficient length to anchor this glycoprotein in the viral membrane is located near the amino-terminus of the protein. Individual cloned cDNAs have been inserted into the genome of adenovirus type 5 replacing the entire ElA and most of the ElB region. […]Nous avons construit une banque de recombinants d'ADNc correspondant aux ARNm spécifiques du virus de la Rougeole (Measles Virus = MV). Les ADNc ainsi clonés ont été identifiés comme représentant (a) l'ARN monocistronique codant pour l'Hémagglutinine virale (HA) et (b) l'ARNm bicistronique P/C dont la traduction au niveau de 2 AUG compris dans des cadres de lecture différents qui se chevauchent conduit à la synthèse des 2 protéines (P et C). La séquence nucléotidique complète de la protéine virale Hemagglutinine (HA) a été déterminée à partir des ADNc clones. Comme le laissait prévoir l'étude de la séquence d'acides aminés, la seule région hydrophobe de longueur suffisante pour permettre l'ancrage de cette protéine a la membrane virale est localisée près de l'extrémité amino-terminale de la protéine. Les différents clones d'ADNc obtenus ont été insères dans le génome d'adénovirus de type 5 remplaçant ainsi la région complète E1A et une partie de la région E1B. […

CCR5Δ32 59537-G/A Promoter Polymorphism Is Associated with Low Translational Efficiency and the Loss of CCR5Δ32 Protective Effects▿

We have recently demonstrated that the CCR5Δ32 protein interacts with CCR5 and CXCR4 and down-modulates their cell surface expression. We have also reported the absence of detectable expression of the truncated CCR5Δ32 protein in four out of six human immunodeficiency virus-infected (HIV+) CCR5−/− individuals. To explain the defect in protein expression in these samples, we cloned and sequenced the promoter regions of the six HIV+ individuals. We have identified several polymorphisms in the CCR5Δ32 promoter region, but these polymorphisms were not associated with significant differences in mRNA levels. Coupled in vitro transcription/translation and polyribosome analysis demonstrated a strong association between a variant genotype designated CCR5Δ32 59537-A/A and a low translation efficiency. Protein analysis indicated that the peripheral blood mononuclear cells from two of the HIV+ CCR5−/− individuals carrying the CCR5Δ32 59537-A/A variant expressed trace amounts of CCR5Δ32 protein compared to the individuals carrying the CCR5Δ32 59537-G/G genotype. The results imply that the absence of CCR5Δ32 protein in two HIV+ individuals is due to a genetic defect in the translation of the protein. Together, these results highlight the importance of the CCR5Δ32 protein as an HIV suppressive factor and provide further insight into the mechanism of the protective effect of the CCR5Δ32 mutation

A Naturally Occurring Splice Variant of CXCL12/Stromal Cell-Derived Factor 1 Is a Potent Human Immunodeficiency Virus Type 1 Inhibitor with Weak Chemotaxis and Cell Survival Activities▿

CXCL12/stromal cell-derived factor 1 is a member of the CXC family of chemokines that plays an important role in hematopoiesis and signals through CXCR4 and CXCR7. Two splice variants of human CXCL12 (CXCL12α and CXCL12β) induce chemotaxis of CXCR4+ cells and inhibit X4 infection. Recent studies described four other novel splice variants of human CXCL12; however, their antiviral activities were not investigated. We constructed and expressed all of the CXCL12 splice variants in Escherichia coli. Recombinant proteins were purified through a His affinity column, and their biological properties were analyzed. All six CXCL12 variants induced chemotaxis of CXCR4+ and CXCR7+ cell lines. Enhancement of survival and replating capacity of human hematopoietic progenitor cells were observed with CXCL12α, CXCL12β, and CXCL12ɛ but not with the other variants. CXCL12γ showed the greatest antiviral activity in X4 inhibition assays and the weakest chemotaxis activity through CXCR4. The order of potency in X4 inhibition assays was as follows: CXCL12γ > CXCL12β > CXCL12α > CXCL12θ > CXCL12ɛ > CXCL12δ. The order of anti-human immunodeficiency virus (HIV) activity was associated with the number of BBXB motifs present in each variant; the most potent inhibitor was CXCL12γ, with five BBXB domains. The results suggest that the different C termini of CXCL12 variants may contain important molecular determinants for the observed differences in antiviral effects and other biological functions. These studies implicate CXCL12γ as a potent HIV-1 entry inhibitor with significantly reduced chemotaxis activity and small or absent effects on progenitor cell survival or replating capacity, providing important insight into the structure-function relationships of CXCL12

CCR5Δ32 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 CCR5Δ32 mutation (CCR5−/−) has rarely been reported, but how the virus overcomes the CCR5Δ32 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 CCR5Δ32 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 CCR5Δ32 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 CCR5Δ32 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 CCR5Δ32 protein conferred resistance to primary X4 but not to lab-adapted X4 virus. These data provide strong support for the hypothesis that the CCR5Δ32 protein actively confers resistance to HIV-1 in vivo and suggest that the loss or reduction of CCR5Δ32 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 CCR5Δ32 protein

Characterization of a cleavage mutant of the measles virus fusion protein defective in syncytium formation

NRC publication: Ye