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R5X4 HIV-1 coreceptor use in primary target cells: implications for coreceptor entry blocking strategies.

Loftin LM, Kienzle M, Yi Y, Collman RG - J Transl Med (2011)

Bottom Line: However, these algorithms were developed to predict coreceptor use in cell lines and not primary cells and, furthermore, are not highly accurate for some classes of viruses.This article focuses on R5X4 HIV-1, the earliest CXCR4-using variants, reviewing the pattern of coreceptor use on primary CD4+ lymphocytes and macrophages, the relationship between primary cell coreceptor use and the two principal approaches to coreceptor analysis (genetic prediction and indicator cell phenotyping), and the implications of primary cell coreceptor use by these strains for treatment with a new class of small molecule antagonists that inhibit CCR5-mediated entry.These are important questions to consider given the development of new CCR5 blocking therapies and the prognosis associated with CXCR4 use.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, 522 Johnson Pavilion, 36th & Hamilton Walk, Philadelphia, PA 19104-6060, USA.

ABSTRACT
Entry coreceptor use by HIV-1 plays a pivotal role in viral transmission, pathogenesis and disease progression. In many HIV-1 infected individuals, there is an expansion in coreceptor use from CCR5 to include CXCR4, which is associated with accelerated disease progression. While targeting HIV-1 envelope interactions with coreceptor during viral entry is an appealing approach to combat the virus, the methods of determining coreceptor use and the changes in coreceptor use that can occur during disease progression are important factors that may complicate the use of therapies targeting this stage of HIV-1 replication. Indicator cells are typically used to determine coreceptor use by HIV-1 in vitro, but the coreceptors used on these cells can differ from those used on primary cell targets. V3 based genetic sequence algorithms are another method used to predict coreceptor use by HIV-1 strains. However, these algorithms were developed to predict coreceptor use in cell lines and not primary cells and, furthermore, are not highly accurate for some classes of viruses. This article focuses on R5X4 HIV-1, the earliest CXCR4-using variants, reviewing the pattern of coreceptor use on primary CD4+ lymphocytes and macrophages, the relationship between primary cell coreceptor use and the two principal approaches to coreceptor analysis (genetic prediction and indicator cell phenotyping), and the implications of primary cell coreceptor use by these strains for treatment with a new class of small molecule antagonists that inhibit CCR5-mediated entry. These are important questions to consider given the development of new CCR5 blocking therapies and the prognosis associated with CXCR4 use.

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Coreceptor use by R5X4 HIV-1 on indicator cell lines and primary CD4+ lymphocytes. (A) Coreceptor use on U87 indicator cells by R5X4 HIV-1. U87 CD4, CD4/CCR5 or CD4/CXCR4 cells were infected with HIV-1 pseudotype viruses (5ng p24 Gag antigen). Three days after infection, cells were lysed and luciferase activity was measured.  Results are means  sem of two experiments performed in triplicate.  (B) Coreceptor-specific entry into CD4+ T lymphocytes.  Purified CD4+ lymphocytes were isolated by negative selection, stimulated with PHA for 3 days, infected with 5ng of HIV-1 pseudotype viruses in the absence or presence of coreceptor antagonists as described in Figure 1, maintained with IL-2 and lysed four days later for measurement of luciferase expression.  Results represent normalized infection through each coreceptor and are means +/- sem of infections done using cells from three different donors, each performed in duplicate.
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Figure 2: Coreceptor use by R5X4 HIV-1 on indicator cell lines and primary CD4+ lymphocytes. (A) Coreceptor use on U87 indicator cells by R5X4 HIV-1. U87 CD4, CD4/CCR5 or CD4/CXCR4 cells were infected with HIV-1 pseudotype viruses (5ng p24 Gag antigen). Three days after infection, cells were lysed and luciferase activity was measured. Results are means sem of two experiments performed in triplicate. (B) Coreceptor-specific entry into CD4+ T lymphocytes. Purified CD4+ lymphocytes were isolated by negative selection, stimulated with PHA for 3 days, infected with 5ng of HIV-1 pseudotype viruses in the absence or presence of coreceptor antagonists as described in Figure 1, maintained with IL-2 and lysed four days later for measurement of luciferase expression. Results represent normalized infection through each coreceptor and are means +/- sem of infections done using cells from three different donors, each performed in duplicate.

Mentions: R5X4 variants have the capacity to use both CCR5 and CXCR4 on macrophages and indicator cell lines (Figs 1 and 2A), but in contrast, the pattern of coreceptor use by R5X4 HIV-1 on CD4+ lymphocytes from peripheral blood is quite different from that seen on those two cell types. Initial reports using a similar coreceptor blocking strategy and prototype strains showed that R5X4 viruses used CXCR4 on lymphocytes but CCR5 use was minimal, and lymphocyte CCR5 use by R5X4 isolates was markedly impaired relative to infection by R5 viruses [11]. Furthermore, unlike macrophages, infection mediated by CXCR4 alone was equivalent to infection when both coreceptors were present, suggesting no additional contribution of CCR5 in the presence of the CXCR4 pathway. More recently, using an expanded panel of R5X4 Envs from diverse sources, we found that that some R5X4 viruses do possess the ability to use CCR5 for entry into CD4+ lymphocytes [29]. A range of CCR5 use was observed among these R5X4 strains, with CCR5 making virtually no contribution to infection by some strains while nearly half the total amount of entry could be mediated by CCR5 for other strains. However, despite more robust CCR5 use by some clones, CXCR4 remained the predominant coreceptor used on CD4+ lymphocytes for all R5X4 viruses. CD4+ lymphocytes in blood are a mixture of different subsets, with CXCR4 expressed on a larger percentage of cells than CCR5 [12,30,31]. Consequently, CXCR4 is likely to be the predominate coreceptor used by the majority of R5X4 strains. Strikingly, there was consistently no difference between infection of unblocked CD4+ T lymphocytes and infection mediated by CXCR4 alone, confirming that even though both pathways can be used, when CXCR4 is available there is no additional contribution to infection made by CCR5 (Fig. 2B).


R5X4 HIV-1 coreceptor use in primary target cells: implications for coreceptor entry blocking strategies.

Loftin LM, Kienzle M, Yi Y, Collman RG - J Transl Med (2011)

Coreceptor use by R5X4 HIV-1 on indicator cell lines and primary CD4+ lymphocytes. (A) Coreceptor use on U87 indicator cells by R5X4 HIV-1. U87 CD4, CD4/CCR5 or CD4/CXCR4 cells were infected with HIV-1 pseudotype viruses (5ng p24 Gag antigen). Three days after infection, cells were lysed and luciferase activity was measured.  Results are means  sem of two experiments performed in triplicate.  (B) Coreceptor-specific entry into CD4+ T lymphocytes.  Purified CD4+ lymphocytes were isolated by negative selection, stimulated with PHA for 3 days, infected with 5ng of HIV-1 pseudotype viruses in the absence or presence of coreceptor antagonists as described in Figure 1, maintained with IL-2 and lysed four days later for measurement of luciferase expression.  Results represent normalized infection through each coreceptor and are means +/- sem of infections done using cells from three different donors, each performed in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Coreceptor use by R5X4 HIV-1 on indicator cell lines and primary CD4+ lymphocytes. (A) Coreceptor use on U87 indicator cells by R5X4 HIV-1. U87 CD4, CD4/CCR5 or CD4/CXCR4 cells were infected with HIV-1 pseudotype viruses (5ng p24 Gag antigen). Three days after infection, cells were lysed and luciferase activity was measured. Results are means sem of two experiments performed in triplicate. (B) Coreceptor-specific entry into CD4+ T lymphocytes. Purified CD4+ lymphocytes were isolated by negative selection, stimulated with PHA for 3 days, infected with 5ng of HIV-1 pseudotype viruses in the absence or presence of coreceptor antagonists as described in Figure 1, maintained with IL-2 and lysed four days later for measurement of luciferase expression. Results represent normalized infection through each coreceptor and are means +/- sem of infections done using cells from three different donors, each performed in duplicate.
Mentions: R5X4 variants have the capacity to use both CCR5 and CXCR4 on macrophages and indicator cell lines (Figs 1 and 2A), but in contrast, the pattern of coreceptor use by R5X4 HIV-1 on CD4+ lymphocytes from peripheral blood is quite different from that seen on those two cell types. Initial reports using a similar coreceptor blocking strategy and prototype strains showed that R5X4 viruses used CXCR4 on lymphocytes but CCR5 use was minimal, and lymphocyte CCR5 use by R5X4 isolates was markedly impaired relative to infection by R5 viruses [11]. Furthermore, unlike macrophages, infection mediated by CXCR4 alone was equivalent to infection when both coreceptors were present, suggesting no additional contribution of CCR5 in the presence of the CXCR4 pathway. More recently, using an expanded panel of R5X4 Envs from diverse sources, we found that that some R5X4 viruses do possess the ability to use CCR5 for entry into CD4+ lymphocytes [29]. A range of CCR5 use was observed among these R5X4 strains, with CCR5 making virtually no contribution to infection by some strains while nearly half the total amount of entry could be mediated by CCR5 for other strains. However, despite more robust CCR5 use by some clones, CXCR4 remained the predominant coreceptor used on CD4+ lymphocytes for all R5X4 viruses. CD4+ lymphocytes in blood are a mixture of different subsets, with CXCR4 expressed on a larger percentage of cells than CCR5 [12,30,31]. Consequently, CXCR4 is likely to be the predominate coreceptor used by the majority of R5X4 strains. Strikingly, there was consistently no difference between infection of unblocked CD4+ T lymphocytes and infection mediated by CXCR4 alone, confirming that even though both pathways can be used, when CXCR4 is available there is no additional contribution to infection made by CCR5 (Fig. 2B).

Bottom Line: However, these algorithms were developed to predict coreceptor use in cell lines and not primary cells and, furthermore, are not highly accurate for some classes of viruses.This article focuses on R5X4 HIV-1, the earliest CXCR4-using variants, reviewing the pattern of coreceptor use on primary CD4+ lymphocytes and macrophages, the relationship between primary cell coreceptor use and the two principal approaches to coreceptor analysis (genetic prediction and indicator cell phenotyping), and the implications of primary cell coreceptor use by these strains for treatment with a new class of small molecule antagonists that inhibit CCR5-mediated entry.These are important questions to consider given the development of new CCR5 blocking therapies and the prognosis associated with CXCR4 use.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, 522 Johnson Pavilion, 36th & Hamilton Walk, Philadelphia, PA 19104-6060, USA.

ABSTRACT
Entry coreceptor use by HIV-1 plays a pivotal role in viral transmission, pathogenesis and disease progression. In many HIV-1 infected individuals, there is an expansion in coreceptor use from CCR5 to include CXCR4, which is associated with accelerated disease progression. While targeting HIV-1 envelope interactions with coreceptor during viral entry is an appealing approach to combat the virus, the methods of determining coreceptor use and the changes in coreceptor use that can occur during disease progression are important factors that may complicate the use of therapies targeting this stage of HIV-1 replication. Indicator cells are typically used to determine coreceptor use by HIV-1 in vitro, but the coreceptors used on these cells can differ from those used on primary cell targets. V3 based genetic sequence algorithms are another method used to predict coreceptor use by HIV-1 strains. However, these algorithms were developed to predict coreceptor use in cell lines and not primary cells and, furthermore, are not highly accurate for some classes of viruses. This article focuses on R5X4 HIV-1, the earliest CXCR4-using variants, reviewing the pattern of coreceptor use on primary CD4+ lymphocytes and macrophages, the relationship between primary cell coreceptor use and the two principal approaches to coreceptor analysis (genetic prediction and indicator cell phenotyping), and the implications of primary cell coreceptor use by these strains for treatment with a new class of small molecule antagonists that inhibit CCR5-mediated entry. These are important questions to consider given the development of new CCR5 blocking therapies and the prognosis associated with CXCR4 use.

Show MeSH
Related in: MedlinePlus