Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cotransfection of selectable marker genes and the gene for the nonstructural proteins NS1 and NS2 of the autonomous parvovirus H-1 failed to produce cell lines that constitutively expressed NS1. A plasmid, pP38NS1cat, was constructed that expressed the NS1-NS2 gene from the H-1 P38 coat protein promoter in place of the natural P4 promoter. The P38 promoter is constitutively weak and is trans-activated by NS1. Stable cell lines were isolated that contained pP38NS1cat that was constitutively silent, but inducible with exogenous NS1 by superinfection or by treatment with sodium butyrate. The cells that were induced for this self-stimulatory genetic circuit did not remain in the culture, suggesting that expression of NS1-NS2 is cytotoxic or that the expression is not sustained. The properties of these cell lines and an example of the construction of a cell line inducible for expression of the viral coat protein gene and the bacterial gene for chloramphenicol acetyltransferase (cat) are described.
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PMID:Construction of a genetic switch for inducible trans-activation of gene expression in eucaryotic cells. 303 74

The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.
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PMID:Influenza virus NS1 protein stimulates translation of the M1 protein. 750 95

The generation of influenza A virus defective interfering (DI) particles was studied by using an NS2 mutant which produces, in a single cycle of virus replication, a large amount of DI particles lacking the PA polymerase gene. The decrease in PA gene replication has been shown to occur primarily at the cRNA synthesis step, with preferential amplification of PA DI RNA species present in a marginal amount in the virus stock. In addition, at the assembly step the PA DI RNAs were preferentially incorporated into virions, resulting in selective reduction in the packaging of the PA gene into virions. Similarly, in cells dually infected with the NS2 mutant and wild-type viruses, packaging of the wild-type PA gene was also greatly suppressed. In contrast, incorporation of other RNA segments, i.e., the PB2 and NS genes, was not affected, suggesting that the PA DI RNAs competed only with the PA gene in a segment-specific manner. Experiments involving rescue of recombinant chloramphenicol acetyltransferase (CAT) RNA flanked by the noncoding regions of the PA (PA/CAT RNA) and PB2 (PB2/CAT RNA) genes into viral particles showed that only PA/CAT RNA was not rescued by infection with the NS2 mutant virus containing the PA DI RNAs. However, recombinant PA/CAT RNA in which either the 3' or 5' noncoding region was replaced with that of the PB2 gene was rescued by the NS2 mutant. These results suggest that the noncoding regions of the PA gene are responsible for the competition with PA DI RNA species at the virus assembly step and that coexistence of the both noncoding regions would be a prerequisite for this phenomenon. Decreased packaging of the progenitor RNA by the DI RNA, in addition to the suppression of cRNA synthesis, is likely involved in the production of DI particles.
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PMID:Segment-specific noncoding sequences of the influenza virus genome RNA are involved in the specific competition between defective interfering RNA and its progenitor RNA segment at the virion assembly step. 903 47

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.
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PMID:Rescue of synthetic minireplicons establishes the absence of the NS1 and NS2 genes from avian pneumovirus. 937 59

It has previously been demonstrated in this laboratory that an influenza virus-like chloramphenicol acetyltransferase (CAT) RNA could be expressed in COS-1 cells that synthesized all ten influenza A virus-encoded proteins from recombinant plasmids. It was also shown that supernatant fluids harvested from these cultures contained virus-like particles (VLPs) that could deliver an enclosed CAT RNA to MDCK cells. Here, it is shown that the levels of expression of the reporter gene in the COS-1 and/or MDCK cells can be altered drastically by modifying the concentrations of the recombinant plasmids transfected in the COS-1 cells. Thus, it was observed that overexpression of NS2 reduced CAT expression in COS-1 cells, whereas overexpression of M2 and NS1 proteins dramatically decreased transmission of the CAT RNA to the MDCK cultures. These results are discussed with reference to the roles of these proteins during virus replication. From these experiments, a ratio of transfected plasmids was found that increased the efficiency of the previously described system by 50-100-fold. Under these optimized conditions, it was demonstrated that VLPs can be formed in the absence of neuraminidase expression and that these VLPs remained aggregated to each other and to cell membranes. Moreover, it is shown that CAT RNA transmission was dependent on specific interactions of the ribonucleoprotein complex with other viral structural polypeptides. These data demonstrate the usefulness of this encapsidation-packaging system for the study of different aspects of the influenza virus life-cycle.
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PMID:Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins. 1042 31

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.
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PMID:Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs. 1131 64

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.
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PMID:Influenza B and C virus NEP (NS2) proteins possess nuclear export activities. 1146 9