Alternative Coreceptor Research Articles

Simian Immunodeficiency Virus Infects Follicular Helper CD4 T Cells in Lymphoid Tissues during Pathogenic Infection of Pigtail Macaques

T follicular helper (Tfh) cells are a specialized subset of memory CD4(+) T cells that are found exclusively within the germinal centers of secondary lymphoid tissues and are important for adaptive antibody responses and B cell memory. Tfh cells do not express CCR5, the primary entry coreceptor for both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), and therefore, we hypothesized that these cells would avoid infection. We studied lymph nodes and spleens from pigtail macaques infected with pathogenic strain SIVmac239 or SIVmac251, to investigate the susceptibility of Tfh cells to SIV infection. Pigtail macaque PD-1(high) CD127(low) memory CD4(+) T cells have a phenotype comparable to that of human Tfh cells, expressing high levels of CXCR5, interleukin-21 (IL-21), Bcl-6, and inducible T cell costimulator (ICOS). As judged by either proviral DNA or cell-associated viral RNA measurements, macaque Tfh cells were infected with SIV at levels comparable to those in other CD4(+) memory T cells. Infection of macaque Tfh cells was evident within weeks of inoculation, yet we confirmed that Tfh cells do not express CCR5 or either of the well-known alternative SIV coreceptors, CXCR6 and GPR15. Mutations in the SIV envelope gp120 region occurred in chronically infected macaques but were uniform across each T cell subset investigated, indicating that the viruses used the same coreceptors to enter different cell subsets. Early infection of Tfh cells represents an unexpected focus of viral infection. Infection of Tfh cells does not interrupt antibody production but may be a factor that limits the quality of antibody responses and has implications for assessing the size of the viral reservoir.

Dual R3R5 tropism characterizes cerebrospinal fluid HIV-1 isolates from individuals with high cerebrospinal fluid viral load

To study the use of major and alternative coreceptors by HIV-1 isolates obtained from paired plasma and cerebrospinal fluid (CSF) samples. Paired plasma and CSF isolates from HIV-1-infected individuals with varying clinical, virologic, and immunologic parameters were assessed for the ability to infect indicator cells expressing a panel of coreceptors with documented expression in the central nervous system (CNS). HIV-1 isolates obtained from plasma and CSF in 28 individuals with varying viral load, CD4 T-cell counts, and with or without AIDS-defining disease were analyzed for the ability to infect NP2.CD4 cells stably expressing a panel of HIV coreceptors (CCR5, CXCR4, CCR3, CXCR6, GPR1, APJ, ChemR23, RDC-1 or BLT1). All isolates from both plasma and CSF utilized CCR5 and/or CXCR4. However, the ability to use both CCR3 and CCR5 (R3R5) was more pronounced in CSF isolates and correlated with high CSF viral load and low CD4 T-cell count. Notably, four out of five CSF isolates of subtype C origin exhibited CXCR6 use, which coincided with high CSF viral load despite preserved CD4 T-cell counts. The use of other alternative coreceptors was less pronounced. Dual-tropic R3R5 HIV-1 isolates in CSF coincide with high CSF viral load and low CD4 T-cell counts. Frequent CXCR6 use by CSF-derived subtype C isolates indicates that subtype-specific differences in coreceptor use may exist that will not be acknowledged when assessing plasma virus isolates. The findings may also bare relevance for HIV-1 replication within the CNS, and consequently, for the neuropathogenesis of AIDS.

Use of (alternative) coreceptors for HIV entry

Although CCR5 and CXCR4 are the predominant coreceptors associated with HIV-1 entry, many more coreceptors can support either infection or viral capture and have been associated with transmission and disease progression. Here, we describe the most recent findings pertinent to which receptors are involved with HIV-1 infection and the proposed consequences. CCR5 or CXCR4 using HIV-1 can differentially utilize their respective coreceptors and influence which cell types are infected. Many alternate coreceptors have been described which expands the pool of cells within which HIV-1 can replicate or reside. A large number of C-type lectins have been described which capture HIV-1 and present the virus to CD4 T cells which can also be considered as receptors involved with transmission or disease course. The molecular mechanism through which gp120 binds host lectins, such as altered N-linked glycosylation profiling, provides further mechanisms influencing HIV-1 capture and transfer. Genetic polymorphisms within the alternative coreceptors have now been shown to associate with disease, implying they can play a significant role in HIV-1 disease. We have summarized the recent findings concerning the multitude of receptors and coreceptors which can interact with HIV-1 and have discussed the consequences for either HIV-1 transmission or disease progression.

The role of the alternative coreceptor GPR15 in SIV tropism for human cells

Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4+, CD8+ and CD19+ peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15+ PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4+, GPR15+ cells is not responsible for the well documented ability of SIV to infect CCR5− blood cells.

Cloning and Analysis of Sooty Mangabey Alternative Coreceptors That Support Simian Immunodeficiency Virus SIVsmm Entry Independently of CCR5

Natural host sooty mangabeys (SM) infected with simian immunodeficiency virus SIVsmm do not develop AIDS despite high viremia. SM and other natural hosts express very low levels of CCR5 on CD4(+) T cells, and we recently showed that SIVsmm infection and robust replication occur in vivo in SM genetically lacking CCR5, indicating the use of additional entry pathways. SIVsmm uses several alternative coreceptors of human origin in vitro, but which molecules of SM origin support entry is unknown. We cloned a panel of putative coreceptors from SM and tested their ability to mediate infection, in conjunction with smCD4, by pseudotypes carrying Envs from multiple SIVsmm subtypes. smCXCR6 supported efficient infection by all SIVsmm isolates with entry levels comparable to those for smCCR5, and smGPR15 enabled entry by all isolates at modest levels. smGPR1 and smAPJ supported low and variable entry, whereas smCCR2b, smCCR3, smCCR4, smCCR8, and smCXCR4 were not used by most isolates. In contrast, SIVsmm from rare infected SM with profound CD4(+) T cell loss, previously reported to have expanded use of human coreceptors, including CXCR4, used smCXCR4, smCXCR6, and smCCR5 efficiently and also exhibited robust entry through smCCR3, smCCR8, smGPR1, smGPR15, and smAPJ. Entry was similar with both known alleles of smCD4. These alternative coreceptors, particularly smCXCR6 and smGPR15, may support virus replication in SM that have restricted CCR5 expression as well as SM genetically lacking CCR5. Defining expression of these molecules on SM CD4(+) subsets may delineate distinct natural host target cell populations capable of supporting SIVsmm replication without CD4(+) T cell loss.

Evidence of extended alternate coreceptor usage by HIV-1 clade C envelope obtained from an Indian patient

HIV-1 clade C tends to exclusively use CCR5 irrespective of disease stages. We previously reported envelopes (Envs) obtained from an Indian patient (VB105) that used CXCR4, CXCR6, CCR2b, CCR3, GPR15, and CX3CR1 as additional coreceptors besides CCR5 for entry. Here we show that the primary VB105 virus was able to replicate in peripheral blood mononuclear cells (PBMCs) in presence of inhibitors that antagonizes all the above seven coreceptors at excess doses. In addition, VB105 Envs were found to efficiently infect CCR5-defective T cells (MOLT-4) in presence of excess TAK-779, AMD3100, vMIP-1 and vMIP-2 further substantiated the usage of additional coreceptors beyond the seven coreceptors as reported earlier by VB105 Env. Interestingly, VB105 Envs showed spontaneous exposure of CD4-induced epitopes and found to be associated with increased infection of macrophages. Information on HIV-1 clade C using alternate coreceptors in primary cells to better understand their impact on pathogenesis and efficacy to future entry inhibitors.

CCR5 Antagonism in HIV Infection: Current Concepts and Future Opportunities

CCR5 antagonists inhibit HIV-1 entry by blocking the interaction of HIV-1 with the CCR5 cellular receptor. In patients with established HIV-1 infection, some viral strains use an alternative coreceptor for HIV-1 entry, CXCR4; CCR5 antagonists are not effective in patients harboring these viral strains. Coreceptor tropism testing of viral strains in an individual patient is necessary prior to treating with a CCR5 antagonist. There is one CCR5 antagonist, maraviroc, that is FDA-approved for treatment of HIV-1 infection. This drug is used most commonly for the treatment of HIV-1 infection in patients who have failed other antiretroviral regimens. In addition to virologic effects, CCR5 antagonists are under investigation for immune-modulating effects and for HIV-1 prevention. Ongoing research will further elucidate the role of CCR5 antagonists in combating HIV disease.

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism.

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.

Alternative Coreceptor Requirements for Efficient CCR5- and CXCR4-Mediated HIV-1 Entry into Macrophages

Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.

Extreme genetic divergence is required for coreceptor switching in HIV-1 subtype C.

Coreceptor switching from CCR5 to CXCR4 is less common in subtype C HIV-1 infection than in subtype B for reasons that are unclear. We have examined sequential virus samples from a subtype C-infected child who had evidence of coreceptor switching. To examine HIV-1 envelope evolution towards CXCR4 usage, env sequences were correlated with phenotypic characteristics determined by entry assays, as well as the ability to use alternative coreceptors such as FPRL1, CCR3, CCR8 and others. The value of a phenotype predictor based on V3 sequences was also assessed. Ninety-three sequences revealed 3 distinct coexistent virus lineages and only some members of one lineage evolved to use CXCR4. These lineages also had diverse alternative coreceptor patterns including the ability to use FPRL1, CCR3, CCR8, APJ, CMKLR1, RDC-1, CXCR6, CCR1, GPCR1, GPR15 and CCR6. Coreceptor switching was associated with extensive and rapid sequence divergence in the V1/V2 region in addition to V3 changes. Furthermore, interlineage recombination within the C2 region resulted in low predictability of a V3 sequence-based phenotype algorithm, and highlighted the importance of V1/V2 and V3 sequences in coreceptor usage. These results suggest that the evolution to coreceptor switching in subtype C infection requires more mutations than other subtypes, and this contributes to the reduced incidence of R5X4 viruses.

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor. Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor. Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

Coreceptor usage by HIV-1 and HIV-2 primary isolates: The relevance of CCR8 chemokine receptor as an alternative coreceptor

The human immunodeficiency virus replication cycle begins by sequential interactions between viral envelope glycoproteins with CD4 molecule and a member of the seven-transmembrane, G-protein-coupled, receptors' family (coreceptor). In this report we focused on the contribution of CCR8 as alternative coreceptor for HIV-1 and HIV-2 isolates. We found that this coreceptor was efficiently used not only by HIV-2 but particularly by HIV-1 isolates. We demonstrate that CXCR4 usage, either alone or together with CCR5 and/or CCR8, was more frequently observed in HIV-1 than in HIV-2 isolates. Directly related to this is the finding that the non-usage of CXCR4 is significantly more common in HIV-2 isolates; both features could be associated with the slower disease progression generally observed in HIV-2 infected patients. The ability of some viral isolates to use alternative coreceptors besides CCR5 and CXCR4 could further impact on the efficacy of entry inhibitor therapy and possibly also in HIV pathogenesis.

A novel CCR5 mutation common in sooty mangabeys reveals SIVsmm infection of CCR5-null natural hosts and efficient alternative coreceptor use in vivo.

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species.

Fitness Epistasis and Constraints on Adaptation in a Human Immunodeficiency Virus Type 1 Protein Region

Fitness epistasis, the interaction among alleles at different loci in their effects on fitness, has potentially important consequences for adaptive evolution. We investigated fitness epistasis among amino acids of a functionally important region of the human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein (gp120). Seven mutations putatively involved in the adaptation of the second conserved to third variable protein region (C2-V3) to the use of an alternative host-cell chemokine coreceptor (CXCR4) for cell entry were engineered singly and in combinations on the wild-type genetic background and their effects on viral infectivity were measured. Epistasis was found to be common and complex, involving not only pairwise interactions, but also higher-order interactions. Interactions could also be surprisingly strong, changing fitness by more than 9 orders of magnitude, which is explained by some single mutations being practically lethal. A consequence of the observed epistasis is that many of the minimum-length mutational trajectories between the wild type and the mutant with highest fitness on cells expressing the alternative coreceptor are selectively inaccessible. These results may help explain the difficulty of evolving viruses that use the alternative coreceptor in culture and the delayed evolution of this phenotype in natural infection. Knowledge of common, complex, and strong fitness interactions among amino acids is necessary for a full understanding of protein evolution.

Resistance to new antiretrovirals (ETV, anti-integrases, entry inhibitors)

HIV is extremely variable and newly arisen variants may be rapidly selected upon drug pressure. New antivirals are available, new therapeutic options are at hand for all patients, either naive or multiexperienced. Individualization is the hallmark of new approaches to antiretroviral therapies. Knowledge of drug resistance is important to guide clinicians in devising treatment regimens and to optimally preserve later treatment options for patients. This presentation will focus on the new technologies, methodologies, models and research tools to enhance detection and interpretation of HIV-1 drug-resistance. Particular attention will be dedicated to the resistance to the new antivirals. The development of the new second-generation NNRTI etravirine, which has a higher genetic barrier than the first-generation drugs, has led to new understanding of the complexity of NNRTI resistance. To date, 17 mutations have been identified as being associated with etravirine resistance and reduced virological response. Integrase inhibitors are very potent drugs providing a great opportunity for clinicians, to construct new regimens for individuals with HIV infection resistant to all three traditional classes of antivirals. However, these drugs have a relatively low genetic barrier to resistance and thus require both the selection of an optimised backbone to ensure durable viral suppression and a clear understanding of their residual activity in a failing integrase regimen. Resistance tests able to sequence and detect mutations in integrase are crucial for their proper use in clinical practice. Viral escape of entry inhibitors may occur through 'classic' viral resistance to the compounds, but also by usage of alternative cell co-receptors. To date the main focus of research on co-receptor antagonists has been on determining and quantifying the CCR5, CXCR4, dual/mixed coreceptor tropism. Recent data begun to emerge on how resistance develops with the accumulation of mutations in gp120 to maraviroc, the first-in-class R5 co-receptor antagonist.

FGFR1 Interaction with Co-Receptor Klotho-Beta at the Plasma Membrane

FGF21/FGFR1 signalling modulates the survival and glucose sensitivity of fat and liver cells, properties that make this signalling pathway a potential target in the treatment of diabetes. The majority of FGFs interact with heparin proteoglycans in the matrix for presentation to high-affinity receptors such as FGFR1. In contrast, FGF21 exhibits negligible affinity for heparin. To activate FGFR1, FGF21 requires expression of an alternative co-receptor, Klotho-beta (KLB). To study the molecular interaction between FGFR1 and KLB at the cell membrane, we created fluorescent protein-tagged constructs of KLB and FGFR1. By using fluorescence recovery after photobleaching (FRAP), we show that KLB has a lower diffusion coefficient and mobile fraction than FGFR1. Subsequent addition of lactose, an inhibitor of non-specific galactoside binding in the matrix, increased mobility of KLB with no effect on FGFR1. To determine whether the addition of FGF21 induces FGFR1/KLB association, we are presently examining whether FGFR1 mobility slows to KLB levels in the presence of FGF21. We are also measuring homo-Forster Resonance Energy Transfer (homoFRET) on a Total Internal Refection Fluorescence (TIRF) microscope to reconcile these results by examining the oligomeric state of KLB and FGFR1 at the plasma membrane. Overall, these studies will determine whether FGFR1 associates with KLB in the presence of FGF21 revealing important mechanistic information of a novel endocrine factor.

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.

Heterosexual Transmission of Human Immunodeficiency Virus Type 1 Subtype C: Macrophage Tropism, Alternative Coreceptor Use, and the Molecular Anatomy of CCR5 Utilization

Human immunodeficiency virus type 1 transmission selects for virus variants with genetic characteristics distinct from those of donor quasispecies, but the biological factors favoring their transmission or establishment in new hosts are poorly understood. We compared primary target cell tropisms and entry coreceptor utilizations of donor and recipient subtype C Envs obtained near the time of acute infection from Zambian heterosexual transmission pairs. Both donor and recipient Envs demonstrated only modest macrophage tropism, and there was no overall difference between groups in macrophage or CD4 T-cell infection efficiency. Several individual pairs showed donor/recipient differences in primary cell infection, but these were not consistent between pairs. Envs had surprisingly broad uses of GPR15, CXCR6, and APJ, but little or no use of CCR2b, CCR3, CCR8, GPR1, and CXCR4. Donors overall used GPR15 better than did recipients. However, while several individual pairs showed donor/recipient differences for GPR15 and/or other coreceptors, the direction of the differences was inconsistent, and several pairs had unique alternative coreceptor patterns that were conserved across the transmission barrier. CCR5/CCR2b chimeras revealed that recipients as a group were more sensitive than were donors to replacement of the CCR5 extracellular loops with corresponding regions of CCR2b, but significant differences in this direction were not consistent within pairs. These data show that sexual transmission does not select for enhanced macrophage tropism, nor for preferential use of any alternative coreceptor. Recipient Envs are somewhat more constrained than are donors in flexibility of CCR5 use, but this pattern is not universal for all pairs, indicating that it is not an absolute requirement.

Receptor Tyrosine Kinase Ror2 Mediates Wnt5a-induced Polarized Cell Migration by Activating c-Jun N-terminal Kinase via Actin-binding Protein Filamin A

The receptor tyrosine kinase Ror2 has recently been shown to act as an alternative receptor or coreceptor for Wnt5a and to mediate Wnt5a-induced migration of cultured cells. However, little is known about the molecular mechanism underlying this migratory process. Here we show by wound-healing assays that Ror2 plays critical roles in Wnt5a-induced cell migration by regulating formation of lamellipodia and reorientation of microtubule-organizing center (MTOC). Wnt5a stimulation induces activation of the c-Jun N-terminal kinase JNK at the wound edge in a Ror2-dependent manner, and inhibiting JNK activity abrogates Wnt5a-induced lamellipodia formation and MTOC reorientation. Additionally, the association of Ror2 with the actin-binding protein filamin A is required for Wnt5a-induced JNK activation and polarized cell migration. We further show that Wnt5a-induced JNK activation and MTOC reorientation can be suppressed by inhibiting PKCzeta. Taken together, our findings indicate that Wnt5a/Ror2 activates JNK, through a process involving filamin A and PKCzeta, to regulate polarized cell migration.