Effectiveness of statins for secondary prevention in elderly patients after acute myocardial infarction: an evaluation of class effect

Background

Clinical trials have shown the benefits of statins after acute myocardial infarction (AMI). However, it is unclear whether different statins exert a similar effect in reducing the incidence of recurrent AMI and death when used in clinical practice.

Methods

We conducted a retrospective cohort study (1997–2002) to compare 5 statins using data from medical administrative databases in 3 provinces (Quebec, Ontario and British Columbia). We included patients aged 65 years and over who were discharged alive after their first AMI-related hospital stay and who began statin treatment within 90 days after discharge. The primary end point was the combined outcome of recurrent AMI or death from any cause. The secondary end point was death from any cause. Adjusted hazard ratios (HRs) for each statin compared with atorvastatin as the reference drug were estimated using Cox proportional hazards regression analysis.

Results

A total of 18 637 patients were prescribed atorvastatin (n = 6420), pravastatin (n = 4480), simvastatin (n = 5518), lovastatin (n = 1736) or fluvastatin (n = 483). Users of different statins showed similar baseline characteristics and patterns of statin use. The adjusted HRs (and 95% confidence intervals) for the combined outcome of AMI or death showed that each statin had similar effects when compared with atorvastatin: pravastatin 1.00 (0.90–1.11), simvastatin 1.01 (0.91– 1.12), lovastatin 1.09 (0.95–1.24) and fluvastatin 1.01 (0.80– 1.27). The results did not change when death alone was the end point, nor did they change after adjustment for initial daily dose or after censoring of patients who switched or stopped the initial statin treatment.

Interpretation

Our results suggest that, under current usage, statins are equally effective for secocondary prevention in elderly patients after AMI.Randomized controlled trials (RCTs) have shown that the use of statins after acute myocardial infarction (AMI) are effective in reducing the incidence of both fatal and nonfatal cardiovascular events.^{1,2,3,4,5,6,7,8} Although these trials have significantly influenced post-AMI treatment,^9,10,11,12 it remains unclear whether all statins are equally effective in preventing recurrent AMI and death. Drugs in the same class are generally thought to be therapeutically equivalent because of similar mechanisms of action (class effect).^13,14,15 However, in the absence of comparative data, this assumption requires evaluation. Statins differ in multiple characteristics, including liver and renal metabolism, half-life, effect on other serum lipid components, bioavailability and potency.^16,17,18,19 These differences could potentially influence the extent to which the drugs are beneficial. Despite limited evidence in support of a differential benefit of statins for secondary prevention, preferential prescribing already occurs in practice and cannot be fully explained by the existing evidence or guidelines.²⁰ Comparative data of statins are thus required to inform health care decision-making.A number of RCTs have directly compared statins using surrogate end points, such as lipid reduction,^21,22,23 markers of hemostasis and inflammation^24,25,26 or reduction in number of atherotic plaques.²⁷ However, the extent to which these results can be extrapolated to clinically relevant outcomes remains to be established. The newly released PROVE IT– TIMI 22 trial²⁸ was the first trial to compare 2 statins for cardiovascular prevention. The study showed that atorvastatin used at a maximal dose of 80 mg (intensive therapy) was better than pravastatin at a dose of 40 mg (standard therapy) in decreasing the incidence of cardiovascular events and procedures. The study was, however, conducted to show the benefit associated with increased treatment intensity. It did not compare the drugs by milligram-equivalent doses or by cholesterol-lowering equivalent doses. Moreover, no difference was detected when death alone or the combined outcome of death or AMI was evaluated. Other than the PROVE IT–TIMI 22 trial, few data are currently available from RCTs that compare statins for cardiovascular prevention.²⁹We conducted a population-based study to examine the relative effectiveness of different statins for long-term secondary prevention after AMI. We used retrospective cohorts of elderly patients prescribed statins after AMI in 3 provinces. Five statins were studied: atorvastatin, pravastatin, simvastatin, lovastatin and fluvastatin. The newest statin, rosuvastatin, was not available during the study period and was not considered in this study.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Characteristics of women undergoing repeat induced abortion

Background

Although repeat induced abortion is common, data concerning characteristics of women undergoing this procedure are lacking. We conducted this study to identify the characteristics, including history of physical abuse by a male partner and history of sexual abuse, of women who present for repeat induced abortion.

Methods

We surveyed a consecutive series of women presenting for initial or repeat pregnancy termination to a regional provider of abortion services for a wide geographic area in southwestern Ontario between August 1998 and May 1999. Self-reported demographic characteristics, attitudes and practices regarding contraception, history of relationship violence, history of sexual abuse or coercion, and related variables were assessed as potential correlates of repeat induced abortion. We used χ² tests for linear trend to examine characteristics of women undergoing a first, second, or third or subsequent abortion. We analyzed significant correlates of repeat abortion using stepwise multivariate multinomial logistic regression to identify factors uniquely associated with repeat abortion.

Results

Of the 1221 women approached, 1145 (93.8%) consented to participate. Data regarding first versus repeat abortion were available for 1127 women. A total of 68.2%, 23.1% and 8.7% of the women were seeking a first, second, or third or subsequent abortion respectively. Adjusted odds ratios for undergoing repeat versus a first abortion increased significantly with increased age (second abortion: 1.08, 95% confidence interval [CI] 1.04–1.09; third or subsequent abortion: 1.11, 95% CI 1.07–1.15), oral contraceptive use at the time of conception (second abortion: 2.17, 95% CI 1.52–3.09; third or subsequent abortion: 2.60, 95% CI 1.51–4.46), history of physical abuse by a male partner (second abortion: 2.04, 95% CI 1.39–3.01; third or subsequent abortion: 2.78, 95% CI 1.62–4.79), history of sexual abuse or violence (second abortion: 1.58, 95% CI 1.11–2.25; third or subsequent abortion: 2.53, 95% CI 1.50–4.28), history of sexually transmitted disease (second abortion: 1.50, 95% CI 0.98–2.29; third or subsequent abortion: 2.26, 95% CI 1.28–4.02) and being born outside Canada (second abortion: 1.83, 95% CI 1.19–2.79; third or subsequent abortion: 1.75, 95% CI 0.90–3.41).

Interpretation

Among other factors, a history of physical or sexual abuse was associated with repeat induced abortion. Presentation for repeat abortion may be an important indication to screen for a current or past history of relationship violence and sexual abuse.Repeat pregnancy termination procedures are common in Canada (where 35.5% of all induced abortions are repeat procedures)^1,2 and the United States (where 48% of induced abortions are repeat procedures).^3,4,5,6,7 Rates of repeat induced abortion increased in both countries for an initial period after abortion was legalized, as a result of an increase in the number of women who had access to a first, and consequently to repeat, legal induced abortion.^1,6,8,9 At present, rates of initial and repeat abortion in Canada and the United States appear to be stabilizing.^2,7Research concerning characteristics of women who undergo repeat induced abortions has been limited in scope. In a literature search we identified fewer than 20 studies in this area published over the past 3 decades. However, available research has shown several consistent findings. Women undergoing repeat abortions are more likely than those undergoing a first abortion to report using a method of contraception at the time of conception.^7,8,10,11 In addition, women seeking repeat abortions report more challenging family situations than women seeking initial abortions: they are more likely to be separated, divorced, widowed or living in a common-law marriage, and to report difficulties with their male partner.^1,5,8,11,12 They also are older,^7,13 have more children^1,5,13 and are more often non-white^7,11,13 than women seeking initial abortions.There is little evidence to suggest that women seeking repeat abortion are using pregnancy termination as a method of birth control.^1,5,6,8,11 Evidence also does not indicate that women seeking repeat abortion are psychologically maladjusted.^8,13Our literature review showed that many studies of repeat abortion are 20 to 30 years old and are based on data collected when abortion was a newly legalized procedure.^5,11 Furthermore, in studies of correlates of repeat abortion the investigators did not examine a range of personality characteristics that are known to influence women''s reproductive health outcomes,^14,15 including attitudes about sexuality,¹⁴ health locus of control,^16,17 degree of social integration,¹⁶ attitudes about contraception^18,19 and history of sexual or physical abuse.^20,21,22 The objective of the current study was to identify characteristics of women who undergo repeat induced abortion.     

Breast size and risk of type 2 diabetes mellitus

Background

Elevated waist circumference and body mass index (BMI), both traditional measures of obesity, are accepted risk factors for type 2 diabetes mellitus. Girls who are obese experience earlier onset of puberty and possibly greater breast development. We sought to evaluate whether a woman''s breast size in late adolescence is associated with an increased risk of type 2 diabetes mellitus in adulthood.

Methods

In conjunction with the ongoing Nurses'' Health Study II, which began to study risk factors for breast cancer among women in 1989, we conducted a prospective cohort study involving 92 106 of the participants. We assessed the risk of type 2 diabetes mellitus in relation to self-reported bra cup sizes, categorized as ≤ A, B, C and ≥ D cups, among participants at age 20.

Results

The mean age of participants at baseline was 38.1 years. A total of 1844 new cases of type 2 diabetes mellitus arose at a mean age of 44.9 years during 886 443 person-years of follow-up. Relative to bra cup size ≤ A, the respective age-adjusted hazard ratios (and 95% confidence intervals [CIs]) were 2.30 (1.99–2.66) for B cup, 4.32 (3.71–5.04) for C cup and 4.99 (4.12–6.05) for ≥ D cup. Upon further adjustments for age at menarche, parity, physical activity, smoking status, diet, multivitamin use, family history of diabetes mellitus, BMI at age 18 and current BMI, the corresponding hazard ratios (and 95% CIs) were 1.37 (1.18–1.59) for B cup, 1.80 (1.53- 2.11) for C cup and 1.64 (1.34–2.01) for ≥ D cup. The addition of waist circumference to this model minimally changed the hazard ratios (and 95% CIs): 1.32 (1.14–1.53) for B cup, 1.71 (1.46–2.01) for C cup and 1.58 (1.29–1.94) for ≥ D cup.

Interpretation

A large bra cup size at age 20 may be a predictor of type 2 diabetes mellitus in middle-aged women. Whether this relation is independent of traditional indicators of obesity remains to be determined.Obesity is an established risk factor for type 2 diabetes mellitus.^1,2 Affected individuals show signs of insulin resistance and hyperinsulinemia, a process that may begin in childhood.^3,4 Pre-adolescent obesity is also an important predictor of age of onset of breast development in young women, and of breast size after puberty.^5,6 Premature onset of puberty is preceded by childhood insulin resistance, hyperinsulinemia and hyperandrogenemia,⁷ which may persist after puberty⁸ and continue into early adulthood.⁹Although an elevated body mass index (BMI)^10,11 and central adiposity¹² are established risk factors for insulin resistance and the onset of type 2 diabetes mellitus, little is known about the contribution of extra-abdominal adipose tissue, including breast tissue, about 60% of which is fatty tissue, to this process.^13,14 We hypothesized that a woman''s breast size in late adolescence reflects her predisposition to insulin resistance and type 2 diabetes mellitus that is both additive to, and independent of, BMI. We explored this hypothesis in conjunction with the Nurses'' Health Study II by relating bra cup size, a proxy for breast size, to the onset of type 2 diabetes mellitus.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Impact of patient communication problems on the risk of preventable adverse events in acute care settings

Background

Up to 50% of adverse events that occur in hospitals are preventable. Language barriers and disabilities that affect communication have been shown to decrease quality of care. We sought to assess whether communication problems are associated with an increased risk of preventable adverse events.

Methods

We randomly selected 20 general hospitals in the province of Quebec with at least 1500 annual admissions. Of the 145 672 admissions to the selected hospitals in 2000/01, we randomly selected and reviewed 2355 charts of patients aged 18 years or older. Reviewers abstracted patient characteristics, including communication problems, and details of hospital admission, and assessed the cause and preventability of identified adverse events. The primary outcome was adverse events.

Results

Of 217 adverse events, 63 (29%) were judged to be preventable, for an overall population rate of 2.7% (95% confidence interval [CI] 2.1%–3.4%). We found that patients with preventable adverse events were significantly more likely than those without such events to have a communication problem (odds ratio [OR] 3.00; 95% CI 1.43–6.27) or a psychiatric disorder (OR 2.35; 95% CI 1.09–5.05). Patients who were admitted urgently were significantly more likely than patients whose admissions were elective to experience an event (OR 1.64, 95% CI 1.07–2.52). Preventable adverse events were mainly due to drug errors (40%) or poor clinical management (32%). We found that patients with communication problems were more likely than patients without these problems to experience multiple preventable adverse events (46% v. 20%; p = 0.05).

Interpretation

Patients with communication problems appeared to be at highest risk for preventable adverse events. Interventions to reduce the risk for these patients need to be developed and evaluated.Patient safety is a priority in modern health care systems. From 3% to 17% of hospital admissions result in an adverse event,^1–8 and almost 50% of these events are considered to be preventable.^3,9–12 An adverse event is an unintended injury or complication caused by delivery of clinical care rather than by the patient''s condition. The occurrence of adverse events has been well documented; however, identifying modifiable risk factors that contribute to the occurrence of preventable adverse events is critical. Studies of preventable adverse events have focused on many factors, but researchers have only recently begun to evaluate the role of patient characteristics.^2,9,12,13 Older patients and those with a greater number of health problems have been shown to be at increased risk for preventable adverse events.^10,11 However, previous studies have repeatedly suggested the need to investigate more diverse, modifiable risk factors.^{3,6,7,10,11,14–16}Language barriers and disabilities that affect communication have been shown to decrease quality of care;^16–20 however, their impact on preventable adverse events needs to be investigated. Patients with physical and sensory disabilities, such as deafness and blindness, have been shown to face considerable barriers when communicating with health care professionals.^20–24 Communication disorders are estimated to affect 5%–10% of the general population,²⁵ and in one study more than 15% of admissions to university hospitals involved patients with 1 or more disabilities severe enough to prevent almost any form of communication.²⁶ In addition, patients with communication disabilities are already at increased risk for depression and other comorbidities.^27–29 Determining whether they are at increased risk for preventable adverse events would permit risk stratification at the time of admission and targeted preventive strategies.We sought to estimate the extent to which preventable adverse events that occurred in hospital could be predicted by conditions that affect a patient''s ability to communicate.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.One of the major challenges to overcome in the development of hepatitis C virus (HCV)-directed antivirals is the high propensity of the virus to mutate. This is due to the lack of proofreading capacity of the HCV NS5B RNA-dependent RNA polymerase (RdRp), which replicates the HCV RNA strand with an error rate of 10⁻² to 10⁻³ nucleotide substitutions per site per year (17). The diversity of HCV has been recognized as six phylogenetically distinct groups, referred to as genotypes and, within each genotype, as subtypes (a, b, c, d, etc.) (44). HCV subtype 1b is the most prevalent genotype in the world, and subtype 1a is widely distributed in northern Europe and in the United States; subtypes 2a and 2b are common in North America, Europe, and Japan, and subtype 2c is found predominantly in Northern Italy; HCV genotype 3a is more prevalent in the Far East and has recently increased in Europe and in the United States, possibly due to the spread of the virus through intravenous drug use (11, 17, 18, 44, 46). Of the other genotypes, genotype 4 is common in Africa and to a lesser extent in Europe (11, 39), whereas genotypes 5 and 6 are found predominantly in southern Africa and Southeast Asia, respectively. Despite the availability of a standard of care for the treatment of hepatitis C, a combination of pegylated alpha interferon and ribavirin, many HCV-infected patients cannot be cured because of the frequent failure of the treatment, particularly in patients with genotype 1 and 4 infections, and perhaps also in those with genotype 6 infections (12, 35). In addition, tolerability issues associated with the standard of care lead to discontinuation of therapy in many patients (31). Therefore, major efforts have been made toward developing novel oral therapeutics that target a specific step of the HCV life cycle (45), with particular attention to HCV subtypes 1a and 1b, as they are the most common genotypes underserved by the current standard of care. Subtypes 1a and 1b are estimated to account for 57% and 17% of the HCV-infected patients in the United States (1) versus approximately 11% and 45% in Europe (11), respectively.The development of HCV polymerase nonnucleoside inhibitors (NNIs) has been successfully validated in phase II clinical trials (21, 24, 41). From the extensive screening of NS5B inhibitors that has been performed to date, several chemotypes have emerged as promising scaffolds, namely, the indole, thiophene, benzothiadiazine, and benzofuran analogs. Each of these NNIs targets four different binding pockets of the HCV polymerase, thumb-1 NNI-1 (10), thumb-2 NNI-2 (29, 48), palm-1 NNI-3 (9), and palm-2 NNI-4 (19), respectively. Historically, the screen for novel NS5B inhibitors was limited to representatives of genotype 1b only (3, 28) because the tools to target other genotypes were not yet available (16, 38, 40). Further assessment of these analogs, using enzyme isolates and intergenotypic chimeric replicons derived from clinical isolates, revealed that the genotypic coverage of the NNI-1 and -4 analogs extends beyond genotype 1, unlike the NNI-2 and -3 derivatives that typically inhibit genotype 1 only (16, 38). An additional drawback stems from the lower genetic barrier of the NNI-2 and -3 analogs in genotype 1 (25) and the reduced susceptibility in subtype 1a of the NNI-3 series (7, 16, 34, 38, 43). This effect was mostly attributed to the Y415F polymorphism observed in the NNI-3 binding site in subtype 1a (38).Here we report the 1a/1b subtype profiling of 1,5-benzodiazepine (1,5-BZD) HCV polymerase inhibitors that bind to the NNI-3 site (32, 36) using a panel of enzyme and chimeric replicons derived from clinical isolates, X-ray crystallography, and surface plasmon resonance (SPR) studies and compare these inhibitors to the four classes of NNIs by thoroughly assessing a representative of each nonnucleoside binding site in subtypes 1a and 1b.     

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient''s pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4⁺ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4⁺ T cells in the presence of APL, with relative sparing of the central memory CD4⁺ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T_CM subset of CD4⁺ T cells and result in improved T cell homeostasis and immune function.Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9, 24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8, 15, 26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23, 35, 49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4, 18, 42, 43, 50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the N′ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13, 31, 32, 46, 52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47, 54).CCR5 antagonists are unusual among antiretroviral agents in that they bind to a host protein rather than a viral target, and therefore the virus cannot directly mutate the drug binding site to evade pharmacologic pressure. Nevertheless, HIV can escape susceptibility to CCR5 antagonists. One mechanism by which this occurs is the use of the alternative HIV coreceptor, CXCR4. In vivo, this has most often been manifest as the outgrowth of R5/X4-tropic HIV isolates that were present in the patient''s circulating viral swarm prior to therapy (17, 27, 55). A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1, 2, 22, 33, 36, 51, 56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21, 48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc. Surprisingly, this antiretroviral-naïve patient harbored Envs resistant to aplaviroc prior to the initiation of therapy. In the present study, we have examined viral and host factors that contribute to aplaviroc resistance and examined the consequences of resistance for viral tropism. Aplaviroc resistance determinants were located within the V3 loop of gp120, although additional residues diffusely spread throughout the gp120 and gp41 proteins modulated the magnitude of drug resistance. The resistant virus displayed altered interactions between gp120 and CCR5 such that the virus became critically dependent upon the N′ terminus of drug-bound CCR5. This differential recognition of CCR5 in the presence of aplaviroc was also associated with increased dependence on a higher CCR5 receptor density for efficient virus infection and a tropism shift toward effector memory cells on primary CD4⁺ T cells.