SEIS: Insight's Seismic Experiment for Internal Structure of Mars.

By the end of 2018, 42 years after the landing of the two Viking seismometers on Mars, InSight will deploy onto Mars' surface the SEIS (Seismic Experiment for Internal Structure) instrument; a six-axes seismometer equipped with both a long-period three-axes Very Broad Band (VBB) instrument and a three-axes short-period (SP) instrument. These six sensors will cover a broad range of the seismic bandwidth, from 0.01 Hz to 50 Hz, with possible extension to longer periods. Data will be transmitted in the form of three continuous VBB components at 2 sample per second (sps), an estimation of the short period energy content from the SP at 1 sps and a continuous compound VBB/SP vertical axis at 10 sps. The continuous streams will be augmented by requested event data with sample rates from 20 to 100 sps. SEIS will improve upon the existing resolution of Viking's Mars seismic monitoring by a factor of ∼ 2500 at 1 Hz and ∼ 200 000 at 0.1 Hz. An additional major improvement is that, contrary to Viking, the seismometers will be deployed via a robotic arm directly onto Mars' surface and will be protected against temperature and wind by highly efficient thermal and wind shielding. Based on existing knowledge of Mars, it is reasonable to infer a moment magnitude detection threshold of M w ∼ 3 at 40 ∘ epicentral distance and a potential to detect several tens of quakes and about five impacts per year. In this paper, we first describe the science goals of the experiment and the rationale used to define its requirements. We then provide a detailed description of the hardware, from the sensors to the deployment system and associated performance, including transfer functions of the seismic sensors and temperature sensors. We conclude by describing the experiment ground segment, including data processing services, outreach and education networks and provide a description of the format to be used for future data distribution. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11214-018-0574-6) contains supplementary material, which is available to authorized users.

Immunologic and virologic analyses of an acutely HIV type 1-infected patient with extremely rapid disease progression.

The immunologic and virologic factors that impact on the rate of disease progression after acute infection with human immunodeficiency virus (HIV) type 1 are poorly understood. A patient with an extraordinarily rapid disease course leading to AIDS-associated death within 6 months of infection was studied intensively for the presence of anti-HIV immune reactivities as well as changes in the genetic and biologic properties of virus isolates. Although altered humoral responses were evident, the most distinctive immunologic feature was a nearly complete absence of detectable HIV-specific CTL responses. In addition to a rapid decline in CD3+CD4+ cells, elevated percentages of CD8+CD45RA+ and CD8+CD57+ cells and diminished CD8+CD45R0+ and CD8+CD28+ cells were evident. Primary viral isolates recovered throughout the course of infection exhibited limited sequence diversity. Cloned viral envelopes were found to have unusually broad patterns of coreceptor usage for cell-cell fusion, although infectivity studies yielded no evidence of infection via these alternative receptors. The infectivity studies demonstrated that these isolates and their envelopes maintained an R5 phenotype throughout the course of disease. The absence of demonstrable anti-HIV CTL reactivities, coupled with a protracted course of seroconversion, highlights the importance of robust HIV-specific immune responses in the control of disease progression.

Sprouting of aberrant neuropeptide Y-immunoreactive sympathetic nerves into neonatally denervated smooth muscle.

Sympathetic nerves normally project ipsilaterally to lateral cranial targets. Following unilateral superior cervical ganglionectomy in neonatal rats, however, neurons from the contralateral superior cervical ganglion sprout into the denervated region. In the present study we examined neuropeptide Y immunoreactivity (NPY-ir) of neurons comprising ipsilateral (control) and denervation-induced contralateral pathways to the superior tarsal smooth muscle of the eyelid. Fluoro-Gold injection of the control muscle retrogradely labelled 133 +/- 18 neurons in the ipsilateral superior cervical ganglion; of these, 21 +/- 3% displayed detectable NPY-ir. Fluoro-Gold injections of the reinnervated muscle labelled 20 +/- 4 neurons in the contralateral superior cervical ganglion, of which 85 +/- 3% contained detectable NPY-ir. Examination of the control tarsal muscle revealed DBH-ir noradrenergic nerves throughout the muscle and vasculature, while NPY-ir nerves were present primarily around blood vessels. In the reinnervated preparation, NPY-ir fibers innervated both blood vessels and tarsal muscle in a pattern similar to that of DBH-ir innervation. Acute excision of the remaining superior cervical ganglion eliminated all DBH-ir fibers bilaterally; NPY-ir was reduced markedly in the reinnervated preparations, though some fibers remained. We conclude that, following neonatal denervation, the tarsal muscle is reinnervated by a subpopulation of sympathetic neurons that differs in neuropeptide phenotype from that of the normal ipsilateral innervation.

Effects of para-chloroamphetamine upon the serotonergic innervation of the rat hippocampus.

The ability of hippocampal serotonergic (5-HT) axons to proliferate in response to damage by para-chloroamphetamine (PCA) was examined in this study. Synaptosomal uptake of 5-HT in the hippocampal formation was decreased to 40% of control 3 days after systemic administration of PCA. Six weeks after PCA, uptake values were 44% of control. Retrograde tracing combined with 5-HT immunocytochemistry showed a significant reduction (18% of control) in the number of 5-HT raphe neurons projecting to the hippocampus 3 days after PCA. The number of 5-HT neurons projecting to the hippocampal formation increased to 69% of control by 6 weeks. The dorsal raphe nucleus was not retrogradely labeled after PCA; the increase in labeled neurons was observed in the median raphe nucleus. PHA-L, injections of the median raphe nucleus demonstrated a reduction of raphe axons in the hippocampal formation after PCA. In rats treated with PCA, raphe axons labeled with PHA-L also appeared to have fewer boutons than raphe axons labeled in control cases. The density of PHA-L containing axons in the hippocampal formation of rats injected 3 days and 6 weeks after PCA was less than control but there was no difference between the experimental groups. Based upon the results from synaptosomal uptake and anterograde tracing experiments, we feel that compensatory proliferation of 5-HT axons does not occur within 6 weeks of PCA-induced damage to the 5-HT plexus of the hippocampal formation. The data derived from the retrograde tracing experiment are thought to reflect reduced uptake and transport of WGA-HRP as an acute effect of PCA.

Early reversal of acidosis and metabolic recovery following ischemia.

Tissue acidosis is believed to be a key element in ischemic injury of neural tissue. The goal of this study was to determine whether persisting postischemic acidosis or the extent of acidosis would affect metabolic recovery following an ischemic event. Intracellular pH (pHi), adenosine triphosphate, phosphocreatine, and lactate levels were measured in the cerebral cortex during the early stages of reperfusion, following either 5 or 10 minutes of global ischemia in both normo- and hyperglycemic gerbils. A total of 130 gerbils were injected with a solution containing 1.5 ml Neutral Red (1%) (+/- 2.5 gm/kg glucose); 30 minutes later, the gerbils were placed under halothane anesthesia, and the carotid arteries were occluded for either 5 or 10 minutes. The brains were frozen in liquid nitrogen at 0, 15, 30, 60, and 120 seconds after reperfusion; they were sectioned and the block face was photographed to determine the pHi by using Neutral Red histophotometry. At the conclusion of the ischemia, the pHi in all groups had decreased significantly from a control value of 7.05 +/- 0.03) (mean +/- standard error of the mean). In normoglycemic brains, the pHi values fell to 6.71 +/- 0.04 and 6.68 +/- 0.11 after 5 and 10 minutes of ischemia, respectively. Hyperglycemic brains were more acidotic; values fell to 6.57 +/- 0.10 and 6.52 +/- 0.24 after 5 and 10 minutes of ischemia, respectively. Lactate levels were approximately fivefold greater than those of control tissue in normoglycemic brains, while lactate levels in hyperglycemic brains were increased eightfold. The adenosine triphosphate and phosphocreatine levels were depleted at the end of ischemia in all groups. After 2 minutes of reflow activity, the pHi levels in both normo- and hyperglycemic brains were restored to those of control values in the '5-minute ischemic group, while the pHi levels remained significantly depressed in the 10-minute ischemic group. Restoration of high-energy phosphates was similar in normoglycemic brains regardless of ischemic duration, recovering to only 20% of the restoration obtained in control tissue at 2 minutes. In hyperglycemic brains, however, there was complete recovery of high-energy phosphates by 2 minutes of reflow activity following 5 minutes of ischemia. Extending the ischemic period to 10 minutes in hyperglycemic brains slowed the rate of metabolic recovery to that observed in normoglycemic brains. The results indicate that the reflow period permits the rapid restoration of pHi levels substantially before the normalization of primary energetic compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucose metabolism and insulin secretion in a patient with ABCC8 mutation and Fanconi-Bickel syndrome caused by maternal isodisomy of chromosome 3.

Fanconi-Bickel syndrome (FBS) is a rare disorder of glucose transport caused by autosomal recessive mutations in GLUT2. Clinically, FBS results in growth failure, hepatomegaly, renal Fanconi syndrome, and abnormal glucose homeostasis. We report a 23 month old female with FBS characterized by more severe and refractory hypoglycemia than typically seen in this disorder. Although previous reports indicate that FBS patients have diminished insulin secretion, our patient showed evidence of hyperinsulinism (HI). Sequence analysis showed that the patient was homozygous for a known null mutation in GLUT2, confirming the clinical diagnosis of FBS. Parental genotyping showed that the mother was heterozygous for the GLUT2 mutation, while the father was wild type. Tandem repeat marker analysis showed that the patient inherited the GLUT2 mutation via maternal isodisomy of chromosome 3. Further molecular testing showed that the patient was heterozygous for a mutation in ABCC8, a known cause of congenital HI. We discuss the patient's biochemical responses in light of the molecular findings.

HIV-1 envelope determinants for cell tropism and chemokine receptor use.

Isolates of human immunodeficiency virus type-1 (HIV-1) display marked differences in their ability to replicate in macrophages and transformed T-cell lines in vitro, a property that has important implications for disease pathogenesis. The restriction in replication between these two CD4-positive cell types is largely at the level of viral entry and is regulated by the viral envelope (env) gene. The envelope protein (Env) is responsible for fusion of the viral and host membranes, and a particular region of Env called the V3-loop has been implicated in regulating viral tropism. However, other regions of Env, such as the V1- and V2-loops, have been shown to modulate the effects of the V3-loop. The discovery that Env initially binds the CD4 molecule on the target cell surface and then makes subsequent interactions with one of several members of the chemokine receptor family has greatly enhanced the molecular understanding of HIV-1 entry. The differential use of chemokine receptors by different viral isolates and their expression in different cell types largely explains viral tropism. The same regions in Env responsible for virus tropism have also been shown to play an important role in mediating chemokine receptor use. The recent crystallization of HIV-1 Env in complex with CD4 illuminates the architecture of the components involved in mediating fusion between the viral and host membranes. The spatial relationship between variable structures of Env previously implicated in tropism and chemokine receptor use and conserved Env structures potentially involved in chemokine receptor binding are discussed.

A biosensor assay for studying ligand-membrane receptor interactions: binding of antibodies and HIV-1 Env to chemokine receptors.

The HIV envelope (Env) protein mediates entry into cells by binding CD4 and an appropriate coreceptor, which triggers structural changes in Env that lead to fusion between the viral and cellular membranes. The major HIV-1 coreceptors are the seven transmembrane domain chemokine receptors CCR5 and CXCR4. The type of coreceptor used by a virus strain is an important determinant of viral tropism and pathogenesis, and virus-receptor interactions can be therapeutic targets. However, Envs from many virus strains interact with CXCR4 and CCR5 with low affinity such that direct study of this important interaction is difficult if not impossible using standard cell-surface binding techniques. We have developed an approach that makes it possible to study ligand binding to membrane proteins, including Env-coreceptor interactions, using an optical biosensor. CCR5, CXCR4, and other membrane proteins were incorporated into retrovirus particles, which were purified and attached to the biosensor surface. Binding of conformationally sensitive antibodies as well as Env to these receptors was readily detected. The equilibrium dissociation constant for the interaction between an Env derived from the prototype HIV-1 strain IIIB for CXCR4 was approximately 500 nM, explaining the difficulty in measuring this interaction using standard equilibrium binding techniques. Retroviral pseudotypes represent easily produced, stable, homogenous structures that can be used to present a wide array of single and multiple membrane-spanning proteins in a native lipid environment for biosensor studies, thus avoiding the need for detergent solubilization, purification, and reconstitution. The approach should have general applicability and can be used to correlate Env-receptor binding constants to viral tropism and pathogenesis.

Effect of nonpolar substitutions of the conserved Phe11 in the fusion peptide of HIV-1 gp41 on its function, structure, and organization in membranes.

The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(11) to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe(11) (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca(2+) or Mg(2+). The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.

HIV type I envelope determinants for use of the CCR2b, CCR3, STRL33, and APJ coreceptors.

The envelope (Env) proteins of primate lentiviruses interact sequentially with CD4 and a coreceptor to infect cells. Changes in coreceptor use strongly influence viral tropism and pathogenesis. We followed the evolution of coreceptor use in pig-tailed macaques that developed severe CD4 T-cell loss during the derivation of a pathogenic simian HIV (SHIV) that contained the tat, rev, vpu, and env genes of the HXBc2 strain of HIV-1 in a genetic background of SIVmac239. The Env from the parental virus as well as one derived from the first macaque to develop AIDS exclusively used CXCR4 as a coreceptor, indicating that CXCR4 can function as a coreceptor in macaques even though it is rarely used by simian immunodeficiency viruses. One Env (Pnb5), obtained from a macrophage-tropic virus isolated from the cerebral spinal fluid, did not use CCR5 or CXCR4. Instead, it used CCR2b and to a lesser extent CCR3, STRL33, and APJ to infect cells. Chimeras between Pnb5 and the parental X4 Env indicated that the V3 loop is the major determinant of CXCR4 use, with other regions of Env influencing the efficiency with which this coreceptor was used. In contrast, the Pnb5 V1/2 and V3 regions in combination were both necessary and sufficient to confer full use of CCR2b, CCR3, STRL33, and APJ to the parental X4 Env protein. These results are consistent with a single, conserved binding site in Env that interacts with multiple coreceptors in conjunction with the V1/2 and V3 loops, and suggest that the V1/2 region plays a more important role in governing the use of CCR2b, CCR3, STRL33, and APJ than for CXCR4.

CCR5 genotypes in sexually active couples discordant for human immunodeficiency virus type 1 infection status.

Persons who are homozygous for the delta32 polymorphism of the CCR5 chemokine receptor gene are highly protected against human immunodeficiency virus type 1 (HIV-1) infection. Previous studies described 54 HIV-1-discordant couples in whom no virus transmission occurred despite extensive sexual contact. The possible role of the delta32 polymorphism in the lack of HIV-1 transmission between these partners was studied. No participants were homozygous for the delta32 allele, but the proportion that was heterozygous was higher among HIV-1-seronegative than HIV-1-seropositive partners (28% vs. 11%, P = .05). This association was seen in heterosexual couples (P = .03) but not in homosexual couples (P = .74). Among white persons, who are most likely to carry the delta32 allele, 38.9% of HIV-1-uninfected and 5.6% of HIV-1-infected heterosexual partners were heterozygous (P = .04). These data are consistent with a possible association between the heterozygous delta32 genotype in heterosexual sex partners and partial protection against HIV-1 infection, and they emphasize the importance of analyzing different risk groups in studies of host factors that influence infection.

Use of GPR1, GPR15, and STRL33 as coreceptors by diverse human immunodeficiency virus type 1 and simian immunodeficiency virus envelope proteins.

Human and simian immunodeficiency viruses (HIV and SIV, respectively) use chemokine receptors as coreceptors along with CD4 to mediate viral entry. Several orphan receptors, including GPR1, GPR15, and STRL33, can also serve as coreceptors for a more limited number of HIV and SIV isolates. We investigated whether these orphan receptors could function as efficient coreceptors for a diverse group of HIV and SIV envelopes (Envs) in comparison with the principal coreceptors CCR5 and CXCR4. We found that a limited number of HIV-1 isolates could mediate inefficient cell-cell fusion with the orphan receptors relative to CCR5 and CXCR4; however, none of the orphan receptors tested could support pseudotype virus infection despite robust infection via CCR5 or CXCR4. All except one of the SIV Envs tested mediated some degree of cell-cell fusion and pseudotype infection, with target cells expressing at least one of these orphan receptors, although CCR5 proved to be the most efficient coreceptor for infection. Only one SIV Env protein, BK28, could mediate infection using GPR1 as a coreceptor, albeit much less efficiently than with CCR5. In addition, use of these coreceptors did not correlate with the published tropism of the SIV clones and was strictly CD4 dependent for both SIV and HIV. We also examined the expression of these molecules in cell lines and primary cells widely used for virus propagation and as targets for infection. All cells examined expressed STRL33, a more limited number expressed GPR15, and GPR1 was much more restricted in its expression pattern. Taken together, our results indicate that GPR15 and STRL33 are rarely used by HIV-1 but are more frequently used by SIV strains, although not in a manner that correlates with SIV tropism.

Characterization of chemokine receptor utilization of viruses in the latent reservoir for human immunodeficiency virus type 1.

Latently infected resting CD4(+) T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4(+) T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4(+) T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.

Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein.

We recently derived a CD4-independent virus from HIV-1/IIIB, termed IIIBx, which interacts directly with the chemokine receptor CXCR4 to infect cells. To address the underlying mechanism, a cloned Env from the IIIBx swarm (8x) was used to produce soluble gp120. 8x gp120 bound directly to cells expressing only CXCR4, whereas binding of IIIB gp120 required soluble CD4. Using an optical biosensor, we found that CD4-induced (CD4i) epitopes recognized by mAbs 17b and 48d were more exposed on 8x than on IIIB gp120. The ability of 8x gp120 to bind directly to CXCR4 and to react with mAbs 17b and 48d in the absence of CD4 indicated that this gp120 exists in a partially triggered but stable state in which the conserved coreceptor-binding site in gp120, which overlaps with the 17b epitope, is exposed. Substitution of the 8x V3 loop with that from the R5 virus strain BaL resulted in an Env (8x-V3BaL) that mediated CD4-independent CCR5-dependent virus infection and a gp120 that bound to CCR5 in the absence of CD4. Thus, in a partially triggered Env protein, the V3 loop can change the specificity of coreceptor use but does not alter CD4 independence, indicating that these properties are dissociable. Finally, IIIBx was more sensitive to neutralization by HIV-positive human sera, a variety of anti-IIIB gp120 rabbit sera, and CD4i mAbs than was IIIB. The sensitivity of this virus to neutralization and the stable exposure of a highly conserved region of gp120 suggest new strategies for the development of antibodies and small molecule inhibitors to this functionally important domain.

Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells.

The mouse leukotriene B4 receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4 (LTB4, Kd = 0.24 +/- 0.03 nM). In competition binding experiments, LTB4 was the most potent competitor (Ki = 0.23 +/- 0.05 nM) followed by 20-hydroxy-LTB4 (Ki = 1.1 +/- 0.2 nM) and by 6-trans-12-epi-LTB4 and LTD4 (Ki > 1 microM). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation.

Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity.

Although infection by human immunodeficiency virus (HIV) typically requires an interaction between the viral envelope glycoprotein (Env), CD4, and a chemokine receptor, CD4-independent isolates of HIV and simian immunodeficiency virus have been described. The structural basis and underlying mechanisms for this phenotype are unknown. We have derived a variant of HIV-1/IIIB, termed IIIBx, that acquired the ability to utilize CXCR4 without CD4. This virus infected CD4-negative T and B cells and fused with murine 3T3 cells that expressed human CXCR4 alone. A functional IIIBx env clone exhibited several mutations compared to the CD4-dependent HXBc2 env, including the striking loss of five glycosylation sites. By constructing env chimeras with HXBc2, the determinants for CD4 independence were shown to map outside the V1/V2 and V3 hypervariable loops, which determine chemokine receptor specificity, and at least partly within an area on the gp120 core that has been implicated in forming a conserved chemokine receptor binding site. We also identified a point mutation in the C4 domain that could render the IIIBx env clone completely CD4 dependent. Mutations in the transmembrane protein (TM) were also required for CD4 independence. Remarkably, when the V3 loop of a CCR5-tropic Env was substituted for the IIIBx Env, the resulting chimera was found to utilize CCR5 but remained CD4 independent. These findings show that Env determinants for chemokine receptor specificity are distinct from those that mediate CD4-independent use of that receptor for cell fusion and provide functional evidence for multiple steps in the interaction of Env with chemokine receptors. Combined with our observation that the conserved chemokine receptor binding site on gp120 is more exposed on the IIIBx gp120 (T. L. Hoffman, C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I. Chaiken, J. A. Hoxie, and R. W. Doms, Proc. Natl. Acad. Sci. USA 96:6359-6364, 1999), the findings from this study suggest novel approaches to derive and design Envs with exposed chemokine receptor binding sites for vaccine purposes.

Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein.

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.

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain.

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and viral entry into the central nervous system.

Derivation and biological characterization of a molecular clone of SHIV(KU-2) that causes AIDS, neurological disease, and renal disease in rhesus macaques.

Previously, we described the derivation of a pathogenic strain of simian-human immunodeficiency virus (SHIV(KU-2)) consisting of the tat, rev, vpu, and env genes of HIV-1 (strain HXB2) in a genetic background of SIV(mac)239 that causes AIDS and productive infection of the CNS in rhesus macaques (Macca mulatta) (Raghavan et al., 1997, Brain Pathol. 7, 851-861). We report here on the characterization of a molecular clone of SHIV(KU-2), designated SHIV(KU-2MC4), that caused CD4(+) T cell loss as well as neurological and renal disease in macaques. DNA sequence analysis of selected SIV regions of SHIV(KU-2MC4) revealed 10 nucleotide changes in the LTR, whereas Gag, Vif, Vpr, Vpx, and Nef had 1, 1, 1, 2, and 13 predicted amino acid substitutions, respectively, compared to SIV(mac)239. DNA sequence analysis of HIV-1 derived regions of SHIV(KU-2MC4) revealed 2, 1, 2, and 18 predicted amino acid substitutions in the Tat, Rev, Vpu, and Env proteins, respectively, when compared to SHIV-4. Unlike the parental SHIV-4, which is not tropic for macrophages, SHIV(KU-2MC4) replicated efficiently in macrophage cultures as determined by p27 assays. However, despite the numerous changes in the Env protein and newly acquired tropism for macrophages, SHIV(KU-2MC4), like the parental SHIV-4, used CXCR4 exclusively as its coreceptor for entry into susceptible cells. Inoculation of SHIV(KU-2MC4) into two rhesus macaques resulted in severe infection in which the numbers of circulating CD4(+) T cells in the blood declined rapidly by 2 weeks postinoculation and virus producing cells in the peripheral blood mononuclear cells were identified throughout the course of infection. At the time of euthanasia (20 and 22 weeks), both macaques had lost a significant amount of weight and had no circulating CD4(+) T cells. In addition, one macaque developed intension tremors and uncoordinated movements. Virological examination of tissues at necropsy revealed active virus replication in both lymphoid and nonlymphoid tissues such as the lung and brain. Histological examination revealed that the induced immunodeficiency was associated with lymphoid depletion of the lymph nodes and spleen, opportunistic infections, lentiviral encephalitis, and severe glomerulosclerosis of the kidney. This molecular clone will serve as the basis for analyzing the molecular determinants through which SHIV(KU-2) causes severe CD4(+) T cell loss, neurological disease, and SHIV nephropathy in rhesus macaques.