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Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b.

ABSTRACT: The nonconditional RNA packaging mutant SE21Q1b contains cis- and trans-acting defects which cause cellular mRNA, rather than viral genomic RNA, to be nonspecifically packaged into SE21Q1b viral particles. Using genomic libraries of the c-SE21Q1b quail cell line, we have been able to construct a molecular clone of the SE21Q1b provirus. Upon transfection into primary quail embryo fibroblasts, the SE21Q1b molecular clone is able to recapitulate the nonspecific RNA packaging phenotype of the c-SE21Q1b cell line. The RNA packaging phenotypes displayed by several SE21Q1b/avian sarcoma-leukemia virus hybrid provirus constructs have further indicated that sequences responsible for the altered RNA packaging phenotype of SE21Q1b are localized in the left third of the SE21Q1b proviral genome. DNA sequence analysis of this region has revealed that the 5' SE21Q1b deletion has removed 179 bp from the SE21Q1b left long terminal repeat and leader regions. Several differences were detected at the carboxyl terminus of the deduced SE21Q1b nucleocapsid protein sequence in comparison with that of Rous sarcoma virus PR-C. Results of site-directed oligonucleotide mutagenesis experiments indicate, however, that the presence of these residues in the nucleocapsid protein alone is not responsible for the decreased RNA packaging specificity of SE21Q1b.

SUBMITTER: Anderson DJ

PROVIDER: S-EPMC238277 | biostudies-other | 1992 Jan

REPOSITORIES: biostudies-other

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Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b.

Anderson D J DJ Lee P P Levine K L KL Sang J S JS Shah S A SA Yang O O OO Shank P R PR Linial M L ML

Journal of virology 19920101 1

The nonconditional RNA packaging mutant SE21Q1b contains cis- and trans-acting defects which cause cellular mRNA, rather than viral genomic RNA, to be nonspecifically packaged into SE21Q1b viral particles. Using genomic libraries of the c-SE21Q1b quail cell line, we have been able to construct a molecular clone of the SE21Q1b provirus. Upon transfection into primary quail embryo fibroblasts, the SE21Q1b molecular clone is able to recapitulate the nonspecific RNA packaging phenotype of the c-SE21 ...[more]

PMID: 1727484

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Project description:PLC (phospholipase C) isoenzymes catalyse the conversion of PtdIns(4,5)P2 into the Ca2+-mobilizing second messenger, Ins(1,4,5)P3, and the protein kinase C-activating second messenger, diacylglycerol. With the goal of identifying additional mammalian PLC isoenzymes, we screened the NCBI non-redundant database using a BLAST algorithm for novel sequences with homology with the conserved PLC catalytic core. Two unique sequences corresponding to two unknown PLC isoenzymes were identified, and one of these, designated PLC-eta2, was cloned and characterized. Most of the coding sequence of PLC-eta2 was constructed from two ESTs (expressed sequence tags), which included an overlapping sequence that was confirmed by multiple ESTs and mRNAs. 5'-RACE (rapid amplification of cDNA ends) also identified an upstream exon not deduced from available EST or mRNA sequences. Sequence analysis of PLC-eta2 revealed the canonical domains of a PLC isoenzyme with an additional long C-terminus that contains a class II PDZ-binding motif. Genomic analyses indicated that PLC-eta2 is encoded by 23 exons. RT-PCR (reverse transcriptase-PCR) analyses illustrated expression of PLC-eta2 in human retina and kidney, as well as in mouse brain, eye and lung. RT-PCR with exon-specific primers also revealed tissue-specific expression of four splice variants in mouse that represent alternative use of sequences in exons 21, 22 and 23. PLC-eta2-specific antisera recognized one of these splice variants as an approx. 155 kDa species when expressed in COS-7 cells; PLC-eta2 natively expressed in 1321N1 human astrocytoma cells also migrated as an approx. 155 kDa species. PLC activity was observed in vitro and in vivo for three different constructs of PLC-eta2, each containing possible alternatively spliced first exons. Co-expression of PLC-eta2 with Gbeta1gamma2 dimers of heterotrimeric G-proteins resulted in marked stimulation of inositol lipid hydrolysis. Thus PLC-eta2 may in part function downstream of G-protein-coupled receptors.

Dataset Information

Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b.

Publications

Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b.

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