scholarly journals Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region.

1984 ◽  
Vol 4 (1) ◽  
pp. 8-16 ◽  
Author(s):  
L Markoff ◽  
B C Lin ◽  
M M Sveda ◽  
C J Lai
Keyword(s):  
Influenza Virus ◽  
Dna Sequences ◽  
Sodium Dodecyl ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Infected Cells ◽  
Late Region ◽  
Na Gene

A full-length double-stranded DNA copy of an influenza A virus N2 neuraminidase (NA) gene was cloned into the late region of pSV2330, a hybrid expression vector that includes pBR322 plasmid DNA sequences and the simian virus 40 early region and simian virus 40 late region promoters, splice sequences, and transcription termination sites. The protein encoded by the cloned wild-type NA gene was shown to be present in the cytoplasm of fixed cells and at the surface of "live" or unfixed cells by indirect immunofluorescence with N2 monoclonal antibodies. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of [35S]methionine-labeled proteins from wild-type vector-infected cells with heterospecific N2 antibody showed that the product of the cloned NA DNA comigrated with glycosylated NA from influenza virus-infected cells, remained associated with internal membranes of cells fractionated into membrane and cytoplasmic fractions, and could form an immunoprecipitable dimer. NA enzymatic activity was detectable after simian virus 40 lysis of vector-infected cells. These properties of the product of the cloned wild-type gene were compared with those of the polypeptides produced by three deletion mutant NA DNAs that were also cloned into the late region of the pSV2330 vector. These mutants lacked 7 (dlk), 21 (dlI), or all 23 amino acids (dlZ) of the amino (N)-terminal variable hydrophobic region that anchors the mature wild-type NA tetrameric structure in the infected cell or influenza viral membrane. Comparison of the phenotypes of these mutants showed that this region in the NA molecule also includes sequences that control translocation of the nascent polypeptide into membrane organelles for glycosylation.

Glycosylation and surface expression of the influenza virus neuraminidase requires the N-terminal hydrophobic region

1984 ◽  
Vol 4 (1) ◽  
pp. 8-16
Author(s):  
L Markoff ◽  
B C Lin ◽  
M M Sveda ◽  
C J Lai
Keyword(s):  
Influenza Virus ◽  
Dna Sequences ◽  
Sodium Dodecyl ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Infected Cells ◽  
Late Region ◽  
Na Gene

A full-length double-stranded DNA copy of an influenza A virus N2 neuraminidase (NA) gene was cloned into the late region of pSV2330, a hybrid expression vector that includes pBR322 plasmid DNA sequences and the simian virus 40 early region and simian virus 40 late region promoters, splice sequences, and transcription termination sites. The protein encoded by the cloned wild-type NA gene was shown to be present in the cytoplasm of fixed cells and at the surface of "live" or unfixed cells by indirect immunofluorescence with N2 monoclonal antibodies. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of [35S]methionine-labeled proteins from wild-type vector-infected cells with heterospecific N2 antibody showed that the product of the cloned NA DNA comigrated with glycosylated NA from influenza virus-infected cells, remained associated with internal membranes of cells fractionated into membrane and cytoplasmic fractions, and could form an immunoprecipitable dimer. NA enzymatic activity was detectable after simian virus 40 lysis of vector-infected cells. These properties of the product of the cloned wild-type gene were compared with those of the polypeptides produced by three deletion mutant NA DNAs that were also cloned into the late region of the pSV2330 vector. These mutants lacked 7 (dlk), 21 (dlI), or all 23 amino acids (dlZ) of the amino (N)-terminal variable hydrophobic region that anchors the mature wild-type NA tetrameric structure in the infected cell or influenza viral membrane. Comparison of the phenotypes of these mutants showed that this region in the NA molecule also includes sequences that control translocation of the nascent polypeptide into membrane organelles for glycosylation.

scholarly journals A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.

1983 ◽  
Vol 3 (8) ◽  
pp. 1381-1388 ◽  
Author(s):  
L P Villarreal ◽  
R T White
Keyword(s):  
Simian Virus 40 ◽  
Splice Junction ◽  
Simian Virus ◽  
The Body ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Wild Type ◽  
Normal Position ◽  
Nuclear Rna ◽  
Late Region

A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.

Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation

1984 ◽  
Vol 4 (8) ◽  
pp. 1661-1663
Author(s):  
L Sompayrac ◽  
K J Danna
Keyword(s):  
Dna Sequences ◽  
Deletion Mutant ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Large Deletion ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Coding Sequence ◽  
Mouse Cells

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.

scholarly journals Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation.

1984 ◽  
Vol 4 (8) ◽  
pp. 1661-1663 ◽  
Author(s):  
L Sompayrac ◽  
K J Danna
Keyword(s):  
Dna Sequences ◽  
Deletion Mutant ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Large Deletion ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Coding Sequence ◽  
Mouse Cells

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.

A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA

1983 ◽  
Vol 3 (8) ◽  
pp. 1381-1388
Author(s):  
L P Villarreal ◽  
R T White
Keyword(s):  
Simian Virus 40 ◽  
Splice Junction ◽  
Simian Virus ◽  
The Body ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Wild Type ◽  
Normal Position ◽  
Nuclear Rna ◽  
Late Region

A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.

The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader

1984 ◽  
Vol 4 (4) ◽  
pp. 813-816
Author(s):  
A Barkan ◽  
J E Mertz
Keyword(s):  
Nucleotide Sequence ◽  
Direct Evidence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
The Body ◽  
Size Distributions ◽  
Wild Type ◽  
Leader Protein ◽  
16S Rna

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products

1991 ◽  
Vol 11 (8) ◽  
pp. 4253-4265
Author(s):  
H G Wang ◽  
G Draetta ◽  
E Moran
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
Physical Interaction ◽  
Resting Cells ◽  
Wild Type ◽  
Quiescent Cells ◽  
Kinase Expression

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants

1986 ◽  
Vol 6 (7) ◽  
pp. 2704-2711 ◽  
Author(s):  
D S Peabody ◽  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Initiation Codon ◽  
Efficient Production ◽  
Translation Efficiency ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Internal Translation ◽  
Late Region

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

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