Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome

Clostridium cellulovorans uses not only cellulose but also xylan, mannan, pectin, and several other carbon sources for its growth and produces an extracellular multienzyme complex called the cellulosome, which is involved in plant cell wall degradation. Here we report a gene for a cellulosomal subunit, pectate lyase A (PelA), lying downstream of the engY gene, which codes for cellulosomal enzyme EngY. pelA is composed of an ORF of 2,742 bp and encodes a protein of 914 aa with a molecular weight of 94,458. The amino acid sequence derived from pelA revealed a multidomain structure, i.e., an N-terminal domain partially homologous to the C terminus of PelB of Erwinia chrysanthemi belonging to family 1 of pectate lyases, a putative cellulose-binding domain, a catalytic domain homologous to PelL and PelX of E. chrysanthemi that belongs to family 4 of pectate lyases, and a duplicated sequence (or dockerin) at the C terminus that is highly conserved in enzymatic subunits of the C. cellulovorans cellulosome. The recombinant truncated enzyme cleaved polygalacturonic acid to digalacturonic acid (G2) and trigalacturonic acid (G3) but did not act on G2 and G3. There have been no reports available to date on pectate lyase genes from Clostridia.

Differences in quantal amplitude reflect GluR4- subunit number at corticothalamic synapses on two populations of thalamic neurons

Low-frequency thalamocortical oscillations that underlie drowsiness and slow-wave sleep depend on rhythmic inhibition of relay cells by neurons in the reticular nucleus (RTN) under the influence of corticothalamic fibers that branch to innervate RTN neurons and relay neurons. To generate oscillations, input to RTN predictably should be stronger so disynaptic inhibition of relay cells overcomes direct corticothalamic excitation. Amplitudes of excitatory postsynaptic conductances (EPSCs) evoked in RTN neurons by minimal stimulation of corticothalamic fibers were 2.4 times larger than in relay neurons, and quantal size of RTN EPSCs was 2.6 times greater. GluR4-receptor subunits labeled at corticothalamic synapses on RTN neurons outnumbered those on relay cells by 3.7 times, providing a basis for differences in synaptic strength.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-β. The ability to maintain growth in TGF-β was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-β resistance may explain our previously described gain of TGF-β resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-β resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

Antisense Expression of the CK2 α-Subunit Gene in Arabidopsis. Effects on Light-Regulated Gene Expression and Plant Growth1

The protein kinase CK2 (formerly casein kinase II) is thought to be involved in light-regulated gene expression in plants because of its ability to phosphorylate transcription factors that bind to the promoter regions of light-regulated genes in vitro. To address this possibility in vivo and to learn more about the potential physiological roles of CK2 in plants, we transformed Arabidopsis with an antisense construct of the CK2 α-subunit gene and investigated both morphological and molecular phenotypes. Antisense transformants had a smaller adult leaf size and showed increased expression of chs in darkness and of cab and rbcS after red-light treatment. The latter molecular phenotype implied that CK2 might serve as one of several negative and quantitative effectors in light-regulated gene expression. The possible mechanism of CK2 action and its involvement in the phytochrome signal transduction pathway are discussed.

Plastome Engineering of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Tobacco to Form a Sunflower Large Subunit and Tobacco Small Subunit Hybrid1

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO2 fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.

RNA tertiary interactions in the large ribosomal subunit: The A-minor motif

Analysis of the 2.4-Å resolution crystal structure of the large ribosomal subunit from Haloarcula marismortui reveals the existence of an abundant and ubiquitous structural motif that stabilizes RNA tertiary and quaternary structures. This motif is termed the A-minor motif, because it involves the insertion of the smooth, minor groove edges of adenines into the minor groove of neighboring helices, preferentially at C-G base pairs, where they form hydrogen bonds with one or both of the 2′ OHs of those pairs. A-minor motifs stabilize contacts between RNA helices, interactions between loops and helices, and the conformations of junctions and tight turns. The interactions between the 3′ terminal adenine of tRNAs bound in either the A site or the P site with 23S rRNA are examples of functionally significant A-minor interactions. The A-minor motif is by far the most abundant tertiary structure interaction in the large ribosomal subunit; 186 adenines in 23S and 5S rRNA participate, 68 of which are conserved. It may prove to be the universally most important long-range interaction in large RNA structures.

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

In skeletal muscle, transcription of the gene encoding the mouse type Iα (RIα) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIα protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIα or RIIα fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIα subunits and requires the amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the full-length RIα–green fluorescent protein template abolishes localization, indicating that dimerization of RIα is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIα at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Iα homologue RCE with AKAPCE and for in vitro binding of RIα to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIα tethering at this site.

Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav 2.2) of N-type calcium channels

N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the α1B subunit (Cav 2.2). The α1B-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to ω-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Mutant G protein α subunit activated by Gβγ: A model for receptor activation?

How receptors catalyze exchange of GTP for GDP bound to the Gα subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's βγ heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Gα. To test this possibility, we designed a mutant Gα that would bind to βγ in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of αs, the α subunit of GS, the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed β1 and γ2, this mutant (αsΔ), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed β protein to interact normally with the lip of the nucleotide binding pocket of αsΔ. We substituted alanine for an aspartate in β1 that binds to a lysine (K206) in the lip of the α subunit's nucleotide binding pocket. Coexpressed with αsΔ and γ2, this mutant, β1-D228A, elevated cAMP much less than did β1-wild type; it did bind to αsΔ normally, however, as indicated by its unimpaired ability to target αsΔ to the plasma membrane. We conclude that βγ can activate αs and that this effect probably involves both a tilt of βγ relative to αs and interaction of β with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.

In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies

A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.

A missense mutation of the Na+ channel αII subunit gene Nav1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in β1 and αI-subunit genes of voltage-gated Na+ channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R187W) in the gene encoding the α-subunit of neuronal voltage-gated Na+ channel type II (Nav1.2) in a patient with FS associated with afebrile seizures. The mutation R187W occurring on Arg187, a highly conserved residue among voltage-gated Na+ channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNav1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na+ channel conductance was not affected. Prolonged residence in the open state of the R187W mutant channel may augment Na+ influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Nav1.2 in a human disease and propose the R187W mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

Requirement of the nicotinic acetylcholine receptor β2 subunit for the anatomical and functional development of the visual system

In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, β2−/− mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the β2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.