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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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R5X4 HIV-1 use CCR5 and CXCR4 on primary macrophages. Monocyte-derived macrophages (MDM) were infected with HIV-1 luciferase-pseudotype viruses (5ng p24 Gag antigen) carrying representative prototype R5X4 envelope glycoproteins, along with control R5 (Bal) and X4 (Tybe) Env-containing viruses.  Infections were carried out without entry blocker or in the presence of the CCR5 antagonist Maraviroc (“CXCR4 pathway”; 5μM), CXCR4 antagonist AMD3100 (“CCR5 pathway”; 5μg/ml) or both inhibitors. Three days after infection, cells were lysed with 0.1% Triton, luciferase assay substrate (Promega) was added and luciferase activity (RLUs) was measured using a Dynex Revelation Luminometer.   Results represent normalized infection mediated by each coreceptor as a percentage of infection in the absence of antagonists and are means ± sem of infections done using cells from two different donors, each performed in triplicate.
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Figure 1: R5X4 HIV-1 use CCR5 and CXCR4 on primary macrophages. Monocyte-derived macrophages (MDM) were infected with HIV-1 luciferase-pseudotype viruses (5ng p24 Gag antigen) carrying representative prototype R5X4 envelope glycoproteins, along with control R5 (Bal) and X4 (Tybe) Env-containing viruses. Infections were carried out without entry blocker or in the presence of the CCR5 antagonist Maraviroc (“CXCR4 pathway”; 5μM), CXCR4 antagonist AMD3100 (“CCR5 pathway”; 5μg/ml) or both inhibitors. Three days after infection, cells were lysed with 0.1% Triton, luciferase assay substrate (Promega) was added and luciferase activity (RLUs) was measured using a Dynex Revelation Luminometer. Results represent normalized infection mediated by each coreceptor as a percentage of infection in the absence of antagonists and are means ± sem of infections done using cells from two different donors, each performed in triplicate.

Mentions: Studies to determine which coreceptors R5X4 viruses use to infect primary macrophages have used replication competent and pseudotype viruses from different clades of HIV-1 [11,28]. Since macrophages express both coreceptors, unlike single coreceptor virus analysis, these studies have largely utilized small molecule antagonists to CCR5 or CXCR4 as a means of evaluating use of the unblocked coreceptor. These studies have shown that in the presence of a CCR5 or CXCR4 antagonist, infection by R5X4 HIV-1 still occurs and infection by these viruses is fully blocked only when both antagonists are present. The proportional contribution of each coreceptor to total infection of macrophages can be determined by comparing entry through that coreceptor to entry in the absence of antagonists. As shown by the results from a representative group of R5X4 viruses in Fig 1 (and expanded upon for R5X4 isolates more broadly in ([11,28]), this analysis reveals that the level of viral entry that occurs through a single coreceptor is reduced relative to infection when both coreceptors are available. Thus, both coreceptors make substantial contributions to the overall infection of macrophages by R5X4 HIV-1, although there are modest differences between isolates in the proportion of total entry mediated by each coreceptor.


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)

R5X4 HIV-1 use CCR5 and CXCR4 on primary macrophages. Monocyte-derived macrophages (MDM) were infected with HIV-1 luciferase-pseudotype viruses (5ng p24 Gag antigen) carrying representative prototype R5X4 envelope glycoproteins, along with control R5 (Bal) and X4 (Tybe) Env-containing viruses.  Infections were carried out without entry blocker or in the presence of the CCR5 antagonist Maraviroc (“CXCR4 pathway”; 5μM), CXCR4 antagonist AMD3100 (“CCR5 pathway”; 5μg/ml) or both inhibitors. Three days after infection, cells were lysed with 0.1% Triton, luciferase assay substrate (Promega) was added and luciferase activity (RLUs) was measured using a Dynex Revelation Luminometer.   Results represent normalized infection mediated by each coreceptor as a percentage of infection in the absence of antagonists and are means ± sem of infections done using cells from two different donors, each performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105503&req=5

Figure 1: R5X4 HIV-1 use CCR5 and CXCR4 on primary macrophages. Monocyte-derived macrophages (MDM) were infected with HIV-1 luciferase-pseudotype viruses (5ng p24 Gag antigen) carrying representative prototype R5X4 envelope glycoproteins, along with control R5 (Bal) and X4 (Tybe) Env-containing viruses. Infections were carried out without entry blocker or in the presence of the CCR5 antagonist Maraviroc (“CXCR4 pathway”; 5μM), CXCR4 antagonist AMD3100 (“CCR5 pathway”; 5μg/ml) or both inhibitors. Three days after infection, cells were lysed with 0.1% Triton, luciferase assay substrate (Promega) was added and luciferase activity (RLUs) was measured using a Dynex Revelation Luminometer. Results represent normalized infection mediated by each coreceptor as a percentage of infection in the absence of antagonists and are means ± sem of infections done using cells from two different donors, each performed in triplicate.
Mentions: Studies to determine which coreceptors R5X4 viruses use to infect primary macrophages have used replication competent and pseudotype viruses from different clades of HIV-1 [11,28]. Since macrophages express both coreceptors, unlike single coreceptor virus analysis, these studies have largely utilized small molecule antagonists to CCR5 or CXCR4 as a means of evaluating use of the unblocked coreceptor. These studies have shown that in the presence of a CCR5 or CXCR4 antagonist, infection by R5X4 HIV-1 still occurs and infection by these viruses is fully blocked only when both antagonists are present. The proportional contribution of each coreceptor to total infection of macrophages can be determined by comparing entry through that coreceptor to entry in the absence of antagonists. As shown by the results from a representative group of R5X4 viruses in Fig 1 (and expanded upon for R5X4 isolates more broadly in ([11,28]), this analysis reveals that the level of viral entry that occurs through a single coreceptor is reduced relative to infection when both coreceptors are available. Thus, both coreceptors make substantial contributions to the overall infection of macrophages by R5X4 HIV-1, although there are modest differences between isolates in the proportion of total entry mediated by each coreceptor.

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