Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection.
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Adult↗
Biomedical subjects
F Kirchhoff
Publications and source records attributed to F Kirchhoff.
Structural and bonding properties of solid tellurium from first-principles calculations.
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Journal Article↗
Energetics and electronic structure of silver chloride.
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Journal Article↗
Kainate activates Ca(2+)-permeable glutamate receptors and blocks voltage-gated K+ currents in glial cells of mouse hippocampal slices.
Glial cells in the CA1 stratum radiatum of the hippocampus of 9- to 12-day-old mice show intrinsic responses to glutamate due to the activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors. In the present study we have focused on a subpopulation of the hippocampal glial cells, the "complex" cells, characterized by voltage-gated Na+ and K+ channels. Activation of glutamate receptors in these cells led to two types of responses, the activation of a cationic conductance, and a longer-lasting blockade of voltage-gated K+ channels. In particular, the transient (inactivating) component of the outwardly rectifying K+ current was diminished by kainate. Concomitantly, as described in Bergmann glial cells, kainate also elevated cytosolic Ca2+. This increase was due to an influx via the glutamate receptor itself. In contrast to Bergmann glial cells, the cytosolic Ca2+ increase was not a link to the K+ channel blockade, since the blockade occurred in the absence of the Ca2+ signal and, vice versa, an increase in cytosolic Ca2+ induced by ionomycin did not block the transient K+ current. We conclude that glutamate receptor activation leads to complex and variable changes in different types of glial cells; the functional importance of these changes is as yet unresolved.
Animals↗
SIVmac expressing hybrid envelope proteins containing HIV-1 V3 and/or C4 sequences is not competent for replication.
The V3- and C4-coding regions in the envelope gene of the infectious, pathogenic SIVmac239 clone were replaced by the corresponding HIV-1 sequences. Viral particles were obtained after transfection of COS-1 cells. Chimeric SIVmac constructs were not replication competent in the human T cell lines CEMx174, AA2, H9, and MT-4 or in primary cultures of rhesus monkey peripheral blood mononuclear cells. The lack of infectivity of the hybrid constructs was associated with inefficient proteolytic processing of the gp160env precursor. Unlike the modular nature of some proteins, gp120 appears to be a highly ordered molecule whose function is dependent on the integration of many discontinuous, interactive regions.
Amino Acid Sequence↗
Normal immune function and inability to isolate virus in culture in an individual with long-term human immunodeficiency virus type 1 infection.
A detailed, longitudinal study was undertaken to investigate the immunological and virological features of an individual with hemophilia infected with human immunodeficiency virus type-1 (HIV-1) for 10 years without disease. Methods applied to serial samples of peripheral blood included Western blot analysis, neutralizing antibody assays, antibody-dependent cell-mediated cytotoxicity (ADCC) titration, HIV-1 specific cytotoxic T lymphocyte (CTL) assays, viral cultures, and PCR with sequence analysis of viral regulatory genes. Strong antibody responses against HIV-1 antigens as measured by Western blot and ADCC assays have persisted throughout infection. Repeated attempts to isolate HIV-1 using sensitive culture techniques and to demonstrate viremia with standard PCR methods have failed. Using the "booster" PCR technique, a period of viremia in peripheral blood mononuclear cells was demonstrated. Concurrent with detection of circulating virus, titers of neutralizing antibodies and circulating HIV-1-specific CTLs became measurable. Sequencing studies of a portion of the viral genome showed no significant abnormalities of the regulatory genes. In this individual, the combination of low viral load in the peripheral blood and a strong, responsive immune system is associated with long-term, disease-free coexistence with HIV-1 infection.
Antibody-Dependent Cell Cytotoxicity↗
Upstream U3 sequences in simian immunodeficiency virus are selectively deleted in vivo in the absence of an intact nef gene.
Major transcriptional control elements are located within the U3 region of the long terminal repeats (LTRs) of lentivirus and other retroviral genomes. The nef auxiliary gene of simian immunodeficiency virus (SIV) and human immunodeficiency virus overlaps about 70% of the 450- to 560-bp-long U3 region present in these primate lentiviruses. We analyzed viral DNA sequences present in rhesus monkeys infected with a mutant of SIVmac containing a 182-bp deletion in the region of nef that does not overlap the LTR. Between 50 and 100% of the viral DNA molecules in eight of nine monkeys infected for 16 or more months contained additional deletions of 111 to 302 bp within the 517-bp U3 region. These deletions were contained within a 334-bp region of U3 that is overlapped by the nef reading frame, and they did not affect the polypurine tract, the NF-kappa B binding site, or other sequence elements in this same region that are important for transcription and replication. Such deletions were not detected in any of 41 PCR amplifications from 8 rhesus monkeys infected with wild-type SIV for 8 to 26 months, nor were they detected in 10 animals infected with vpr, vpx or vpr-vpx deletion mutants. These results indicate that, in the absence of an intact nef gene, these upstream U3 sequences are not advantageous for the virus.
Animals↗
The "V3" domain is a determinant of simian immunodeficiency virus cell tropism.
Thirty-one different mutant forms of simian immunodeficiency virus (SIVmac) were created with changes in the region of env corresponding to the V3 domain of HIV-1. Sixteen of these mutants had one amino acid change, 12 had two changes, two had three changes, and one had four changes in the SIVmac "V3" loop. The ability of the mutant viruses to replicate in CEMx174 cells, rhesus monkey peripheral blood mononuclear cells, and rhesus monkey alveolar macrophages was investigated. Ten of the mutant viruses replicated with approximately wild-type kinetics in all three cell types. Of the 31 mutants, 22 were able to replicate in one or more of the cell types. Thus, this region of SIVmac gp120 is quite tolerant to change. Nine of the mutants replicated poorly or not at all in any of the cells tested. The lack of replication competence of some of the mutants was associated with inefficient proteolytic processing of the gp160 precursor. Some mutations had dramatic differential effects in different cell types. For example, changing P to S at position 321 and M to I at position 325 drastically reduced replication in macrophages and CEMx174 cells but had no effect on replication in peripheral blood mononuclear cells. Mutants with altered tropism were blocked at an early stage that includes virus entry into cells. Thus, sequences in SIVmac that correspond to V3 in HIV-1 can affect virus entry and cell tropism in a manner analogous to that of HIV-1 V3.
Amino Acid Sequence↗
Myelin-associated glycoprotein is phosphorylated by protein kinase C.
The myelin-associated glycoprotein (MAG) is a neural recognition molecule involved in heterophilic interactions between myelin-forming cells and neurons. To characterize the molecular mechanisms underlying post-translational modifications which may be instrumental in signal transduction following the recognition event, we have studied the stimuli leading to modification of 32P-orthophosphate incorporation into MAG in cultures of oligodendrocytes or transformed differentiated Schwann cells. Here we show that in oligodendrocytes both the 67 and 72 kD isoforms of MAG were phosphorylated exclusively on serine, while in the transformed Schwann cells only the 67 kD isoform was found to be present and phosphorylated. The phorbol ester phorbol-12-myristoyl-13-acetate (PMA) did not affect biosynthesis of the protein backbone, but enhanced incorporation of phosphate by a factor of 2-3, indicating the involvement of protein kinase C. Exclusive phosphorylation of serine residues was also observed, when purified MAG was incubated with protein kinase C in the presence of [gamma-32P]ATP. In searching for the physiological stimuli which may trigger phosphorylation of MAG, cultures of oligodendrocytes were exposed to extracellular signals, such as coculture with dorsal root ganglion and spinal cord neurons carrying the MAG receptor, to membrane fractions of these neurons, monoclonal MAG antibody 513 binding to the recognition site of MAG, or platelet-derived growth factor. None of these additives modified the phosphorylation of MAG. These observations point to the possibility that phosphorylation of MAG is controlled by yet unknown intracellular cues rather than by extracellular signals interacting with cell surface receptors of oligodendrocytes.
Amino Acid Sequence↗
Functional topography of the myelin-associated glycoprotein. I. Mapping of domains by electron microscopy.
The functional topography of the myelin-associated glycoprotein (MAG) was investigated by electron microscopic analysis of rotary-shadowed molecules of a MAG fragment (MAG 90) comprising the five immunoglobulin-like domains of the extracellular part of the molecule. MAG 90 molecules appeared as rod-like structures (18.5 +/- 1.2 nm long and 4.0 +/- 0.8 nm wide) with a globular domain at one end. Antibodies directed against the amino- and carboxy-terminus of MAG 90 interacted with the non-globular terminal region, indicating that the molecule is bent in the globular region with the amino- and carboxy-terminal arms in close apposition to each other. An antibody which interferes with the binding of MAG to neurons interacted predominantly with the globular domain of MAG 90. The fibril-forming collagen types I, III and V bound mainly to the non-globular terminal region of MAG 90, whereas the majority of heparin molecules interacted with the globular region of the molecule. The L2/HNK-1 carbohydrate structure was localized at the non-globular region in the protein fragment comprising the fourth and fifth immunoglobulin-like domains.
Animals↗
Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain.
Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high-affinity binding sites with respective KD and Bmax values of 0.43 nM and 6.9 fmol/2.7 x 10(5) cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: 1) electrophysiological responses and 2) single cell measurements of changes in [Ca2+]i. In that fraction of cells responding (20-70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y-induced membrane depolarization. As detected by single cell microfluorimetric (fura-2) measurements neuropeptide Y induced an increase of [Ca2+]i which was caused by the entry of extracellular Ca2+. Both the [Ca2+]i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin.
Adenosine Diphosphate Ribose↗
Evidence for the cooperation of gp120 amino acids 322 and 448 in SIVmac entry.
The substitution of glycine for valine at amino acid 448 in the C4 domain of SIVmac239 envelope glycoprotein gp120 resulted in little or no viral infectivity for CEMX174 cells and rhesus monkey peripheral blood mononuclear cells. The block to viral replication was located at an early stage including virus entry into cells since the mutant virus was severely impaired in its ability to form newly synthesized viral DNA during the initial 14 hr after exposure to cells. Envelope glycoprotein with the 448V-->G mutation bound to soluble CD4 in a manner similar to wild type in an in vitro assay and was also translated, processed, and cleaved in a manner similar to wild-type envelope protein. When CEMx174 cells were transfected with 448V-->G mutant proviral DNA and held for longer than 40 days, a variant appeared in one culture that was able to replicate with near wild-type kinetics. Sequencing of viral DNA from cultures infected with this variant revealed that the original 448V-->G mutation was retained in eight of eight clones analyzed but that six of eight clones analyzed had an additional mutation of V-->I at amino acid 322 of gp120. Amino acid 322 in SIVmac gp120 is located in a region which corresponds to V3 of HIV. Analysis of site-specific mutants demonstrated that the mutation of V-->I at position 322 can indeed compensate for the 448V-->G mutation, resulting in efficient virus entry and replication by the double mutant. These results identify single amino acids in gp120 that are critical for early events in SIVmac replication and suggest that the "V3" and C4 domains of SIVmac gp120 cooperate to effect virus entry.
Amino Acid Sequence↗
A PCR-derived library of random point mutations within the V3 region of simian immunodeficiency virus.
Oligonucleotide primers corresponding to variable region 3 (V3) of simian immunodeficiency virus (SIV) were randomly mutagenized during synthesis by doping each of the four nucleoside phosphoramidites with a small amount of the other three. PCR was then used to incorporate the altered sequences into larger, clonable DNA fragments by spliced overlap extension (SOE). With the composition of the phosphoramidites used, 53 of the 100 clones analyzed were unique, having one or more point mutation within the 84-bp target sequence. These 53 unique clones contained an average of 2.1 nucleotide substitutions and 1.5 amino acid substitutions per clone within the target V3 sequence. Of the internal 25 amino acid positions within the V3 domain, 23 were changed at least once. This method should be generally useful for the construction of libraries of random point mutations within a defined target DNA sequence.
Amino Acid Sequence↗
Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene.
Vaccine protection against the human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV) in animal models is proving to be a difficult task. The difficulty is due in large part to the persistent, unrelenting nature of HIV and SIV infection once infection is initiated. SIV with a constructed deletion in the auxiliary gene nef replicates poorly in rhesus monkeys and appears to be nonpathogenic in this normally susceptible host. Rhesus monkeys vaccinated with live SIV deleted in nef were completely protected against challenge by intravenous inoculation of live, pathogenic SIV. Deletion of nef or of multiple genetic elements from HIV may provide the means for creating a safe, effective, live attenuated vaccine to protect against acquired immunodeficiency syndrome (AIDS).
Animals↗
A fast perfusion system for single cell physiology optimized for microscopes with water immersion objectives.
A perfusion system was constructed which allows the fast application of different solutes underneath a water immersion objective. The perfusion system is mounted into the immersion objective by milling a slot into the frontal metal plate of the lens holder. It consists of a five-channel pipette fixed to the objective and solution reservoirs gated by computer controlled magnetic valves. Up to five different solutions can be applied to the specimen under study. The solution between objective and specimen is completely exchanged after 1-2 s as determined from fluorescence measurements. This arrangement is optimized for [Ca2+] measurements with a fluorescence measurement system in tissue slices, where upright microscopes are required. It offers the advantage of saving a micromanipulator for the perfusion pipette and facilitates a fast, reproducible and precise positioning of the perfusion system.
Animals↗
Morphogenesis of recombinant HIV-2 gag core particles.
The gag-pol coding region of the HIV-2BEN genome was expressed in CV-1 cells infected with four recombinant vaccinia viruses (VV). These recombinant VV encoded either the whole gag-pol region or the gag gene including the protease-coding region of the pol gene or the gag gene truncated at its 3'-end or only the pol gene. The HIV-2BEN gag precursor p55, its mature cleavage products p24 and p17 as well as the pol reverse transcriptase (RT) p66 were detected in VV-infected CV-1 cells. The p55 and two intermediate cleavage products p40 and p35 were myristilated. Comparison to lysates of permanently HIV-2BEN-infected Molt 4 clone 8 cells revealed that several additional gag and pol proteins were present in the VV-infected CV-1 cells. Deletion of the gag and pol overlapping region coding for the viral protease prevented cleavage of the recombinant gag precursor. Electron microscopy of VV-infected CV-1 cells revealed budding structures and immature as well as mature retroviral particles formed by the recombinant gag proteins. Striking differences in the ability to form complete particles were observed between the different recombinant VV. Expression of the truncated gag gene led to the formation of budding structures, but completely budded circular particles were not detectable. Such particles were produced by expression of the whole gag gene and the protease. Mature virions with an internal core structure were only detected in VVgagpol-infected cells. From these findings we conclude that the 3'-end of the gag gene coding for the p16 protein is essential for the formation of complete HIV-2 particles and that the pol proteins support the assembly of the viral core.
Base Sequence↗
Antibody response to the negative regulatory factor (nef) in experimentally infected macaques: correlation with viremia, disease progression, and seroconversion to structural viral proteins.
The antibody response to structural and regulatory viral proteins was studied in 14 rhesus (Macaca mulatta) and 6 cynomolgus (Macaca fascicularis) macaques experimentally infected with HIV-2 or SIVMAC. To investigate the humoral antibody response to the negative regulatory factor (nef), the recombinant protein was expressed to high levels with recombinant vaccinia virus (VV). nef-specific antibodies were detected in 14 of 20 infected macaques (70%). In sera of all infected monkeys antibodies directed to the structural proteins gp120, p56, and p24 appeared 2 to 6 weeks postinfection. In contrast, the extent and the appearance of nef-specific antibodies during the course of infection varied considerably between individual animals. However, only in sera of four animals (20%) were nef-specific antibodies detectable as early as those against the core proteins p24 and p56. In SIVMAC-infected rhesus macaques at different clinical stages, the antibody response towards nef neither correlated with the development of viral latency nor to disease progression or viremia. Our data indicate that in macaques experimentally infected with SIV or HIV-2 antibody formation against nef is not a useful diagnostic marker either for early detection of viral infection or of disease progression.
AIDS-Related Complex↗
Down-regulation of myelin-associated glycoprotein on Schwann cells by interferon-gamma and tumor necrosis factor-alpha affects neurite outgrowth.
To investigate the influence of inflammatory cytokines on the potential of peripheral nerves to regenerate, we analyzed the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the ability of immortalized Schwann cells to mediate outgrowth of neurites from primary DRG neurons. We found that IFN-gamma and TNF-alpha synergistically inhibited the neurite outgrowth-promoting properties of the Schwann cells by specifically down-regulating myelin-associated glycoprotein (MAG) at the levels of mRNA and cell surface protein by approximately 60%. Antibodies to MAg inhibited the outgrowth of neurites on Schwann cells to the same extent as treatment with the two cytokines. Since MAG appears to be involved in both neurite outgrowth and myelination, our findings may provide evidence for a mechanism, by which inflammatory cytokines interfere with Schwann cell-neuron interactions.
Animals↗