Efficient selection for high-expression transfectants with a novel eukaryotic vector

Niwa, Hitoshi; Yamamura, Ken‐ichi; Miyazaki, Junichi

doi:10.1016/0378-1119(91)90434-d

Cited by 4,733 publications

(998 citation statements)

References 27 publications

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“…The cDNA of HaloTag was amplified by PCR using the pFC14A (HaloTag 7) cytomegalovirus (CMV) Flexi vector (Promega, Madison, WI) as the template. The cDNA of monomerized enhanced green fluorescent protein (mEGFP), HaloTag, or the GGSGGS linker was inserted into the pCAGGS, pCXN2, pCX4puro, or pCX4neo vector (17,18) to generate pCAGGS-HaloTag-GGSGGS-mEGFP, pCAGGS-mEGFP, pCAGGS-FLAG-mEGFP, pCXN2-mEGFP, pCX4puro-HaloTag, pCX4neo-mEGFP, pCAGGS-FLAG-HaloTag, and pCXN2-HaloTag. pDONR223-RPS6KA1 (RSK1), pDONR223-RPS6KA2 (RSK3), and pDONR223-KSR (KSR1) were gifts from William Hahn (19) (Addgene plasmids 23860, 23530, and 23443, respectively).…”

Section: Methodsmentioning

confidence: 99%

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

Sadaie

Harada

Aoki

2014

Molecular and Cellular Biology

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Section: Methodsmentioning

confidence: 99%

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

Sadaie

Harada

Aoki

2014

Molecular and Cellular Biology

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“…Construction details and sequence files of the plasmids are available upon request. MuV NP, P, and L genes were cloned into the pCAGGS expression vector (10). A plasmid containing firefly luciferase (pFF-Luc) and the MuV minigenome plasmid (BH526/pMG-RLuc), containing Renilla and driven by the T7 promoter, were used in the MuV minigenome assays.…”

Section: Methodsmentioning

confidence: 99%

Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

Pickar

Elson

et al. 2014

J Virol

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Mumps virus (MuV), a paramyxovirus containing a negative-sense nonsegmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel antiviral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced and mutating residue T at 101 to A (T101A) significantly enhanced activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis. IMPORTANCE Mumps virus (MuV) is a reemerging paramyxovirus that caused large outbreaks in the UnitedStates, where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. This work leads to a better understanding of viral RNA synthesis as well as to potential novel strategies to control mumps. Mumps virus (MuV) is a human pathogen that causes acute parotitis and is highly neurotropic (1). Invasion of the central nervous system is evident in almost half of all clinical cases, with asceptic meningitis occurring in approximately 10% of cases and encephalitis in less than 1% (1). Even though the mumps vaccine has dramatically reduced disease incidence, large outbreaks have recently occurred in vaccinated populations (2, 3). Over 5,700 mumps cases were reported in a 2006 outbreak that originated at a university in Iowa and spread to 10 other states (2). A mumps outbreak occurred in New York and New Jersey in 2009 to 2010 where 88% of the patients had one dose of mumps vaccine and 75% of patients had two doses (3). There is no antiviral drug for MuV infection. Understanding functions of viral proteins will aid development of antiviral strategies. In this study, a strain of MuV from a recent outbreak in Iowa in 2006 (4), MuV Iowa/US/06 (referred to here as MuV), was used to examine the role of phosphorylation of the M...

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“…Plasmid-based reverse genetics for virus generation were performed as previously described (22). Briefly, eight RNA polymerase I plasmids (for the synthesis of the eight influenza A viral RNAs), together with plasmids for the expression of the viral PB2, PB1, PA, and NP proteins derived from an influenza A virus strain A/WSN/33 (H1N1) (23), were transfected into 293T cells using Trans-IT 293 (Mirus). At 48 h posttransfection, culture supernatants were harvested and inoculated into MDCK cells for virus propagation.…”

mentioning

confidence: 99%

Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

Yamayoshi

Yamada

Fukuyama

et al. 2014

J Virol

111

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Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals. (1). To date, 143 confirmed cases of A(H7N9) virus infection and 45 associated deaths have been reported. This represents a case-fatality rate of ca. 31.5%; however, this rate may actually be lower because the number of mild cases is unknown (2). Phylogenetic analysis has revealed that the hemagglutinin (HA) and neuraminidase (NA) genes of the A(H7N9) viruses share the greatest sequence identities with Eurasian avian A(H7N3) viruses and N9 viruses of different HA subtypes, respectively (3-6). The other six viral genes are highly related to A(H9N2) viruses that have circulated in poultry in China (1, 3-7). These findings indicate that A(H7N9) viruses are reassortant viruses with genes from several avian isolates. IMPORTANCE Novel avian-origin influenzaWe (8) and others (9-14) recently reported that prototype A(H7N9) viruses replicate efficiently in mammalian cells, mice, and ferrets, and transmit among subsets of pairs of ferrets. A(H7N9) viruses isolated from humans possess several amino acid changes already known to facilitate infection of mammals, including leucine at position 226 of HA (H3 HA numbering), which confers increased binding to human-type receptors (15), and lysine at position 627 (PB2-627K) (16,17) and asparagine at position 701 (PB2-701N) (18,19) in the polymerase protein PB2, which increase the polymerase activity in mammalian cells. Notably, the PB2-627K and PB2-701N markers have been detected in almost all human, but not avian or environmental, A(H7N9) isolates (6).In addition to the known mammalian-adapting amino acid changes in the PB2 and HA proteins, additional amino acid changes in A(H7N9) proteins may have enabled these viruses to infect mammals. We therefore undertook a comprehensive analysis of A(H7N9) sequences and identified several amino acids in the polymerase PA protein that are typically found in human (but not avian) influenza A viruses or are characteristic of A(H7N9) viruses (i.e., not commonly detected among human or avian influenza viruses). Here, we evaluated these distinguishing amino acid changes in vivo and in vitro. Cells. Madin-Darby canine kidney ...

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Efficient selection for high-expression transfectants with a novel eukaryotic vector

Cited by 4,733 publications

References 27 publications

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses

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