Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system

Mena, Ignacio; Luna, S de la; Albó, Carmen; Martı́n, Javier; Nieto, Amelia; Ortı́n, Juan; Portela, Agustı́n

doi:10.1099/0022-1317-75-8-2109

Cited by 62 publications

(79 citation statements)

References 26 publications

(26 reference statements)

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“…To generate plasmids pHHVicPB1, pHHVicPB2, pHHVicPA, pHHVicNP, and pHHVicNS, their viral cDNAs were amplified from the corresponding pGEM plasmids (17,55) by using the Expand PCR system from Roche and cloned into plasmid pHH21 as described previously (57).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids expressing the A/Victoria/3/75 polymerase and NP genes under control of the cytomegalovirus promoter (pCMVPB1, pCMVPB2, pCMVPA, and pCMVNP) were constructed by subcloning the cDNAs derived from the corresponding pGEM plasmids (17,55) into the pCMV vector. For the construction of mutant plasmids pHHVicNS1-81 and pHHVicNS1-110, the mutant cDNAs were amplified by using the corresponding pGEM constructs as templates (51) and cloned into the pHH21 vector as indicated above.…”

Section: Methodsmentioning

confidence: 99%

“…The expression of virus proteins, at least NP, leads to the shift from transcription to the synthesis of complete positive-polarity RNAs (cRNAs) (29,73), which serve as templates for the synthesis of virion RNAs (vRNAs). Transcription and replication of vRNA take place in the nucleus of the infected cell (30,34) and require at least the activity of the three subunits of the polymerase (PB1, PB2, and PA) and the NP (9,31,38,55,64). The syntheses of the various vRNAs are not simultaneous during the infection cycle.…”

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Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein

Falcón

Marión

Zürcher

et al. 2004

J Virol

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Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.The genome of influenza A virus consists of eight singlestranded RNA molecules of negative polarity associated with nucleoprotein (NP) molecules and the polymerase in the form of ribonucleoprotein (RNP) complexes (for reviews, see references 40, 43, and 66). The first step in viral gene expression is primary transcription from the incoming viral RNPs (28). The expression of virus proteins, at least NP, leads to the shift from transcription to the synthesis of complete positive-polarity RNAs (cRNAs) (29, 73), which serve as templates for the synthesis of virion RNAs (vRNAs). Transcription and replication of vRNA take place in the nucleus of the infected cell (30,34) and require at least the activity of the three subunits of the polymerase (PB1, PB2, and PA) and the NP (9,31,38,55,64). The syntheses of the various vRNAs are not simultaneous during the infection cycle. Thus, NS1 or NP vRNAs are replicated earlier than M1 or hemagglutinin (HA) vRNAs (72). Since transcription is coupled to replication at the beginning of vRNA synthesis, the NS1 protein and NP are expressed earlier than the M1 protein and HA (72). However, later in the process of vRNA synthesis, transcription is discontinued and viral protein synthesis rests on previously synthesized mRNAs (72). In the course of the infection, viral gene expression takes over the cell machinery, leading to the shutoff phenomenon. Several alterations induced by the virus in the infected cell may be connected to shutoff: nuclear retention and degradation of polymerase II transcripts in the nucleus (35), inhibition of cellular pre-mRNA cleavage and polyadenylation (56, 74), cytoplasmic degradation of preexisting cellular mRNAs (3, 32), and preferential utilization of the translation machinery by the virus-specific mRNAs (36).Influenza A virus encodes a nonstructural protein (NS1) that is translated from the unspliced transcript of segment 8 (33, 44). NS1 is a nuclear protein, both in the infected cell (5, 41) and when expressed from cDNA (23,45,6...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein

Falcón

Marión

Zürcher

et al. 2004

J Virol

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“…The simian virus 40 (SV40) recombinant virus SVPA, which expresses the influenza virus polymerase PA subunit of strain A\Victoria\3\75, has been described elsewhere (de la Luna et al, 1993 ;Nieto et al, 1992). The pGEM3 recombinant plasmid encoding the influenza virus PA polymerase subunit (pGPA) has been previously described (Mena et al, 1994).…”

Section: Methodsmentioning

confidence: 99%

The PA influenza virus polymerase subunit is a phosphorylated protein.

Sanz‐Ezquerro

Santarén

Sierra

et al. 1998

Journal of General Virology

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The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by twodimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [ 32 P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine

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“…19 Indeed, minireplicon systems have been available since long ago, in which a single virus-like RNA serves as a model template that can replicate and transcribe in vivo with the sole trans-action of the polymerase and NP. [20][21][22][23] When isolated from purified virions, the genomic RNPs appear as ribbon-like, closed superhelical structures. [24][25][26][27] The structural features of these RNPs are in part due to the NP, as in vitro RNA-NP complexes resemble native RNPs 28 and even …”

Section: The Virus Rna Synthesis Machinementioning

confidence: 99%