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We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II−/− (CII−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII−/−, CD40−/−, or CD80/86−/− mice, compared with that in wild-type or CD28/CTLA4−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen which is closely related to Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) (17, 64). Intranasal administration of MHV-68 to mice results in acute productive infection of lung epithelial cells and a latent infection in various cell types, including B lymphocytes, dendritic cells, epithelial cells, and macrophages (18, 19, 52, 53, 61, 65). The virus induces an inflammatory infiltrate in the lungs, lymph node enlargement, splenomegaly, and mononucleosis comprising increased numbers of activated CD8 T cells in the blood (53, 58). It has also been reported to induce lymphoproliferative disease/lymphoma in immunocompromised mice (30, 55, 60). Thus, the pathogenesis resembles that of EBV in humans, although structurally, the virus is more closely related to KSHV.Infectious MHV-68 is cleared from the lungs by a T-cell-dependent mechanism 10 to 15 days after infection (18, 53, 56). In wild-type mice, the lungs remain clear of replicating virus thereafter. Although CD4 T cells are not essential for primary clearance of replicating virus, they are required for effective long-term control (11). Thus, major histocompatibility complex (MHC) class II−/− mice that lack CD4 T cells or mice rendered CD4 deficient by antibody treatment initially clear infectious virus from the lungs. However, infectious virus reactivates in the lungs 10 to 15 days later and gradually increases in titer (11, 43). The infected CD4-deficient mice eventually die, apparently from long-term lung damage due to continuing lytic viral replication (11). MHC class II−/− mice do not produce antibody to T-dependent antigens (10). Cytotoxic T-lymphocyte (CTL) epitopes have been identified in open reading frame (ORF) 6 (p56, H-2Db-restricted), and ORF 61 (p79, H-2Kb-restricted) gene products, which appear to encode early lytic-phase proteins (32, 49). The epitopes are presented during two distinct phases during MHV-68 infection, which changes the pattern of CTL dominance (32, 51). However, there is no significant difference in the numbers of CD8 T cells specific for each epitope in wild-type mice and CD4 T-cell-deficient mice (4, 50). In addition, CTL activity measured in vitro does not differ substantially in the lungs of wild-type mice or CD4 T-cell-deficient mice (4, 11, 50). Furthermore, postexposure vaccination with the p56 epitope failed to prevent viral reactivation in class II−/− mice, despite dramatically expanding the number of CD8 T cells specific for the peptide (5). In contrast, vaccination of wild-type mice against these epitopes reduced lytic viral titers in the lung dramatically on subsequent challenge with MHV-68. B-cell-deficient mice clear MHV-68 with the kinetics of wild-type mice and do not show viral reactivation in the lungs (13, 61), suggesting that antibody is not essential for control of the virus. Depletion of CD4 T cells during the latent phase of infection in B-cell-deficient mice does not induce viral reactivation, whereas depletion of both CD4 and CD8 T-cell subsets provokes viral reactivation in the lungs (52). Short-term depletion of both CD4 and CD8 T-cell subsets during the latent phase of infection in wild-type mice does not lead to viral reactivation probably due to the presence of neutralizing antibody (11). Taken together, these results suggest that CD4 and CD8 T cells and B cells play overlapping roles in preventing or controlling reactivation of MHV-68 during the latent phase of infection. However, the B-cell- and CD8 T-cell-mediated control mechanisms do not develop in the absence of CD4 T cells.We, and others, have previously shown that the costimulatory molecule CD28 is not required for long-term control of MHV-68 (28, 29). However, interestingly, mice lacking both of the ligands for CD28, CD80 and CD86, show viral reactivation in the lung (21, 35). Our previously published data showed that agonistic antibodies to CD40 could substitute for CD4 T-cell function in the long-term control of MHV-68 (46). CD8 T-cell receptor-positive (TCR+) cells were required for this effect, while antibody production was not restored (45, 46). MHV-68-infected CD40L−/− mice (7) and CD40−/− mice (29) also showed viral reactivation in the lungs. However, no change in CD8 CTL activity was detected in in vitro assays following anti-CD40 treatment (46). A key question was whether anti-CD40 treatment (or CD4 T-cell help) caused a direct change in CD8 T-cell function or whether both CD8 T cells and an independent anti-CD40-sensitive step were required for viral control. To address this question, we used adoptive transfer of CD8 T cells from MHV-68-infected wild-type mice, anti-CD40-treated mice, or control MHC class II−/− mice to MHV-68-infected class II−/− recipients. We also investigated whether anti-CD40 treatment prolonged survival in addition to reducing lung viral titers. The heterodimeric molecule CD94/NKG2A has been implicated in negatively regulating the CD8 T-cell response to polyomavirus (38) and herpes simplex virus (HSV) (54), while the inhibitory receptor PD-1 (programmed death 1) has been implicated in T-cell exhaustion following infection with several other persistent viruses (2, 15, 20, 22, 26, 36, 39-41, 57, 67). In the present study, we investigated the effect of signaling via various costimulatory molecules on the expression of NKG2A and PD-1 and how these molecules influenced viral control.  相似文献   

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The c-Jun NH2-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1−/− Jnk2−/− ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.The c-Jun NH2-terminal kinase (JNK) is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. JNK is encoded by two ubiquitously expressed genes (Jnk1 and Jnk2) and by a third gene (Jnk3) that is selectively expressed in neurons (14). Gene disruption studies demonstrate that mice without Jnk1 or Jnk2 are viable, but compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality (14). Murine embryonic fibroblasts (MEFs) isolated from Jnk1−/− Jnk2−/− mice exhibit a severe growth retardation phenotype (54). The markedly reduced growth of Jnk1−/− Jnk2−/− MEFs is consistent with the finding that JNK is critically required for the regulation of AP1-dependent gene expression (56) that is implicated in cellular proliferation (26). Thus, Jnk1−/− Jnk2−/− MEFs express low levels of AP1 proteins (e.g., c-Jun and JunD) and exhibit marked defects in AP1 target gene expression (34, 56). This loss of AP1 function is mediated, in part, by reduced phosphorylation of the activation domain of Jun family proteins and ATF2 (56).More recent studies using a conditional gene ablation strategy have demonstrated that compound JNK deficiency causes rapid senescence (12). This conclusion was confirmed by using chemical genetic analysis with MEFs isolated from mice with a germ line mutation that sensitizes JNK to inhibition by a predesigned small-molecule drug (12, 25). This form of senescence was found to be p53 dependent (12) and resembles the p53-dependent senescence of c-Jun−/− MEFs (49). These data indicate that JNK plays a critical role in cellular proliferation. Indeed, it is possible that the p53-dependent senescence observed in JNK-deficient cells may contribute to aging. This is because altered p53 function is established to be an important determinant of early aging (36, 55). Importantly, this role of p53 in aging appears to be distinct from p53-mediated tumor suppression and DNA damage responses (21, 39, 43).One aspect of the aging process is a reduction in the regenerative capacity of stem cells (50). Indeed, it has been established that altered p53 activity associated with aging causes decreased stem cell function (8, 18, 42) and that disruption of the p53 pathway can increase stem cell function (1). Since JNK can influence p53-dependent senescence (12), these data indicate that JNK may be important for stem cell proliferation and self-renewal potential.Embryonic stem (ES) cells proliferate and are capable of both self-renewal and differentiation to multiple cell types. Indeed, murine ES cells can differentiate to create all tissues within a mouse. The profound growth retardation and rapid p53-dependent senescence of Jnk1−/− Jnk2−/− MEFs (12) suggests that JNK may play a critical role in the normal function of ES cells, including self-renewal and differentiation potential. The purpose of the present study was to test this hypothesis. Our approach was to isolate ES cells from wild-type and JNK-deficient mice. We demonstrate that JNK is not required for self-renewal or the proliferation of ES cells. However, JNK is required for ES cell differentiation.  相似文献   

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Accumulation of tau into neurofibrillary tangles is a pathological consequence of Alzheimer''s disease and other tauopathies. Failures of the quality control mechanisms by the heat shock proteins (Hsps) positively correlate with the appearance of such neurodegenerative diseases. However, in vivo genetic evidence for the roles of Hsps in neurodegeneration remains elusive. Hsp110 is a nucleotide exchange factor for Hsp70, and direct substrate binding to Hsp110 may facilitate substrate folding. Hsp70 complexes have been implicated in tau phosphorylation state and amyloid precursor protein (APP) processing. To provide evidence for a role for Hsp110 in central nervous system homeostasis, we have generated hsp110/ mice. Our results show that hsp110/ mice exhibit accumulation of hyperphosphorylated-tau (p-tau) and neurodegeneration. We also demonstrate that Hsp110 is in complexes with tau, other molecular chaperones, and protein phosphatase 2A (PP2A). Surprisingly, high levels of PP2A remain bound to tau but with significantly reduced activity in brain extracts from aged hsp110/ mice compared to brain extracts from wild-type mice. Mice deficient in the Hsp110 partner (Hsp70) also exhibit a phenotype comparable to that of hsp110/ mice, confirming a critical role for Hsp110-Hsp70 in maintaining tau in its unphosphorylated form during aging. In addition, crossing hsp110/ mice with mice overexpressing mutant APP (APPβsw) leads to selective appearance of insoluble amyloid β42 (Aβ42), suggesting an essential role for Hsp110 in APP processing and Aβ generation. Thus, our findings provide in vivo evidence that Hsp110 plays a critical function in tau phosphorylation state through maintenance of efficient PP2A activity, confirming its role in pathogenesis of Alzheimer''s disease and other tauopathies.Diseases like Alzheimer''s disease (AD) and other tauopathies are defined by the expression of neurofibrillary tangles (NFTs) deposited mainly in neurons. The NFTs are aggregates of the hyperphosphorylated tau (p-tau) (3, 74). Normal tau increases microtubule stability, but tau can be hyperphosphorylated under disease conditions and released from microtubules (3, 5, 6). The molecular mechanisms involved in the formation of NFTs are not completely understood. However, accumulation of abnormal p-tau and NFTs causes neurodegeneration (3). A number of protein kinases, including glycogen synthase kinase 3 (GSK3) and cyclin-dependent protein kinase 5 (CDK5), have been shown to phosphorylate tau at Thr231 and Ser262 as well as several other sites that flank the microtubule binding repeat, leading to tangles of paired helical filaments (PHFs) similar to those observed in the brains of patients with AD (54, 72). Evidence shows that GSK3 physically interacts with tau and is thought to be the main contributor to the formation of NFTs and amyloid β (Aβ) plaques in AD patients (18, 53, 54). Phosphorylation of GSK3a/b at S9/S21 which is inhibitory to its activity during insulin signaling, leads to phosphorylation of tau in neurons (80). GSK3a/b phospho-S9/S21, p-tau, and 14-3-3zeta have been isolated in a 500-kDa complex, and the interaction has been shown to result in tau phosphorylation by GSK3 (1, 80). Although not well characterized, p-tau has been shown to be dephosphorylated by the B family regulatory subunit of the heterotrimeric PP2A holoenzyme (76). There are two protein phosphatase 2A (PP2A) binding sites on microtubule tau binding repeats, perhaps allowing tau to be more efficiently dephosphorylated by PP2A catalytic subunit (76).Both GSK3 and CDK5 are also known to be involved in the phosphorylation of amyloid precursor protein (APP) at Thr668 and APP processing and Aβ production (53, 58). Studies suggest that amyloid peptide can activate GSK3 signaling, and the increase in GSK3 activity can then contribute to abnormal APP processing. Indeed, reduction in GSK3 activity reduces amyloid peptide production in murine AD models (18, 53, 57, 71). Reduction in PP2A activity leads to altered APP regulation as well (26, 43). Additional molecules that affect tau hyperphosphorylation and APP processing are the peptidyl prolyl isomerases (9, 36, 51). Deletion of Pin1 isomerase in vivo leads to p-tau and neurodegeneration (42). Crossing Pin1-deficient mice with transgenic mice expressing mutant APP (APPβsw) leads to abnormal APP processing and accumulation of toxic amyloid β42 (Aβ42) species. Pin1, therefore, is implicated in isomerization of tau, perhaps facilitating its dephosphorylation (42). The presence of Pin1 has been implicated in promoting nonamyloidogenic processing of APP and reduction in toxic Aβ42 production (51).Hsp70/Hsc70 has been shown to preferentially bind to a hyperphosphorylated form of tau in the diseased human brain (49). Cross talk between the ubiquitin proteasome system (UPS) and molecular chaperones might also be critical in regulating the deposition and toxicity of tau (8, 16). These results suggest that the activity of Hsp70 and Hsp90 preserve the native structure and function of tau protein. Hsp70 and the C-terminal Hsp70-interacting protein (Chip) have been shown to regulate tau ubiquitination and degradation (11, 12, 21, 52, 65). Interestingly, Chip and βAPP interact, and Chip and Hsp70/90 expression have been shown to lower the cellular levels of Aβ and reduce Aβ toxicity in vitro (39). Misfolded proteins are either degraded through the UPS or are folded, at least in part, by the Hsps (4, 7).Eukaryotic cells possess a class of heat shock proteins (Hsps) related to the Hsp70 family. This Hsp100 family of proteins contains Hspa41 (Apg1 or OSP94), Hsp94 (Apg2), and Hsp110 (2, 17, 28, 61, 70, 77, 78). They were initially considered to be “holdases” that keep denatured proteins in solution, and no client proteins have been described for them (14, 15, 56, 62). Hsp110 interacts with Hsp70 and increases its ATPase activity (15, 56, 62). The main function of Hsp110 appears to be a nucleotide exchange factor (NEF) for Hsp70 (14, 64). In general, Hsp110 is known to induce suppression of aggregation and protein refolding, and it protects proteins from the damaging effects of various stresses; however, its physiological function in mammalian cells remains unknown (15, 60). In these studies, we examined the role of Hsp110 in central nervous system (CNS) homeostasis in vivo. We have found that hsp110/ mice exhibit an age-dependent accumulation of p-tau that is associated with pathological features, such as the appearance of NFTs and neurodegeneration. We also show that lack of Hsp110 leads to accelerated pathology as evidenced by the early appearance of senile plaques containing Aβ42 (a major toxic species [46]) in an AD transgenic mouse model. At the biochemical level, we show that Hsp110 interacts with tau, a number of Hsps, GSK3, Pin1, and PP2A. Furthermore, tau immunocomplexes pulled down from hsp110/ brain extracts possess elevated levels of PP2A, but the pulled-down PP2A has significantly lower activity than the PP2A from wild-type mice. Our studies therefore suggest a critical role for Hsp110 in maintaining the proper folding environment that is required for phosphorylation and dephosphorylation of tau and APP processing in vivo.  相似文献   

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Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.Cell migration is essential for morphogenesis during embryonic development and for epithelial homeostasis and wound healing throughout life. As myosin II is involved in all aspects of cell migration, from cell polarization and adhesion to protrusion and tail retraction (34, 48), the signaling pathways regulating myosin-dependent cytoskeletal contraction are of particular interest. Myosin contraction is regulated by phosphorylation of myosin regulatory light chain (MRLC) at Thr18/Ser19. Although a number of kinases have been identified which phosphorylate these sites, the principal kinases in most cells are myosin light chain kinase (MLCK), a calcium/calmodulin-regulated enzyme, and Rho kinase (ROCK), a downstream effector of the Rho family GTPase RhoA. To provide the stringent control of cytoskeletal contraction needed for migration, RhoA is subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1 (4, 21), and negative regulation by GTPase-activating proteins (GAPs), such as the Src-regulated protein p190RhoGAP (1, 3, 10, 13). An additional level of regulation is provided by guanine nucleotide dissociation inhibitors, which bind to inactive RhoA and other Rho family GTPases, sequestering them in the cytosol (3). Two major downstream effectors of RhoA with regard to the cytoskeleton are the mammalian homologue of diaphanous, involved in actin polymerization (43), and ROCK, which phosphorylates MRLC and myosin phosphatase (20).Cdk5, a serine/threonine kinase, is an atypical member of the well-known family of cyclin-dependent kinases (Cdks). Unlike the other Cdks, it has no known function in cell cycle regulation and is activated by one of two noncyclin proteins, p35 or p39 (16, 41). Phosphorylation of Cdk5 at Y15 increases its activity severalfold (36, 49). Although Cdk5 is most abundant in neuronal cells, where it regulates migration, cytoskeletal dynamics, and membrane trafficking (37, 38, 45), a growing body of evidence indicates that Cdk5 has similar functions in nonneuronal cells (35). In particular, Cdk5 has been shown to strengthen cell-to-matrix adhesion and regulate migration in lens epithelial cells (28), corneal epithelial cells (11, 12, 40), keratinocytes (27), and CHO-K1 cells (15). The effects of Cdk5 on adhesion and migration have been linked, at least in part, to Cdk5-dependent phosphorylation of talin, which strengthens adhesion by slowing the rate of focal adhesion turnover (15). However, we have observed that Cdk5 not only binds to focal adhesions, where talin is located, but also to stress fibers (33). Moreover, in spreading cells, Cdk5 exerts its greatest effect on adhesion 1 to 2 h after plating (28), when stress fiber contraction is pronounced and Cdk5 activity is maximum (33). Therefore, we hypothesized that Cdk5 might regulate the MRLC phosphorylation necessary for stress fiber contraction and stability. To test this possibility, we examined the relationship of Cdk5 activity to MRLC phosphorylation and cytoskeletal contraction in spreading human lens epithelial cells.  相似文献   

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Rag2−/− γC−/− mice transplanted with human hematopoietic stem cells (DKO-hu-HSC mice) mimic aspects of human infection with human immunodeficiency virus type 1 (HIV-1), including sustained viral replication and CD4+ T-cell decline. However, the extent of HIV-1 evolution during long-term infection in these humanized mice, a key feature of the natural infection, has not been assessed fully. In this study, we examined the types of genotypic and phenotypic changes in the viral env gene that occur in the viral populations of DKO-hu-HSC mice infected with the CCR5-tropic isolate HIV-1JRCSF for up to 44 weeks. The mean rate of divergence of viral populations in mice was similar to that observed in a cohort of humans during a similar period of infection. Many amino acid substitutions were common across mice, including losses of N-linked glycosylation sites and substitutions in the CD4 binding site and in CD4-induced epitopes, indicating common selective pressures between mice. In addition, env variants evolved sensitivity to antibodies directed at V3, suggesting a more open conformation for Env. This phenotypic change was associated with increased CD4 binding efficiency and was attributed to specific amino acid substitutions. In one mouse, env variants emerged that exhibited a CXCR4-tropic phenotype. These sequences were compartmentalized in the mesenteric lymph node. In summary, viral populations in these mice exhibited dynamic behavior that included sequence evolution, compartmentalization, and the appearance of distinct phenotypic changes. Thus, humanized mice offer a useful model for studying evolutionary processes of HIV-1 in a complex host environment.Animal models of HIV-1 infection are important tools for studying transmission, replication, and pathogenesis, as well as therapeutic intervention, of HIV-1 infection. Nonhuman primates such as rhesus macaques, infected with simian or chimeric simian/human immunodeficiency viruses (SIV or SHIV, respectively), represent well-characterized and highly relevant models; however, key limitations include expense, genetic variability of the host animals, and the fact that SIV, while closely related, is distinct from HIV-1. Therefore, small animal models that support HIV-1 infection and recapitulate many aspects of the human infection have been sought using several approaches.Recent approaches have involved the use of genetically immunodeficient mice that have been reconstituted using human-derived hematopoietic stem cells (HSC) (known as humanized mice). Several models have been developed based on this approach, including Rag2−/− γC−/− (DKO) and NOD/SCID/γC−/− (NOG or NSG) mice transplanted with human HSC (DKO-hu-HSC or NOG-hu-HSC mice) (40, 92) and the NOD/SCID mouse with transplanted human fetal thymus and liver tissue in addition to HSC (62). These models all support HIV-1 infection (1, 3, 6, 30, 87, 96, 102; for a review of these models, see the work of Denton and Garcia [22]). The DKO-hu-HSC mouse lacks both recombination activating gene 2 (Rag2) and the cytokine receptor common gamma chain (γC), and as a result, it does not generate murine T, B, and natural killer (NK) cells but supports engraftment of HSC and differentiation of human myeloid and lymphoid lineages. Immune reconstitution in this model likely involves education of human T cells in the mouse thymus and dissemination of differentiated human lymphoid subsets into the peripheral blood and to multiple lymphoid tissues, including lymph nodes, spleen, and bone marrow (92). The DKO-hu-HSC mouse, along with the other humanized mouse models, has been used in studies of transmission (5, 21), pathogenesis (43), and viral inhibition (16, 21, 53, 88, 94).One important feature of HIV-1 infection is the diversification and evolution of the viral genome over the course of infection. Diversification occurs most prominently in the envelope (env) gene, which encodes the viral surface glycoprotein (Env). Env mediates viral entry into cells through attachment to the primary receptor CD4, which primes Env for engagement with a coreceptor, either CCR5 or CXCR4, triggering virion fusion with the cellular plasma membrane (54). HIV-1 infection is typically established by one or a few CCR5-tropic (R5) variants that give rise to an initially homogenous viral population, which then diversifies over the course of chronic infection (45, 84). Diversification of Env results from immune selective pressures (27), isolation in or adaptation to different cellular and anatomical compartments (20, 28, 33, 46, 51), and selection for altered CD4 affinity (72, 90, 95) and coreceptor tropism (26, 39). In many cases, during late-stage infection, variants emerge from the R5 virus population that are CXCR4 tropic (X4), an event that is often associated with accelerated CD4 T-cell loss and progression to AIDS (9, 18, 89). In an effort to determine if any of these aspects of HIV-1 evolution are exhibited in the humanized mouse model, we examined the extent of HIV-1 diversification and the types of evolutionary changes that occur in env in mice infected with CCR5-tropic HIV-1 for up to 44 weeks.Sampling of viral env variants from the peripheral blood plasma over the course of the infection revealed increasing diversity and divergence of the viral population at rates similar to those observed in natural infection. Mutations were identified that affected Env conformation and sensitivity to neutralizing antibodies, CXCR4 coreceptor use, and potential N-linked glycosylation sites. Other mutations potentially affecting the Env phenotype were identified in CD4 binding sites and CD4-induced epitopes. The patterns of substitutions indicated that certain sites were under selection, particularly in cases where the same substitution was identified in multiple mice.This study demonstrates the potential for studying HIV-1 evolution in the DKO-hu-HSC mouse model and also gives insight into the types of selective pressures driving HIV-1 env evolution in this host environment. These findings, while highlighting some of the limitations of this model, will help to inform its appropriate use for studying different aspects of HIV-1 infection, such as the evolutionary constraints placed on HIV-1 during natural infection and in the face of pharmacological and immunological inhibition.  相似文献   

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