The major human rhinovirus receptor is ICAM-1

The major human rhinovirus receptor has been identified with monoclonal antibodies that inhibit rhinovirus infection. These monoclonal antibodies recognize a 95 kd cell surface glycoprotein on human cells and on mouse transfectants expressing a rhinovirus binding phenotype. Purified 95 kd protein binds to rhinovirus in vitro. Protein sequence from the 95 kd protein showed an identity with that of intercellular adhesion molecule-1 (ICAM-1); a cDNA clone obtained from mouse transfectants expressing the rhinovirus receptor had essentially the same sequence as ICAM-1. Thus, the major human rhinovirus receptor is ICAM-1. The gene for this receptor maps to human chromosome 19, which also contains the genes for a number of other picornavirus receptors.     

Autophagy of cytoplasmic bulk cargo does not require LC3

To investigate the role of LC3 in bulk autophagy we compared its autophagic-lysosomal processing (using an improved quantitative immunoblotting method) with autophagic-lysosomal bulk cargo flux (measured by our established LDH [lactate dehydrogenase] sequestration assay) in amino acid-starved rat hepatocytes treated with cycloheximide to prevent new LC3 influx. Block-release experiments with the reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) showed that while only 3MA suppressed phagophoric LC3 attachment (lipidation), both inhibitors prevented phagophore closure (cargo sequestration). Upon release from closure blockade, some autophagic-lysosomal LC3 flux was resumed even in the presence of 3MA, i.e., without an accompanying bulk cargo flux. Conversely, whereas the autophagic-lysosomal flux of LC3 halted within ～100 min of cycloheximide treatment, the bulk cargo flux continued at a high rate. siRNA-mediated knockdown of LC3 family proteins in LNCaP prostate carcinoma cells confirmed that autophagy of cytoplasmic bulk cargo was completely LC3 independent also in these cells, and in the absence of cycloheximide. However, a strong requirement for GABARAP family proteins was evident. Since bulk autophagy of cytoplasm (macroautophagy) and autophagic-lysosomal LC3 processing may apparently be mutually independent, LC3 would seem to be unsuitable as a general indicator of autophagy.     

Viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in ICAM-1-negative cells

Major receptor group common cold virus HRV89 was adapted to grow in HEp-2 cells, which are permissive for minor group human rhinoviruses (HRVs) but which only marginally support growth of major-group viruses. After 32 blind passages in these cells, each alternating with boosts of the recovered virus in HeLa cells, HRV89 acquired the capacity to effectively replicate in HEp-2 cells, attaining virus titers comparable to those in HeLa cells although no cytopathic effect was observed. Several clones were isolated and shown to replicate in HeLa cells whose ICAM-1 was blocked with monoclonal antibody R6.5 and in COS-7 cells, which are devoid of ICAM-1. Blocking experiments with recombinant very-low-density lipoprotein receptor fragments and enzyme-linked immunosorbent assays indicated that the mutants bound a receptor different from that used by minor-group viruses. Determination of the genomic RNA sequence encoding the capsid protein region revealed no changes in amino acid residues at positions equivalent to those involved in the interaction of HRV14 or HRV16 with ICAM-1. One mutation was within the footprint of a very-low-density lipoprotein receptor fragment bound to minor-group virus HRV2. Since ICAM-1 not only functions as a vehicle for cell entry but has also a "catalytic" function in uncoating, the use of other receptors must have important consequences for the entry pathway and demonstrates the plasticity of these viruses.     

OxLDL-mediated survival of macrophages does not require LDL internalization or signalling by major pattern recognition receptors

Macrophages play a key role in the pathogenesis of atherosclerosis, in part by destabilizing plaques. We and others have shown that low concentrations of oxidized LDL (oxLDL) inhibit macrophage apoptosis. As oxLDL is present in lesions, this may be a mechanism by which macrophage populations in the intima are expanded. We have previously shown that oxLDL activates prosurvival signalling pathways such as the phosphoinositide 3-kinase (PI3K) pathway in bone marrow derived macrophages (BMDMs). However, little is known about more upstream signalling events especially at the receptor level. The endocytic pattern recognition receptors (PRRs), scavenger receptor A (SR-A) and CD36, are the main receptors on macrophages for uptake of oxLDL and are therefore important in foam cell formation. The signalling PRRs such as toll-like receptor (TLR) 2 and 4 also bind some types of oxLDL. This study was done to determine if any of the known PRRs are required for the anti-apoptotic effects of oxLDL in BMDMs. To do this, we tested the effect of oxLDL on viability of BMDMs lacking both SR-A and CD36 or lacking TLR2, TLR4, CD14, FcγRIIb, or RAGE. Our results indicate that none of these receptors are essential for activating the oxLDL prosurvival pathway. Furthermore, we show that the anti-apoptotic effect is not dependent on the uptake of oxLDL.     

Modeling of the human intercellular adhesion molecule-1, the human rhinovirus major group receptor

A model has been built of the amino-terminal domain of the intercellular adhesion molecule-1 (ICAM-1), the receptor for most human rhinovirus serotypes. The model was based on sequence and presumed structural homology to immunoglobulin constant domains. It fits well into the putative receptor attachment site, the canyon, on the human rhinovirus-14 (HRV14) surface in a manner consistent with most of the mutational data for ICAM-1 (Staunton, D. E., Dustin, M. L., Erickson, H. P., Springer, T. A. Cell, in press, 1989) and HRV14 (Colonno, R. J., Condra, J. H., Mizutani, S., Callahan, P. L., Davies, M. E., Murcko, M. A. Proc. Natl. Acad. Sci. U.S.A. 85: 5449-5453, 1988).     

Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity.     

Induction of autophagy does not affect human rhinovirus type 2 production

Induction of autophagy has been shown to be beneficial for the replication of poliovirus, a phenomenon that might also apply for other picornaviruses. We demonstrate that de novo synthesis of human rhinovirus type 2 (HRV2), an HRV of the minor receptor group, is unaffected by tamoxifen, rapamycin, and 3-methyladenine (3-MA), drugs either stimulating (tamoxifen and rapamycin) or inhibiting (3-MA) autophagic processes. Furthermore, LC3-positive vesicles (i.e., autophagosomes) are not induced upon infection. Therefore, multiplication of this particular picornavirus is not dependent on autophagy.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly ¹⁵N and ¹³C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the ¹H, ¹³C, and ¹⁵N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

DNA curvature does not require bifurcated hydrogen bonds or pyrimidine methyl groups.

Short tracts of the homopolymer dA.dT confer intrinsic curvature on the axis of the DNA double helix. This phenomenon is assumed to be a consequence of such tracts adopting a stable B'-DNA conformation that is distinct from B-form structure normally assumed by other DNA sequences. The more stable B' structure of dA.dT tracts has been attributed to several possible stabilizing factors: (1) optimal base stacking interactions consequent upon the high propeller twist, (2) bifurcated hydrogen bonds between adjacent dA.dT base-pairs, (3) stacking interactions involving the dT methyl groups, and finally (4) a putative spine of ordered water molecules in the minor groove. DNA oligodeoxynucleotides have been synthesized that enable these hypotheses to be tested; of particular interest is the combination of effects due to bifurcation (2) and methylation of the pyrimidines nucleotides (3). The data indicate that neither bifurcated hydrogen bonds nor pyrimidine methyl groups nor both are essential for DNA curvature. The data further suggest that the influence of the minor groove spine of hydration on the B'-formation is small. The experiments favor the hypothesis that base stacking interactions are the dominant force in stabilizing the B'-form structure.     

Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Nonsense-mediated mRNA decay in yeast does not require PAB1 or a poly(A) tail

Eukaryotic mRNAs harboring premature translation termination codons are recognized and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. The mechanism for discriminating between mRNAs that terminate translation prematurely and those subject to termination at natural stop codons remains unclear. Studies in multiple organisms indicate that proximity of the termination codon to the 3' poly(A) tail and the poly(A) RNA-binding protein, PAB1, constitute the critical determinant in NMD substrate recognition. We demonstrate that mRNA in yeast lacking a poly(A) tail can be destabilized by introduction of a premature termination codon and, importantly, that this mRNA is a substrate of the NMD machinery. We further show that, in cells lacking Pab1p, mRNA substrate recognition and destabilization by NMD are intact. These results establish that neither the poly(A) tail nor PAB1 is required in yeast for discrimination of nonsense-codon-containing mRNA from normal by NMD.     

Adenovirus homologous recombination does not require expression of the immediate-early E1a gene.

To investigate whether early genes other than those involved directly in DNA replication are required for efficient adenovirus recombination, pairs of viruses with deletions in E1a, E1b 496R, E1b 196R, or E4 and containing differing restriction site markers were used to infect both permissive and non- or semipermissive cells. Recombination was assayed among intracellular and extracellular genomes by restriction digestion and blot hybridization. Recombination was delayed in infections of nonpermissive cells with E1a- viruses until a time consistent with the late onset of DNA replication characteristic of the cell type. This shows that E1a expression is not absolutely required for adenovirus recombination. Similar tests with deletion mutations in E1b and E4 also show that these genes are not required for efficient recombination. Taken together with earlier results showing that recombination depends on DNA replication, it is likely that adenovirus recombination is a consequence of cellular repair functions acting on the substrates produced by replication.     

Proteolysis of the McpA chemoreceptor does not require the Caulobacter major chemotaxis operon

The degradation of the McpA chemoreceptor in Caulobacter crescentus accompanies the swarmer cell to the stalked-cell differentiation event. To further analyze the requirements for its degradation, we have constructed a series of strains that have deletions in the mcpA gene and in the mcpA chemotaxis operon. Internal deletions of the mcpA gene demonstrate that the highly conserved domain (signalling unit) and the methylation domains are not required for cell cycle-regulated proteolysis. The deletion of the chemotaxis operon, which is absolutely required for chemotaxis and McpA chemoreceptor methylation, has no effect on McpA proteolysis.     

Regulation of extracellular signal-regulated kinase activity by p120 RasGAP does not involve its pleckstrin homology or calcium-dependent lipid binding domains but does require these domains to regulate cell proliferation.

The gene encoding for p120 RasGAP, has been disrupted in mice (M. Henkemeyer et al., Nature (Lond.), 377: 695-701, 1995).In this study, using fibroblasts derived from these mouse embryos (Gap-/-; P. van der Geer et al., Mol. Cell Biol., 17: 1840-1847, 1997), we demonstrate that mitogen-activated protein kinase (MAPK) activation is prolonged after epidermal growth factor (EGF), but not lysophosphatidic acid, stimulation as compared with wild-type cells. Furthermore, these cells exhibited a moderate increase in their proliferative rate and saturation density, as well as a limited ability to form colonies in soft agar. Stable cell lines expressing full-length p120GAP not only restored the ability to down-regulate MAPK after EGF stimulation but also lowered their saturation densities. Similarly, expression of p120GAP, missing either its pleckstrin homology (PH) or its calcium-dependent lipid binding (CaLB)/C2 domain, restored MAPK down-regulation and retained the ability to associate with p190 RhoGAP and to be phosphorylated by v-src but exhibited higher saturation densities similar to Gap-/- cells. Our results, therefore, suggest that p120GAP functions not only by down-regulating the Ras/MAPK pathway after growth factor stimulation but is also important in regulating cell proliferation that involves its PH and CaLB domains.     

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.     

ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling

ANG II type 1 (AT₁) receptors respond to sustained exposure to ANG II byundergoing downregulation of absolute receptor numbers. It has beenassumed previously that downregulation involves endocytosis. Thepresent study hypothesized that AT₁ receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT₁ receptors with carboxy-terminal deletionsinternalized <5% of radioligand compared with 65% for wild-typeAT₁ receptors. The truncated AT₁ receptorsretained the ability to undergo downregulation. These data suggest theexistence of an alternative pathway to AT₁ receptordegradation that does not require endocytosis, per se. Point mutationsin either the second transmembrane region or second intracellular loopimpaired G protein (G_q) coupling. These receptors exhibiteda biphasic pattern of downregulation. The earliest phase ofdownregulation (0-2 h) was independent of coupling toG_q, but no additional downregulation was observed after2 h of ANG II exposure in the receptors with impaired coupling toG_q. These data suggest that coupling to G_q isrequired for the later phase (2-24 h) of AT₁ receptor downregulation.

     

Nucleosome sliding by Chd1 does not require rigid coupling between DNA‐binding and ATPase domains

Chromatin remodellers are ATP‐dependent motor proteins that physically reposition and reorganize nucleosomes. Chd1 and Iswi‐type remodellers possess a DNA‐binding domain (DBD) needed for efficient nucleosome mobilization; however, it has not been clear how this domain physically contributes to remodelling. Here we show that the Chd1 DBD promotes nucleosome sliding simply by tethering the remodeller to nucleosome substrates. Nucleosome sliding activity was largely resistant to increasing length and flexibility of the linker connecting the DBD and ATPase motor, arguing that the ATPase motor does not shift DNA onto the nucleosome by pulling on the DBD.     

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.     

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.