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R Levis

Publications and source records attributed to R Levis.

At least 19 recordsLinked to original sources
PubMed2005

Cells of human aminopeptidase N (CD13) transgenic mice are infected by human coronavirus-229E in vitro, but not in vivo.

Aminopeptidase N, or CD13, is a receptor for serologically related coronaviruses of humans, pigs, and cats. A mouse line transgenic for the receptor of human coronavirus-229E (HCoV-229E) was created using human APN (hAPN) cDNA driven by a hAPN promoter. hAPN-transgenic mice expressed hAPN mRNA in the kidney, small intestine, liver, and lung. hAPN protein was specifically expressed on epithelial cells of the proximal convoluted renal tubules, bronchi, alveolar sacs, and intestinal villi. The hAPN expression pattern within transgenic mouse tissues matched that of mouse APN and was similar in mice heterozygous or homozygous for the transgene. Primary embryonic cells and bone marrow dendritic cells derived from hAPN-transgenic mice also expressed hAPN protein. Although hAPN-transgenic mice were resistant to HCoV-229E in vivo, primary embryonic cells and bone marrow dendritic cells were infected in vitro. hAPN-transgenic mice are valuable as a source of primary mouse cells expressing hAPN. This hAPN-transgenic line will also be used for crossbreeding experiments with other knockout, immune deficient, or transgenic mice to identify factors, in addition to hAPN, that are required for HCoV-229E infection.

Animals↗
PubMed1997

Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts.

Aedes↗
PubMed1996

Feline aminopeptidase N serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup I.

Two members of coronavirus serogroup I, human respiratory coronavirus HCV-229E and porcine transmissible gastroenteritis virus (TGEV), use aminopeptidase N (APN) as their cellular receptors. These viruses show marked species specificity in receptor utilization, as HCV-229E can utilize human but not porcine APN, while TGEV can utilize porcine but not human APN. To determine whether feline APN could serve as a receptor for two feline coronaviruses in serogroup I, feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FeCV), we cloned the cDNA encoding feline APN (fAPN) by PCR from cDNA isolated from a feline cell line and stably expressed it in FIPV- and FeCV-resistant mouse and hamster cells. The predicted amino acid sequence of fAPN shows 78 and 77% identity with human and porcine APN, respectively. When inoculated with either of two biologically different strains of FIPV or with FeCV, fAPN-transfected mouse and hamster cells became infected and viral antigens developed in the cytoplasm. Infectious FIPV was released from hamster cells stably transfected with fAPN. The fAPN-transfected mouse and hamster cells were challenged with other coronaviruses in serogroup I including canine coronavirus, porcine coronavirus TGEV, and human coronavirus HCV-229E. In addition to serving as a receptor for the feline coronaviruses, fAPN also served as a functional receptor for each of these serogroup I coronaviruses as shown by development of viral antigens in the cytoplasm of infected mouse or hamster cells stably transfected with fAPN. In contrast, fAPN did not serve as a functional receptor for mouse hepatitis virus (MHV-A59), which is in serogroup II and utilizes mouse biliary glycoprotein receptors unrelated to APN. Thus, fAPN serves as a receptor for a much broader range of group I coronaviruses than human and porcine APNs. The human, porcine, and canine coronaviruses in serogroup I that are able to use fAPN as a receptor have previously been shown to infect cats without causing disease. Therefore, host factors in addition to receptor specificity apparently affect the virulence and transmissibility of nonfeline serogroup I coronaviruses in the cat.

3T3 Cells↗
PubMed1995

Multiple receptor-dependent steps determine the species specificity of HCV-229E infection.

Human coronavirus (HCV)-229E causes disease only in humans and grows in human cells and in cells of other species that express recombinant human aminopeptidase N (hAPN), the receptor for HCV-229E. We compared the species specificity of HCV-229E infection with the species specificity of virus binding using immunofluorescence, assay of virus yields, fluorescence activated cell sorting and a monoclonal antibody directed against hAPN that blocks infection. We found that HCV-229E binds to intestinal brush border membranes (BBM) and to membranes of cell lines from cats, dogs, pigs, and humans, however the virus only infects two of these species. HCV-229E will not bind to BBM or to membranes from cell lines derived from hamster or mice. Animal coronaviruses related to HCV-229E, including FIPV, CCV, and TGEV bind to cell membranes from cats, dogs, cows, pigs and humans (but not mice), while each virus infects cells from only a subset of these species. Infectious genomic HCV-229E RNA, can infect cells of all of these species. These data suggest that the species-specificity of infection for this serogroup of coronaviruses is determined at the levels of virus binding and penetration. Since binding of viral spike glycoprotein to cellular receptors is not the only limiting factor, we suggest that one or more steps associated with virus penetration may determine the species specificity of infection with the HCV-229E serogroup of coronaviruses.

Animals↗
PubMed1993

Effects of perchlorate on the molecules of excitation-contraction coupling of skeletal and cardiac muscle.

To understand the nature of the transmission process of excitation-contraction (EC) coupling, the effects of the anion perchlorate were investigated on the voltage sensor (dihydropyridine receptor, DHPR) and the Ca release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum (SR). The molecules, from rabbit skeletal muscle, were either separated in membrane vesicular fractions or biochemically purified so that the normal EC coupling interaction was prevented. Additionally, the effect of ClO4- was investigated on L-type Ca2+ channel gating currents of guinea pig ventricular myocytes, as a native DHPR not in the physiological interaction of skeletal muscle. At 20 mM, ClO4- had minor effects on the activation of ionic currents through Ca channels from skeletal muscle transverse tubular (T) membranes fused with planar bilayers: a +7-mV shift in the midpoint voltage, V, with no change in kinetics of activation or deactivation. This is in contrast with the larger, negative shift that ClO4- causes on the distribution of intramembrane charge movement of skeletal muscle. At up to 100 mM it did not affect the binding of the DHP [3H]PN200-110 to triad-enriched membrane fractions (TR). At 8 mM it did not affect the kinetics or the voltage distribution of gating currents of Ca channels in heart myocytes. These negative results were in contrast to the effects of ClO4- on the release channel. At 20 mM it increased several-fold the open probability of channels from purified RyR incorporated in planar bilayers and conducting Ba2+, an effect seen on channels first closed by chelation of Ca2+ or by the presence of Mg2+. It significantly increased the initial rate of efflux of 45Ca2+ from TR vesicles (by a factor of 1.75 at 20 mM and 4.5 at 100 mM). ClO4- also increased the binding of [3H]ryanodine to TR fractions. The relative increase in binding was 50-fold at the lowest [Ca2+] used (1 microM) and then decayed to much lower values as [Ca2+] was increased. The increase was due entirely to an increase in the association rate constant of ryanodine binding. The chaotropic ions SCN- and I- increased the association rate constant to a similar extent. The binding of ryanodine to purified RyR protein reconstituted into liposomes had a greater affinity than to TR fractions but was similarly enhanced by ClO4-. The reducing agent dithiothreitol (5 mM) did not reduce the effect of ClO4-, and 5% polyethylene glycol, with an osmolarity equivalent to 20 mM ClO4-, did not change ryanodine binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor.

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately -30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)-dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.

Animals↗
PubMed1992

Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes.

Intramembrane charge movement was recorded in guinea pig ventricular myocytes at 19-22 degrees C using the whole-cell patch clamp technique. From a holding potential of -110 mV, the dependence of intramembrane charge moved on test voltage (Q(V)) followed the sum of two Boltzmann components. One component had a transition voltage (V) of -48 mV and a total charge (Qmax) of congruent to 3 nC/microF. The other had a V of -18 mV and a Qmax of 11 nC/microF. Ba2+ currents through Ca channels began to activate at -45 mV and peaked at congruent to -15 mV. Na+ current peaked at -35 to -30 mV. Availability of charge (in pulses from -70 to +10 mV) depended on the voltage of conditioning depolarizations as two Boltzmann terms plus a constant. One term had a V of -88 mV and a Qmax of 2.5 nC/microF; the other had a V of -29 mV and a Qmax of 6.3 nC/microF. From the Q(V) dependence, the voltage dependence of the ionic currents, and the voltage dependence of the availability of charge, the low voltage term of Q(V) and availability was identified as Na gating charge, at a total of 3.5 nC/microF. The remainder, 11 nC/microF, was attributed to Ca channels. After pulses to -40 mV and above, the OFF charge movement had a slow exponentially decaying component. Its time constant had a bell-shaped dependence on OFF voltage peaking at 11 ms near -100 mV. Conditioning depolarizations above -40 mV increased the slow component exponentially with the conditioning duration (tau approximately equal to 480 ms). Its magnitude was reduced as the separation between conditioning and test pulses increased (tau approximately equal to 160 ms). The voltage distribution of the slow component of charge was measured after long (5 s) depolarizations. Its V was -100 mV, a shift of -80 mV from the value in normally polarized cells. This voltage was the same at which the time constant of the slow component peaked. Qmax and the steepness of the voltage distribution were unchanged by depolarization. This indicates that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

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Publishing↗
PubMed1990

Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.

Animals↗
PubMed1989

Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.

Aedes↗
PubMed1989

A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis.

The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been proposed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of Sindbis virus from which infectious RNA transcripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied after RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction region into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independent revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region showed that all were pseudorevertants, with only two containing potentially compensating changes in the junction region. An assay was developed to identify revertants with second-site changes in trans-acting viral components involved in subgenomic RNA synthesis. At least two such revertants were identified. Mapping of these and other second-site compensating mutations may provide genetic clues as to which virus-specific protein(s) is responsible for interaction with the conserved junction region to promote subgenomic RNA synthesis.

Amino Acid Sequence↗
PubMed1987

Engineered defective interfering RNAs of Sindbis virus express bacterial chloramphenicol acetyltransferase in avian cells.

We are investigating the feasibility of using the positive-strand RNA virus Sindbis virus and its defective interfering (DI) particles as vectors for introducing foreign genes into cells. In previous work we showed by deletion mapping of a cloned cDNA derived from one of the DI RNAs that only nucleotides at the 3' and 5' termini of the RNA are essential for the DI RNA to be amplified after it is transfected into cells in the presence of helper virus. As a first step in developing a vector we replaced 75% of the internal nucleotides of this DI cDNA with foreign sequences including the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. DI RNAs transcribed from this cDNA were replicated and packaged by helper Sindbis virus and became a major viral RNA species in infected cells by the third passage after transfection. They were also translated to produce enzymatically active CAT. CAT activity was measured at passage 3 but could also be detected in transfected cells. DI RNAs containing the CAT gene were translated in vivo and in vitro to produce two polypeptides immunoprecipitable by anti-CAT antibodies. One polypeptide was identical in size to the authentic CAT polypeptide; the other was the size expected for a protein initiated at an upstream, viral-specific AUG in frame with the CAT AUG. These studies establish that DI genomes of Sindbis virus can tolerate the insertion and direct the expression of at least one foreign gene.

Acetyltransferases↗
PubMed1987

Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.

We constructed full-length cDNA clones of Sindbis virus that can be transcribed in vitro by SP6 RNA polymerase to produce infectious genome-length transcripts. Viruses produced from in vitro transcripts are identical to Sindbis virus and show strain-specific phenotypes reflecting the source of RNA used for cDNA synthesis. The cDNA clones were used to confirm the mapping of the causal mutation of ts2 to the capsid protein. A general strategy for mapping Sindbis virus mutations is described and was used to identify two lethal mutations in an original full-length construct which did not produce infectious transcripts. An XbaI linker was inserted in the cDNA clone near the transcriptional start of the subgenomic mRNA; the resulting virus retains the XbaI recognition sequence, thus providing formal evidence that viruses are derived from in vitro transcripts of cDNA clones. The potential applications of the cDNA clones are discussed.

Animals↗
PubMed1986

Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging.

Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes.

Base Sequence↗
PubMed1985

Separable cis-acting control elements for expression of the white gene of Drosophila.

The white gene of Drosophila is required for the pigmentation of the eyes, ocelli, Malpighian tubules and testis sheath. We have examined the regulation of white expression in germ line transformants that carry the mRNA-coding sequences of white flanked by varying amounts of the sequences normally located adjacent to them. A segment with as little as 1.9 kb of 5'- and 2.1 kb of 3'-flanking DNA was expressed to give essentially wild-type pigmentation in all of these tissues and interacted in trans with the zeste-1 allele. In contrast, transformants having segments with 1.1 kb of 5'-flanking DNA had reduced levels of pigment in their eyes, no detectable pigment in their testes and did not interact with zeste-1. The eyes, Malpighian tubules and ocelli of transformants with as little as 0.4 kb of 5'-flanking DNA were pigmented and the eye pigmentation exhibited dosage compensation. We conclude that there are sequence elements present in the region from 1.1 to 1.9 kb upstream from the 5' end of the white transcript which are required for expression in the testes and interaction of white and zeste-1. The same or other elements in this region appear to augment the pigmentation of the eye.

Animals↗
PubMed1985

Effects of genomic position on the expression of transduced copies of the white gene of Drosophila.

The white gene of Drosophila is expressed normally when introduced at many different sites in the genome by P-element-mediated DNA transformation, but is expressed abnormally when inserted at two particular genomic positions. It is now demonstrated that the mutant expression in these two cases is caused by the surrounding chromosomal region into which the white gene has been inserted. The white gene could be moved from these two positions, where it confers a mutant phenotype, to other positions in the genome where it confers a wild-type phenotype. However, flies in which white has been moved to one new location have an unusual mosaic phenotype.

Animals↗
PubMed1984

DNA sequence of the white locus of Drosophila melanogaster.

The DNA sequence of the white locus of Drosophila melanogaster is presented. This 14,100 base-pair sequence includes the region of the locus required for wild-type levels of expression and control of expression. We also report the sequence of a complementary DNA clone which established the position of the 3' end of the white RNA on this genomic sequence. The probable exon-intron structure of the gene has been predicted from the DNA sequence of the regions known to be represented in the RNA. The amino acid sequence of the protein which would be produced by translation of this RNA suggests that the white locus gene product may be a membrane protein. The DNA sequence rearrangements associated with seven insertion mutants (white-dominant-zeste-like (wDZL), white-spotted (wsp), white-honey (wh), white-zeste-mottled (wzm), white-apricot (wa), white-buff (wbf) and white-hd81b11 (whd81b11)), one deletion mutant (white-spotted 4 (wsp4)) and one internal duplication mutant (white-ivory (wi)) have been determined and positioned on the wild-type sequence. The positions of these insertions and those of previously characterized insertions associated with six other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.

Animals↗