Preferential Use of CXCR4 by R5X4 Human Immunodeficiency Virus Type 1 Isolates for Infection of Primary Lymphocytes

Abstract

Coreceptor specificity of human immunodeficiency virus type 1 (HIV-1) strains is generally defined in vitro in cell lines expressing CCR5 or CXCR4, but lymphocytes and macrophages are the principal targets in vivo. CCR5-using (R5) variants dominate early in infection, but strains that use CXCR4 emerge later in a substantial minority of subjects. Many or most CXCR4-using variants can use both CXCR4 and CCR5 (R5X4), but the pathways that are actually used to cause infection in primary cells and in vivo are unknown. We examined several R5X4 prototype and primary isolates and found that they all were largely or completely restricted to CXCR4-mediated entry in primary lymphocytes, even though lymphocytes are permissive for CCR5-mediated entry by R5 strains. In contrast, in primary macrophages R5X4 isolates used both CCR5 and CXCR4. The R5X4 strains were also more sensitive than R5 strains to CCR5 blocking, suggesting that interactions between the R5X4 strains and CCR5 are less efficient. These results indicate that coreceptor phenotyping in transformed cells does not necessarily predict utilization in primary cells, that variability exists among HIV-1 isolates in the ability to use CCR5 expressed on lymphocytes, and that many or most strains characterized as R5X4 are functionally X4 in primary lymphocytes. Less efficient interactions between R5X4 strains and CCR5 may be responsible for the inability to use CCR5 on lymphocytes, which express relatively low CCR5 levels. Since isolates that acquire CXCR4 utilization retain the capacity to use CCR5 on macrophages despite their inability to use it on lymphocytes, these results also raise the possibility that a CCR5-mediated macrophage reservoir is required for sustained infection in vivo. CD4ϩ T cells and macrophages are the two principal targets for productive human immunodeficiency virus type 1 (HIV-1) infection in vivo. Viral entry is initiated by the viral envelope glycoprotein gp120 binding to cell surface CD4, followed by interactions with a chemokine receptor. All naturally occurring strains of HIV-1 use CCR5 or CXCR4 or both, and both coreceptors are expressed by primary CD4 ϩ T lymphocytes and macrophages. Early in infection, viral variants are almost uniformly CCR5 dependent (R5 variants), but CXCR4-using variants emerge later in a substantial minority of infected people, and their appearance is strongly associated with accelerated disease progression (7,23). In most of these individuals, the late-stage variants retain the capacity to use both CCR5 and CXCR4 (R5X4), although some may harbor only CXCR4-dependent (X4) variants and others may harbor a mix of R5 and X4 variants (7, 23, 28).Chemokine receptor selectivity of HIV-1 isolates is typically characterized on the basis of cell-cell fusion or infection of CD4-expressing cell lines transfected with CCR5 or CXCR4. CXCR4 utilization can also be defined based on the syncytiainducing (SI) capacity in MT-2 cells, which reflects CXCR4-dependent infection, although this method cannot distinguish between X4 and R5X4 ...

Genetic and Functional Analysis of R5X4 Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Derived from Two Individuals Homozygous for the CCR5Δ32 Allele

“…2A), use CXCR4 exclusively for entry into PBMC. These results are consistent with the results of recent studies that demonstrated preferential use of CXCR4 by R5X4 HIV-1 isolates for infection of primary T lymphocytes (51). The data also indicate that DR-17 and C2-16 Envs, which are dual-tropic for T lymphocytes and macrophages ( Fig.…”

“…2A). Consistent with previous studies, ADA and 89.6 Envs entered PBMC and MDM, whereas HXB2 Env entered PBMC but not MDM (18,19,34,42,43,(49)(50)(51). All C2 and DR Envs entered PBMC, but only C2-16 and DR-17 Envs entered MDM.…”

Baseline Resistance of Primary Human Immunodeficiency Virus Type 1 Strains to the CXCR4 Inhibitor AMD3100

“…The lysates were then incubated at 60°C for 2 h and 98°C for 15 min. For each reaction, 1 l of DNA lysate was transferred to a MicroAmp fast optical 96-well reaction plate with a barcode (Applied Biosystems) containing 12.5 l TaqMan Universal PCR Master Mix (Applied Biosystems), 9 l of sterile water (Fisher Scientific), and 1.5 l of the primer/probe sets recognizing an early HIV replication product (the first strong stop or long terminal repeat [LTR]) or the cellular GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene (61). RealTime PCR was then performed using the Applied Biosystems 7500 Fast RealTime PCR System.…”

“…While many HIV-1 strains can use either CCR5 or CXCR4 (R5X4 viruses) to infect cell lines, the efficiency with which a given virus uses each coreceptor for infection can vary widely and does not always predict the mechanism of entry into human T cells or macrophages (18). Thus, some R5X4 viruses use only CXCR4 to infect certain primary cells, others use only CCR5, and some viruses use both coreceptors to infect multiple cell types (16,17,26,36,(59)(60)(61). The use of specific and potent coreceptor antagonists can be utilized to prevent entry via one coreceptor, revealing the efficiency with which the alternative coreceptor can support virus infection.…”

Tropism-independent protection of macaques against vaginal transmission of three SHIVs by the HIV-1 fusion inhibitor T-1249

“…SHIV-89.6P therefore behaves in PBMCs as if it were an X4 virus (12). However, SHIV-89.6P can use either CCR5 or CXCR4 to enter indicator cell lines, and some dual-tropic viruses that use only CXCR4 to enter T cells can also use CCR5 for replication in macrophages (17,18). Our unpublished studies suggest that CMPD-167 or AMD3465 can each inhibit SHIV-89.6P vaginal transmission to macaques, albeit inconsistently.…”