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.     

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.     

Effect of B-Cell Depletion on Viral Replication and Clinical Outcome of Simian Immunodeficiency Virus Infection in a Natural Host

Simian immunodeficiency virus (SIV)-infected African nonhuman primates do not progress to AIDS in spite of high and persistent viral loads (VLs). Some authors consider the high viral replication observed in chronic natural SIV infections to be due to lower anti-SIV antibody titers than those in rhesus macaques, suggesting a role of antibodies in controlling viral replication. We therefore investigated the impact of antibody responses on the outcome of acute and chronic SIVagm replication in African green monkeys (AGMs). Nine AGMs were infected with SIVagm.sab. Four AGMs were infused with 50 mg/kg of body weight anti-CD20 (rituximab; a gift from Genentech) every 21 days, starting from day −7 postinfection up to 184 days. The remaining AGMs were used as controls and received SIVagm only. Rituximab-treated AGMs were successfully depleted of CD20 cells in peripheral blood, lymph nodes (LNs), and intestine, as shown by the dynamics of CD20⁺ and CD79a⁺ cells. There was no significant difference in VLs between CD20-depleted AGMs and control monkeys: peak VLs ranged from 10⁷ to 10⁸ copies/ml; set-point values were 10⁴ to 10⁵ SIV RNA copies/ml. Levels of acute mucosal CD4⁺ T-cell depletion were similar for treated and nontreated animals. SIVagm seroconversion was delayed for the CD20-depleted AGMs compared to results for the controls. There was a significant difference in both the timing and magnitude of neutralizing antibody responses for CD20-depleted AGMs compared to results for controls. CD20 depletion significantly altered the histological structure of the germinal centers in the LNs and Peyer''s patches. Our results, although obtained with a limited number of animals, suggest that humoral immune responses play only a minor role in the control of SIV viral replication during acute and chronic SIV infection in natural hosts.In marked contrast to pathogenic human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections of humans and macaques, which are characterized by the constant progression to AIDS in a variable time frame (26), African monkey species naturally infected with SIV are generally spared from any signs of disease (reviewed in references 53 and 71).There are currently three animal models of SIV infection in natural hosts: SIVagm infection of African green monkeys (AGMs), SIVsmm infection of sooty mangabeys, and SIVmnd-1 and SIVmnd-2 infection of mandrills (53, 71). SIV infection in natural hosts is characterized by the following: (i) active viral replication, with set-point viral loads (VLs) similar to or even higher than those found in pathogenic infections (44-46, 49, 50, 52, 61-63); (ii) transient depletion of peripheral CD4⁺ T cells during primary infection, which rebound to preinfection levels during chronic infection (12, 30, 44-46, 49, 62); (iii) significant CD4⁺ T-cell depletion in the intestine, which can be partially restored during chronic infection in spite of significant viral replication (21, 48); (iv) low levels of CD4⁺ CCR5⁺ cells in blood and tissues (47); (v) transient and moderate increases in immune activation and T-cell proliferation during acute infection, with a return to baseline levels during the chronic phase (44-46, 49, 50, 52, 61-63), as a result of an anti-inflammatory milieu which is rapidly established after infection (14, 30); and (vi) no significant increase in CD4⁺ T-cell apoptosis during either acute or chronic infection (37, 48), thus avoiding enteropathy and microbial translocation, which control excessive immune activation and prevent disease progression by allowing CD4⁺ T-cell recovery in the presence of high VLs (21, 48). Hence, the current view is that the main reason behind the lack of disease progression in natural African hosts lies in a better adaptation of the host in response to the highly replicating virus. A better understanding of the mechanisms underlying the lack of disease in natural hosts for SIV infection may provide important clues for understanding the pathogenesis of HIV infection (53, 71).To date, it is still unknown whether or not immune responses are responsible for the lack of disease progression in natural hosts, since data are scarce. Studies of cellular immune responses are significantly more limited than is the case with pathogenic infection, and although not always in agreement (3, 13, 28, 29, 73, 76), their convergence point is that cellular immune responses are not essentially superior to those observed in pathogenic infections (3, 13, 28, 29, 73, 76). This observation is not surprising in the context of the high viral replication in natural hosts. Data are even scarcer on the role of humoral immune responses in the control of disease progression in natural hosts. However, several studies reported that anti-SIV antibody titers are lower in SIV infections of natural hosts, with a lack of anti-Gag responses being characteristic of natural SIV infections in African nonhuman primates (1, 6, 24, 25, 42, 43, 71). Because the viral replication in SIVagm-infected AGMs is of the same magnitude or higher than that in pathogenic infections of rhesus macaques (RMs), it has been hypothesized that these high VLs may be a consequence of the lower antibody titers. Moreover, a recent study has also shown that B cells in lymph nodes (LNs) of AGMs are activated at an earlier time point than is the case for SIVmac251-infected RMs, which implies that humoral immune responses may be important in controlling SIV replication in the natural hosts (9). Conversely, it has been shown that passively transferring immunoglobulins from animals naturally infected with SIVagm prior to infection with a low dose of SIVagm did not prevent infection in AGMs (42, 60), which is in striking contrast to results in studies of pathogenic infections, which convincingly demonstrated with animal models that intravenously administered or topically applied antibodies can protect macaques against intravenous or mucosal simian-human immunodeficiency virus challenge (34-36, 54, 72).Previous CD20⁺ B-cell-depletion studies during pathogenic RM infections have indicated that humoral immune responses may be important for controlling both the postpeak VL and disease progression (38, 57). However, these studies used strains that are highly resistant to neutralization (SIVmac251 and SIVmac239), making it difficult to assess the role that antibodies have in controlling SIV replication and disease progression. Moreover, our recent results suggested a limited impact of humoral immune responses in controlling replication of a neutralization-sensitive SIVsmm strain in rhesus macaques (18).To investigate the effect that CD20⁺ B cells and antibodies have on SIV replication in natural hosts, we have depleted CD20⁺ B cells in vivo in AGMs infected with SIVagm.sab92018. We assessed the impact of humoral immune responses on the control of viral replication and other immunological parameters, and we report that ablating humoral immune responses in SIVagm-infected AGMs does not significantly alter the course of virus replication or disease progression.     

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).     

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.     

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

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.     

Proliferation Capacity and Cytotoxic Activity Are Mediated by Functionally and Phenotypically Distinct Virus-Specific CD8 T Cells Defined by Interleukin-7Rα (CD127) and Perforin Expression

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127⁺ perforin⁻/CD127⁻ perforin⁺, and CD127⁻/perforin⁻ CD8 T cells, respectively. CD127⁻/perforin⁻ and CD127⁻/perforin⁺ cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin⁺/IL-2⁻ or perforin⁻/IL-2⁺ cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127⁺/IL-2-secreting) and cytotoxic (perforin⁺) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.Cytotoxic CD8 T cells are a fundamental component of the immune response against viral infections and mediate an important role in immunosurveillance (7, 10, 55), and the induction of vigorous CD8 T-cell responses after vaccination is thought to be a key component of protective immunity (37, 41, 49, 50, 58, 60, 69). Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules containing perforin (pore-forming protein) and several granule-associated proteases, including granzymes (Grms) (5, 15, 20, 44). Several studies have recently advanced the characterization of the mechanism of granule-dependent cytotoxic activity and performed a comprehensive investigation of the content of cytotoxic granules in human virus-specific CD8 T cells (2, 19, 29, 44, 53).Heterogeneous profiles of cytotoxic granules have been identified in different virus-specific memory CD8 T cells and associated with distinct differentiation stages of memory CD8 T cells (2, 19, 29, 44). Furthermore, we have observed a hierarchy among the cytotoxic granules in setting the efficiency of cytotoxic activity and demonstrated that perforin (and to a lesser extent GrmB) but not GrmA or GrmK were associated with cytotoxic activity (29). Recently, a novel mechanism of perforin-dependent granule-independent CTL cytotoxicity has also been demonstrated (45).Major advances in the characterization of antigen (Ag)-specific CD4 and CD8 T cells have been made recently and have aimed at identifying functional profiles that may correlate with protective CD8 T-cell responses (1, 3, 4, 12, 13, 24, 28, 36-38, 40, 41, 49, 50, 56-58, 60, 64, 68). In particular, the functional characterization of antigen-specific T cells was mainly performed on the basis of (i) the pattern of cytokines secreted (i.e., gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], or macrophage inflammatory protein 1β [MIP-1β]), (ii) the proliferation capacity, and (iii) the cytotoxic capacity (13, 28, 59). Of note, degranulation activity (i.e., CD107a mobilization following specific stimulation) has been used as a surrogate marker of cytotoxic activity (11, 13).The term “polyfunctional” has been used to define T-cell immune responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, or MIP-1β and cytotoxic activity (measured by the degranulation capacity), comprise distinct T-cell populations able to secrete IL-2 and retain proliferation capacity (13, 28, 49, 50). Some evidence indicates that a hallmark of protective immune responses is the presence of polyfunctional T-cell responses (59). Furthermore, the ability to secrete IL-2 was shown to be linked to proliferation capacity, and both factors have been associated with protective antiviral immunity (13, 28, 49, 50). Although a lack of correlation between degranulation activity and GrmB expression was reported in mice (65), the relationship between degranulation activity and perforin expression has never been comprehensively investigated in mice and in humans.The private α chain of the IL-7 receptor (IL-7Rα, also called CD127) has been suggested to selectively identify CD8 T cells that will become long-lived memory cells (6, 34, 36). Moreover, it was shown in mice (34, 36) and humans (14, 48, 63) that the CD127^high memory-precursor CD8 T cells produced IL-2 in contrast to CD127^low effector CD8 T cells. Of interest, CD127 expression has also been shown to correlate with Ag-specific proliferation capacity in mice (34, 36). A similar correlation was observed in humans, although only for polyclonal stimulations (48). With the exception of studies performed in HIV-1 infection, where an association between CD127 expression and HIV-1 viremia has been shown (21, 22, 42, 48, 54), very limited information is available on the CD127 expression in human virus-specific CD8 T cells other that HIV-1.Although cytotoxic activity and proliferation capacity are key components of the antiviral cellular immune response, the relationship between these functions has been only investigated in nonprogressive HIV-1 infection (46), where these two functions were shown to be related. However, it still remains to be determined whether these functions are mediated by the same or by different T-cell populations.In the present study, we performed a comprehensive characterization of virus-specific CD8 T-cell responses against HIV-1, cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus (Flu) in order to (i) analyze the degree of concordance between degranulation activity and perforin/Grm expression; (ii) identify the relevance of CD127 in identifying virus-specific CD8 T cells endowed with proliferation capacity; (iii) delineate the relationship between proliferation capacity, cytotoxic activity, activation/differentiation stage, and level of exhaustion of CD8 T cells; and (iv) determine the influence of antigen exposure in shaping the functional composition of virus-specific CD8 T cells.Our data indicate that cytotoxic (as defined by perforin expression) and proliferating (as defined by CD127 expression or IL-2 secretion) virus-specific CD8 T cells are contained within distinct CD8 T-cell populations. Furthermore, the proportion of proliferating and cytotoxic T cells within a given virus-specific CD8 T-cell population appears to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferative capacity, differentiation stage, and Ag exposure of memory CD8 T cells.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

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.     

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from,but Overlaps with,the Receptor Complexes of HTLV-1 and HTLV-2

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4⁺ and CD8⁺ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4⁺ T cells, and HTLV-2 SU, which primarily binds to activated CD8⁺ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4⁺ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.The primate T-cell lymphotropic virus (PTLV) group of deltaretroviruses consists of three types of human T-cell lymphotropic viruses (HTLVs) (HTLV-1, HTLV-2, HTLV-3), their closely related simian T-cell lymphotropic viruses (STLVs) (STLV-1, STLV-2, STLV-3), an HTLV (HTLV-4) for which a simian counterpart has not been yet identified, and an STLV (STLV-5) originally described as a divergent STLV-1 (5-7, 30, 35, 37, 38, 45, 51, 53). HTLV-1 and HTLV-2, which have a 70% nucleotide homology, differ in both their pathobiology and tropism (reviewed in reference 13). While HTLV-1 causes a neurological disorder (tropical spastic paraparesis/HTLV-1-associated myelopathy) and a hematological disease (adult T-cell leukemia/lymphoma) (15, 42, 55), HTLV-2 is only rarely associated with tropical spastic paraparesis/HTLV-1-associated myelopathy-like disease and is not definitively linked to any lymphoproliferative disease (12, 20). In vivo, both HTLV-1 and HTLV-2 infect T cells. Although HTLV-1 is primarily found in CD4⁺ T cells, other cell types in the peripheral blood of infected individuals have been found to contain HTLV-1, including CD8⁺ T cells, dendritic cells, and B cells (19, 29, 33, 36, 46).Binding and entry of retroviruses requires specific interactions between the Env glycoproteins on the virus and cell surface receptor complexes on target cells. For HTLV-1, three molecules have been identified as important for entry, as follows: heparan sulfate proteoglycans (HSPGs), neuropilin-1 (NRP-1), and glucose transporter 1 (GLUT-1) (16, 22, 26, 28, 29, 34, 39, 44). Recent studies support a model in which HSPG and NRP-1 function during the initial binding of HTLV-1 to target cells, and GLUT-1 functions at a postattachment stage, most likely to facilitate fusion (29, 34, 49). Efficient HTLV-2 binding and entry requires NRP-1 and GLUT-1 but not HSPGs (16, 26, 39, 49).This difference in the molecules required for binding to target cells reflects differences in the T-cell tropisms of these two viruses. Activated CD4⁺ T cells express much higher levels of HSPGs than CD8⁺ T cells (26). In infected individuals, HTLV-1 is primarily found in CD4⁺ T cells, while HTLV-2 is primarily found in CD8⁺ T cells (21, 43, 46). In vitro, HTLV-1 preferentially transforms CD4⁺ T cells while HTLV-2 preferentially transforms CD8⁺ T cells, and this difference has been mapped to the Env proteins (54).We and others have reported the discovery of HTLV-3 in two Cameroonese inhabitants (6, 7, 53). We recently uncovered the presence of a third HTLV-3 strain in a different population living several hundred kilometers away from the previously identified groups (5), suggesting that this virus may be common in central Africa. Since the HTLV-3 sequences were obtained by PCR amplification of DNA isolated from peripheral blood mononuclear cells (PBMCs) of infected individuals, little is known about its tropism and pathobiology in vivo. Based on the correlation between HSPG expression levels and viral tropisms of HTLV-1 and HTLV-2, we reasoned that knowledge about the HTLV-3 receptors might provide insight into the tropism of this virus. We therefore generated vectors expressing HTLV-3 Env proteins and used them to begin to characterize the receptor complex used by HTLV-3 to bind and enter cells.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.