Biomedical subjects

R J Gorelick

Publications and source records attributed to R J Gorelick.

26 records · Page 2Linked to original sources

PubMed1994

Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus.

We have analyzed RNA packaging by a series of mutants altered in the nucleocapsid (NC) protein of Moloney murine leukemia virus (Mo-MuLV). We found that mutants lacking residues 8 through 11 or 44 through 60 of NC package Mo-MuLV RNA with virtually the same efficiency as wild-type Mo-MuLV. In contrast, point mutants altered at the conserved cysteines in the cysteine array (residues 26 and 29) and a mutant lacking residues 16 through 23 packaged Mo-MuLV RNA with approximately 1% of the efficiency of wild-type Mo-MuLV. The deficiency in packaged RNA was observed not only in Northern (RNA) analysis but also in an RNA-PCR assay, which would detect degraded as well as intact RNA. One of the cysteine array mutants was also shown to be defective with respect to encapsidation of hygromycin phosphotransferase mRNA containing a Mo-MuLV packaging signal. We suggest that a central region of NC, consisting of the cysteine array and flanking basic residues, is required for RNA packaging in Mo-MuLV.

Amino Acid Sequence↗

PubMed1993

The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent.

The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.

Amino Acid Sequence↗

PubMed1990

Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA.

All retroviruses contain, in the nucleocapsid domain of the Gag protein, one or two copies of the sequence Cys-X2-Cys-X4-His-X4-Cys. We have generated a series of mutants in the two copies of this motif present in human immunodeficiency virus type 1. These mutants encoded virus particles that were apparently composed of the normal complement of viral proteins but contained only 2 to 20% of the normal level of genomic RNA. No infectivity could be detected in the mutant particles, while 10(5) infectious U were present in an equivalent amount of wild-type particles. Thus, the mutants have another defect in addition to the inefficiency with which they encapsidate genomic RNA. Our results show that both copies of the motif are required for normal RNA packaging and for infectivity. Mutants of this type may have important applications, including nonhazardous materials for research, immunogens in vaccine and immunotherapy studies, and diagnostic reagents.

Amino Acid Sequence↗

PubMed1989

Suppression of UAA and UGA termination codons in mutant murine leukemia viruses.

Genomes of mammalian type C retroviruses contain a UAG termination codon between the gag and pol coding regions. The pol region is expressed in the form of a gag-pol fusion protein following readthrough suppression of the UAG codon. We have used oligonucleotide-directed mutagenesis to change the UAG in Moloney murine leukemia virus to UAA or UGA. These alternate termination codons were also suppressed, both in infected cells and in reticulocyte lysates. Thus, the signal or context inducing suppression of UAG in wild-type Moloney murine leukemia virus is also effective with UAA and UGA. Further, mammalian cells and cell extracts contain tRNAs capable of translating UAA and UGA as amino acids. To our knowledge, this is the first example of natural suppression of UAA in higher eucaryotes.

Animals↗

PubMed1988

Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence.

All retroviruses encode a nucleic acid-binding or nucleocapsid protein that is believed to be associated with RNA in the virion. Further, all retroviral nucleocapsid proteins contain either one or two copies of the sequence Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys. The conservation of this sequence suggested that it is important for virus replication, and its resemblance to the "zinc-finger" sequences found in eukaryotic transcription factors raised the possibility that it recognizes specific sequences in viral RNA during retrovirus assembly. We used oligonucleotide-directed mutagenesis to generate a series of mutations in the nucleocapsid protein-coding region of Moloney murine leukemia virus. These mutations changed single amino acids, including each of the cysteines, to serine. The mutant viral genomes direct the synthesis of virus particles; these particles lack detectable viral RNA but do contain significant levels of cellular RNAs. Thus it appears that the mutations have destroyed the ability of the viral proteins to specifically package viral RNA during virus assembly. We propose that the conserved sequence in retroviral nucleocapsid proteins functions in RNA sequence recognition and suggest that it is evolutionarily related to the zinc fingers that recognize specific sequences in double-stranded DNA.

Amino Acid Sequence↗

PubMed1986

Electron-transferring flavoprotein from pig kidney: flavin analogue studies.

Apo-electron-transferring flavoprotein from pig kidney (apo-ETF) has been prepared by an acid ammonium sulfate procedure and reconstituted with FAD analogues to probe the flavin binding site. The 8-position of the bound flavin is accessible to solvent as judged by the reaction of 8-Cl-FAD-ETF with sodium sulfide and thiophenol. A series of 8-alkylmercapto-FAD analogues containing increasingly bulky substituents bind tightly to apo-ETF and can be reduced to the dihydroflavin level by octanoyl-CoA in the presence of catalytic levels of the medium-chain acyl-CoA dehydrogenase. Bulky substituents severely slow the rate of these interflavin electron-transfer reactions. In the case of the 8-cyclohexylmercapto derivative, this decrease reflects a sizable increase in the Km for ETF (approximately 14-fold) with only a 20% decrease in Vmax. Reduction of all of these 8-substituted derivatives involves the accumulation of ETF anion radical intermediates. Dihydro-5-deaza-FAD dehydrogenase, unlike the corresponding 1-deazaflavin substitution, is unable to reduce native ETF despite a strongly favorable redox potential difference. These results, together with data from the native proteins, are consistent with obligatory 1-electron transfer between dehydrogenase and ETF possibly involving the exposed dimethylbenzene edge of ETF. Irradiation of apo-ETF reconstituted with the photoaffinity analogue 8-azidoflavin leads to approximately 10% covalent incorporation of the flavin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of apo-ETF labeled with tritiated 8-azido-FAD shows preferential labeling of the smaller subunit (88%, Mr 30,000 subunit; 12%, Mr 33,000 subunit).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Interflavin oxidation-reduction reactions between pig kidney general acyl-CoA dehydrogenase and electron-transferring flavoprotein.

The mechanism of interflavin electron transfer between pig kidney general acyl-CoA dehydrogenase (GAD) and its physiological acceptor, electron-transferring flavoprotein (ETF), has been studied by static and stopped-flow absorbance and fluorescence measurements. At 3 degrees C, pH 7.6, reoxidation of the dehydrogenase (stoichiometrically reduced by octanoyl-CoA) by ETF is multiphasic, consisting of two rapid phases (t1/2 of about 20 and 50 ms), a slower phase half-complete in about 1 s, and a final reaction with a half-time of 20 s. Only the two most rapid phases are significant in turnover. This complicated reaction course was dissected by examining the rates of plausible individual steps, e.g., GAD2e X P + ETF1e, GAD1e X P + ETFox, and GAD1e X P + ETF1e (where P represents the product, octenoyl-CoA, and the subscripts indicate the redox state of the flavin). Rapid reaction and static fluorescence measurements, in all cases, showed that the final equilibrium mixture included appreciable levels of oxidized ETF. This was confirmed by measuring the reverse reactions, e.g., ETF1e + GADox X P, ETF1e + GAD1e X P, and ETF2e + GADox X P. These data support the following overall scheme for the reaction of GAD2e X P with ETFox: The first and second phases correspond to reoxidation of GAD2e X P in two successive one-electron steps requiring two molecules of ETFox. This results in a rapid rise in absorbance at 370 nm where the red anionic radicals of both product-complexed dehydrogenase and ETF absorb strongly.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl-CoA Dehydrogenases↗

PubMed1982

Purification and properties of electron-transferring flavoprotein from pig kidney.

Electron-transferring flavoprotein has been isolated from pig kidney by a simple procedure with a 7-fold higher yield over a previous method using pig liver. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, amino acid analysis, peptide mapping, and measurement of flavin content indicate that pig kidney electron-transferring flavoprotein contains nonidentical subunits (Mr 30 000 and 33 000) with one flavin adenine dinucleotide per dimer. These data contrast with reports that the liver protein is a dimer of identical subunits containing two flavin molecules. Dithionite and ferricyanide titrations indicate that flavin is the only redox-active moiety in pig kidney electron-transferring flavoprotein. Disproportionation of the anionic semiquinone is very slow, requiring about 10 h for half-completion. In contrast to results obtained with the liver protein, pig kidney electron-transferring flavoprotein does not bind crotonyl coenzyme A (crotonyl-CoA) significantly, and the semiquinone form is not reoxidized by crotonyl-CoA directly. These data do not support recent suggestions for a broader role of electron-transferring flavoprotein in beta oxidation.

Amino Acids↗