We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.The human immunodeficiency virus (HIV) envelope protein is a trimeric type I integral membrane protein in which each monomer consists of a heavily glycosylated surface subunit (gp120) noncovalently associated with a transmembrane (TM) domain subunit (gp41) (reviewed in reference 62). The gp120 subunit contains highly conserved domains involved in CD4 and coreceptor binding (46). However, parts of these domains, particularly the bridging sheet, are poorly immunogenic, due in part to shielding by N-linked carbohydrate structures, the V3 loop, and the V1-V2 region (61). For its membrane fusion potential to be realized, Env must first bind CD4, which induces the exposure or formation of a highly conserved domain in gp120 that is important for coreceptor binding (33,46,56,60). Binding to a coreceptor, most often the CCR5 or CXCR4 chemokine receptor (reviewed in reference 13), triggers the final conformational changes in Env that ultimately result in fusion between the viral and cellular membranes.While the gp120 subunit mediates binding to cell surface receptors as well as attachment factors, such as DC-SIGN (reviewed in reference 4), the membrane-spanning gp41 subunit plays a critical role in the actual membrane fusion process (reviewed in references 9 and 62). The gp41 subunit contains at its N terminus a hydrophobic fusion peptide that is thought to insert into the membrane of the cell, thus linking the cellular membrane with that of the virus. A peculiar feature of gp41 is its unusually long cytoplasmic domain, ...

The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1␣ and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.The discovery of the receptors used by human immunodeficiency virus type 1 (HIV-1) to infect cells, coupled with a greater understanding of the membrane fusion-inducing conformational changes undergone by the viral envelope protein (Env) upon receptor binding, has identified several promising drug and vaccine targets (reviewed in reference 12). The viral Env protein binds cell surface CD4 with a high affinity, resulting in conformational changes that enable Env to bind a coreceptor (32,54,56). Coreceptor binding is thought to trigger the ability of Env to mediate fusion between the viral and cellular membranes. The major HIV-1 coreceptors are the chemokine receptors CCR5 and CXCR4 (reviewed in reference 11). The R5 virus strains that use CCR5 with CD4 to infect cells are largely responsible for virus transmission and are typically macrophage tropic. The accrual of mutations in Env can lead to X4 virus strains that use CXCR4 in place of CCR5 or R5X4 virus strains that can use both receptors. While X4 virus strains do not always evolve in infected individuals, their emergence is a harbinger of progression to AIDS (51, 52).While virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by receptor concentration (21,29,30,43,48), affinity between Env and receptors (28), and potenti...

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.

Abstract-Inherited restrictive cardiomyopathy (RCM) is a debilitating disease characterized by a stiff heart with impaired ventricular relaxation. Mutations in cardiac troponin I (cTnI) were identified as causal for RCM. Acute genetic engineering of adult cardiac myocytes was used to identify primary structure/function effects of mutant cTnI. Studies focused on R193H cTnI owing to the poor prognosis of this allele. Compared with wild-type cTnI, R193H mutant cTnI more effectively incorporated into the sarcomere, where it exerted dose-dependent effects on basal and dynamic contractile function. Under loaded conditions, permeabilized myocyte Ca 2ϩ sensitivity of tension was increased, whereas the passive tension-extension relationship was not altered by R193H cTnI. Normal rod-shaped myocyte morphology acutely transitioned to a "short-squat" phenotype in concert with progressive stoichiometric incorporation of R193H in the absence of altered diastolic Ca 2ϩ . The specific myosin inhibitor blebbistatin fully blocked this transition. Heightened Ca 2ϩ buffering by the R193H myofilaments, and not alterations in Ca 2ϩ handling by the sarcoplasmic reticulum, slowed the decay rate of the Ca 2ϩ transient. Incomplete mechanical relaxation conferred by R193H was exacerbated at increasing pacing frequencies independent of elevated diastolic Ca 2ϩ . R193H cTnI-dependent mechanical tone caused acute remodeling to a quasicontracted state not elicited by other Ca 2ϩ -sensitizing proteins and is a direct correlate of the stiff heart characteristic of RCM in vivo. These results point toward targets downstream of Ca 2ϩ handling, notably thin filament regulation and actin-myosin interaction, in designing therapeutic strategies to redress the primary cell morphological and mechanical underpinnings of RCM. (Circ Res. 2007;100:1494-1502.) Key Words: cardiomyopathy Ⅲ troponin Ⅲ contraction I nherited cardiomyopathies represent a clinically diverse group of progressive heart muscle diseases that can be caused by mutations in specific sarcomeric genes. 1 Cardiomyopathies are classified into several distinct clinical subtypes based on a range of morphological and functional criteria. 1 The most malignant and least studied of the subtypes is restrictive cardiomyopathy (RCM). 2 The distinguishing clinical features of RCM patients include markedly impaired ventricular filling, pronounced diastolic dysfunction from an extremely stiff heart, 1,2 and the potential for rapid progression to overt heart failure. 2,3 Recently, mutations were identified in the gene encoding cardiac troponin I (cTnI), TNNI3, in human patients with autosomal dominant RCM. 3 RCM-linked mutant cTnIs result in amino acid substitutions in the most highly conserved regions of cTnI. 4 cTnI is known to function as a molecular switch within the sarcomere by regulating the Ca 2ϩ dependent cardiac muscle contraction. 4 During diastole, myocyte intracellular Ca 2ϩ is low and cTnI binds tightly to actin inhibiting strong actin-myosin interactions. Elevation of intracellular Ca...

Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a twoto fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4-and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.

A highly reproducible quantitative PCR (Q-PCR) assay was used to study the stability of human papillomavirus (HPV) in undifferentiated keratinocytes that maintain viral episomes. The term "stability" refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway from that of PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and small interfering RNAs (siRNAs) acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia Rad3-related (ATR) pathways significantly reduced viral episomes, suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological inhibition of ATM proteins had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episome levels, suggesting that ATM proteins are playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a littleunderstood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.

Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against 3 different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (tested in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), 4 polyamides were identified that possessed apparent IC50s ≤ 150 nM with no evidence of cytotoxicity and we report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as an antiviral compound for treatment of HPV-related disease, including cervical dysplasia. PA1 and related polyamides offer promise as antiviral agents and the ability to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to PA1, is accompanied by significant cellular toxicity.

DC-SIGN, a type II membrane protein with a C-type lectin binding domain that is highly expressed on mucosal dendritic cells (DCs