Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Correspondence
Paul Digard
pd1{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Present address: British Columbia Research Institute, 950 West 28th Avenue, Rm 318, Vancouver BC, Canada, V5Z 4H4.
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INTRODUCTION |
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In vitro lysis of purified virus releases transcriptionally competent RNPs that, given the appropriate substrates, synthesize capped and polyadenylated mRNAs (Plotch & Krug, 1977; Plotch et al., 1981
). However, the same RNPs do not synthesize cRNA except in the context of an infected cell and then only after a round of mRNA transcription and subsequent protein expression (Hay et al., 1977b
; Skorko et al., 1991
). Whilst it is possible that cellular factors are neccesary to render the input RNPs competent for replication and candidate proteins have been identified (Momose et al., 2001
, 2002
), definitive proof of such a mechanism is lacking. However, it is well-established that the viral NP is essential for genome synthesis. Several NP temperature-sensitive (ts) mutants have been isolated that are defective for replicative transcription at the non-permissive temperature (reviewed by Portela & Digard, 2002
) and some deliberate NP mutations affect genome replication and mRNA transcription differentially (Mena et al., 1999
). Furthermore, several studies have shown that, in contrast to detergent-disrupted virus, nuclear extracts from infected cells catalyse both viral mRNA synthesis and genome replication (Beaton & Krug, 1984
; del Rio et al., 1985
; Takeuchi et al., 1987
). Depletion of the fraction of NP that is not already bound into RNPs from these extracts abolishes both c- and vRNA synthesis (Beaton & Krug, 1986
; Shapiro & Krug, 1988
). Nevertheless, the reasons that NP is required for genome replication remain uncertain. Both v- and cRNA are encapsidated to form functional RNPs (Pons, 1971
; Hay et al., 1977b
), so this provides part of the explanation (Shapiro & Krug, 1988
). Functional analysis of two ts NP mutants that are specifically defective for genome replication supports this hypothesis, because both mutants lose the ability to bind RNA at the non-permissive temperature (Medcalf et al., 1999
). However, NP may also play a direct regulatory role in controlling genome replication. Such a role would be particularly likely for positive-strand RNA synthesis, where two types of RNA, differing in their mode of transcription initiation and termination, are made from the same template. One possibility is that newly synthesized NP alters the structure of the RNA promoter sequences found at the termini of each genome segment, thereby biasing the polymerase towards replicative transcription (Hsu et al., 1987
; Fodor et al., 1994
; Klumpp et al., 1997
). Alternatively, NP might exert such an effect by direct proteinprotein interactions with the PB1 and/or PB2 subunits of the RNA polymerase (Biswas et al., 1998
; Medcalf et al., 1999
; Poole et al., 2004
).
Irrespective of the possible mechanisms by which NP may control viral replicative transcription, there is a long-standing hypothesis that its intracellular concentration is important. It is proposed that, at low NP levels, mRNA transcription is favoured, but as translation of these messages proceeds and NP concentration rises, the balance of RNA synthesis tips towards genome replication. Experimental evidence supporting this scheme has been found in non-segmented, negative-strand virus systems (Blumberg et al., 1981; Arnheiter et al., 1985
). Although the absence of functional NP clearly precludes genome replication, this hypothesis has not been tested directly for influenza virus. Here, we show that, contrary to this hypothesis, variation of intracellular NP levels in a purely recombinant setting and in the context of virus infection does not lead to higher levels of replicative transcription, but instead causes a slight decrease in genomic RNA accumulation.
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METHODS |
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Plasmids pcDNA-PB2, -PB1 and -PA were created by ligation of fragments excised from pAPR102, pAPR206 and pAPR303 (Young et al., 1983) with BamHI, HindIII or EcoRI, respectively, into similarly linearized pcDNA3. Construction of plasmid pcDNA-NP is described elsewhere (Carrasco et al., 2004
). pPolI()NS.CAT.RT, containing an antisense chloramphenicol acetyltransferase (CAT) gene flanked by the non-coding sequences of influenza A/WSN/33 virus segment 8, under control of a human RNA polymerase I promoter (Pol-I) and upstream of a hepatitis
ribozyme sequence, was generously provided by Ervin Fodor (University of Oxford) and is described elsewhere (Pleschka et al., 1996
; Fodor et al., 2002
). A positive-sense CAT reporter [pPol-I(+)NS.CAT] was constructed similarly by PCR amplification from pPolI()NS.CAT.RT using primers PD211 (5'-GCATGCTCTTCTATTAGCAAAAGCAGGGTGAC-3') and PD212 (5'-GCATGCTCTTCGGCCAGTAGAAACAAGGGTG-3'). PCR products were digested with SapI and ligated into the vector pPolI.SapI.Rib, kindly provided by Ervin Fodor. Plasmids pT7(+)NS.CAT and pT7()NS.CAT contained positive- and negative-sense CAT gene sequences flanked by non-coding sequences from influenza A virus segment 8, under control of a T7 promoter in a pUC19 background. The sequences were amplified from the appropriate pPol-I.NS.CAT constructs by PCR using primers PD237 (5'-GCGCAAGCTTAATACGACTCACTATAAGTAGAAACAAGGGTG-3') and PD238 (5'-GGCTCTAGACGCTCGAGAGCAAAAGCAGGGTGAC-3') for pT7()NS.CAT and PD213 (5'-GCGCAAGCTTAATACGACTCACTATAAGCAAAAGCAGGGTGAC-3') and PD214 (5'-GGCTCTAGACGCTCGAGAGTAGAAACAAGGGTG-3') for pT7(+)NS.CAT. The products were digested by HindIII and XbaI and cloned into similarly digested pUC19. Digestion of these plasmids with HgaI allowed T7 in vitro transcription of an RNA with the correct 3' and 5' termini.
Rabbit polyclonal antisera to influenza A virus RNP have been described previously (Mahy et al., 1977). Anti-PR8 PB2 sera were prepared by immunizing rabbits with the fusion protein MBPPB2-C (Poole et al., 2004
). Antiserum against human Nup62 was purchased from Transduction Laboratories, anti-rabbit IgG antibody conjugated to fluorescein isothiocyanate from DAKO A/S and Alexa 594-conjugated anti-mouse IgG antibodies from Molecular Probes.
Influenza virus gene-expression assay.
1x106 cells per 35 mm well in 1 ml complete medium were transfected in suspension with plasmid DNA by using cationic liposomes (Lipofectin; Gibco-BRL). To reconstitute RNPs, 0·25 µg each of pcDNA-PB1, -PB2 and -PA and 50 ng of either pPol-I(+)NS.CAT or pPol-I()NS.CAT were transfected with varying amounts of pcDNA-NP. The total amount of DNA in each transfection mix was normalized by the addition of pcDNA3 vector. Optimal amounts of plasmids other than pcDNA-NP were established by separate titration experiments (data not shown). Following incubation at 37 °C for 3 days, CAT accumulation was quantified by a commercial ELISA (Roche Diagnostics).
Primer-extension analysis of viral RNA.
Total cellular RNA was isolated by using a commercial kit (SV Total RNA Isolation System; Promega) according to the manufacturer's instructions. After spectrophotometric quantification and normalization, RNA (1·5 µg) was reverse-transcribed by using avian myeloblastosis virus reverse transcriptase (Promega) and 0·4 pmol each of the appropriate DNA oligonucleotides that had been 5' end-labelled with [-32P]ATP. Following 30 min incubation at 42 °C, samples were heated to 90 °C for 10 min and separated by 6 % urea-PAGE. Radiolabelled products were detected by autoradiography and quantified by densitometry using NIH Image software (http://rsb.info.nih.gov/nih-image/). The oligonucleotides used were 5'-GGCGATTCAGGTTCATCATGCCG-3' (for detection of vRNA-sense CAT transcripts), 5'-ATGTTCTTTACGATGCGATTGGG-3' (positive-sense CAT transcripts), 5'-CGTCCAATTCCACCAATC-3' (positive-sense segment 5), 5'-GTCTTCGAGCTCTCGGACG-3' (segment 5 vRNA), 5'-TCAAGTCTCGGTGCGATCTCG-3' (positive-sense segment 7) and 5'-ACCGTCGCTTTAAATACGG-3' (segment 7 vRNA). The predicted size of primer-extension product for each oligonucleotide was validated by comparison with matching DNA sequencing ladders (CAT; see Results section, segments 5 and 7; data not shown). Primers were designed so that the reverse-transcription products from m-, c- and vRNA were distinguishable by gel electrophoresis. Preliminary experiments established that simultaneous detection of positive- and negative-sense RNA species gave equivalent results to separate reactions carried out with individual primers (data not shown).
Protein analyses.
For Western blots, cell lysates were separated by 10 % SDS-PAGE and transferred to nitrocellulose. Blots were probed with primary followed by secondary antibodies conjugated to horseradish peroxidase (DAKO) and developed by chemiluminescence (ECL reagent; Amersham Biosciences). For immunofluorescence, subconfluent cells on glass coverslips were fixed with PBS containing 4 % formaldehyde for 20 min and washed in PBS containing 2 % FCS. Cells were permeabilized with 0·2 % Triton X-100 before incubation with primary antibodies, followed by fluorophore-conjugated secondary antibodies in PBS/FCS. Coverslips were mounted in Citifluor (Agar Scientific) and examined under a Leica TCS NT confocal microscope.
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RESULTS |
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It has been demonstrated previously that, when NP is expressed by itself, higher levels of expression bias it towards cytoplasmic accumulation (Digard et al., 1999; Elton et al., 2001
). To determine whether the same trend was apparent in cells producing functional RNPs, parallel samples from each transfection were examined by indirect immunofluorescence for NP and Nup62, a component of the nuclear pore complex. No NP staining was detected in control cells [Fig. 4
a(i)]. When pcDNA-NP-transfected cells were examined 1 day post-transfection, the majority of NP was found in the nucleus at all plasmid doses tested (data not shown). At 3 days post-transfection, cells transfected with the lowest dose of NP plasmid (11 ng) showed a mixture of nuclear and cytoplasmic staining [Fig. 4a
(ii)]. When replicate experiments were stained and scored for NP localization, it was found that, on average, approximately half of the transfected cells still contained nuclear NP (Fig. 4b
). As the amount of transfected plasmid was raised, fewer cells contained nuclear NP until, at the very highest dose, the protein was almost exclusively cytoplasmic [Fig. 4a
(iii and iv) and b]. Similar results were obtained when experiments were carried out by using a plasmid expressing CAT cRNA (data not shown). Therefore, NP exhibits the same dose-dependent localization pattern when expressed alone or with vRNA, PB1, PA and PB2: a shift from nuclear to cytoplasmic localization with increased NP expression. In light of the preceding experiments, this suggests that a reduction in synthesis of replicative RNA types may correlate with increased or earlier cytoplasmic localization of NP.
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DISCUSSION |
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Although plasmid-based systems for reconstituting RNPs have been used widely to study influenza virus RNA synthesis, they probably do not reproduce all facets of authentic virus transcription, particularly with regard to its temporal regulation. During virus infection, cRNA is only synthesized at early times p.i., following which vRNA and mRNA amplification occurs. Whilst vRNA synthesis continues later in infection, mRNA transcription peaks and then declines (Fig. 5a; Hay et al., 1977b
; Barrett et al., 1979
; Smith & Hay, 1982
; Shapiro et al., 1987
). In contrast, time-course experiments analysing viral RNA synthesis in the recombinant system used here show no evidence of any such synthetic cascade, with the amounts of all three RNA species simply increasing over time (data not shown). However, like authentic virus infection, cRNA remains a relatively low-abundance species. As such, the system can be considered to be a partially deregulated model of viral transcription, which perhaps lessens the confidence that we can place in our finding that increased concentrations of NP do not stimulate genome replication. However, consistent results were obtained from infected cells that were manipulated to contain varying amounts of NP prior to virus inoculation. No significant alteration was seen in the overall amounts of cRNA for segments 5 and 7, or segment 5 vRNA. Prior expression of NP did cause a slight alteration in the ratio of segment 7 vRNA to mRNA but, as with the recombinant CAT segment, this was a decrease, rather than an increase. The infection system relies on transfection of the cells with an NP-expressing plasmid before subsequent virus infection. Although virtually every cell would be expected to be infected, the same would not be true of transfection, leading to a background of virus transcription taking place in the absence of extra NP. Accordingly, we monitored transfection efficiencies by immunofluorescence to establish the size of this background, finding that approximately one-third of cells were transfected successfully (data not shown). Statistical analysis of the densitometry data suggests that an overall twofold change in the ratio of genome or antigenomic RNAs to mRNA would be detected reliably, because the less than twofold change in ratio of segment 7 vRNA to mRNA was partially statistically significant. Therefore, for a constant amount of mRNA, a fourfold change in v- or cRNA accumulation would be required in the transfected cells to achieve an overall twofold increase in the ratio of genomic or antigenomic RNA species to mRNA. As the only change seen was a minor decrease in the relative amount of segment 7 vRNA, we can exclude any major (greater than fourfold) stimulatory effect of increased NP expression on virus genome replication.
If the relative rate of genome replication is not directly proportional to the intracellular concentration of NP, then what explains the clear necessity for NP for replicative RNA synthesis? One model is that other factors (e.g. the polymerase and/or cellular factors) regulate RNA synthesis, keeping mRNA as the predominant positive-sense RNA. NP is nonetheless an essential cofactor, because of a requirement for co-transcriptional encapsidation of nascent c- and vRNA segments. This model is consistent with the heterogeneous size of short vRNA products that are synthesized by infected-cell extracts depleted of NP (Shapiro & Krug, 1988) and the finding that ts NP mutants that are specifically defective for replicative transcription lose RNA-binding activity at the elevated temperature (Medcalf et al., 1999
). It is also consistent with the observation that NP is not required in vitro for unprimed transcription initiation (Lee et al., 2002
). Indeed, these findings and our current data are consistent with a recent study (published while this manuscript was under review) that suggests that there is no switch mechanism regulating cRNA synthesis and that the requirement for protein synthesis during genome replication reflects degradation of naked cRNA that is not protected by encapsidation into an RNP (Vreede et al., 2004
). On the other hand, identification of NP mutations that affect v- and cRNA synthesis differentially is highly suggestive of a direct regulatory role for the protein (Thierry & Danos, 1982
; Markushin & Ghendon, 1984
; Mena et al., 1999
). This regulation could be exerted through NPpolymerase interactions (Biswas et al., 1998
; Mena et al., 1999
; Medcalf et al., 1999
; Poole et al., 2004
) or through NP-mediated modification of the panhandle promoter structure (Fodor et al., 1994
; Klumpp et al., 1997
; Lee et al., 2003
). To be consistent with the findings reported here, one would have to postulate that only small amounts of NP are required to enable replicative synthesis and that some other factor prevents cRNA synthesis from rising above 510 % of positive-sense transcription.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Barrett, T., Wolstenholme, A. J. & Mahy, B. W. J. (1979). Transcription and replication of influenza virus RNA. Virology 98, 211225.[CrossRef][Medline]
Beaton, A. R. & Krug, R. M. (1984). Synthesis of the templates for influenza virion RNA replication in vitro. Proc Natl Acad Sci U S A 81, 46824686.[Abstract]
Beaton, A. R. & Krug, R. M. (1986). Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end. Proc Natl Acad Sci U S A 83, 62826286.[Abstract]
Biswas, S. K., Boutz, P. L. & Nayak, D. P. (1998). Influenza virus nucleoprotein interacts with influenza virus polymerase proteins. J Virol 72, 54935501.
Blumberg, B. M., Leppert, M. & Kolakofsky, D. (1981). Interaction of VSV leader RNA and nucleocapsid protein may control VSV genome replication. Cell 23, 837845.[Medline]
Carrasco, M., Amorim, M. J. & Digard, P. (2004). Lipid raft-dependent targeting of the influenza A virus nucleoprotein to the apical plasma membrane. Traffic (in press). doi: 10.1111/j.1600-0854.2004.00237.x
del Rio, L., Martinez, C., Domingo, E. & Ortín, J. (1985). In vitro synthesis of full-length influenza virus complementary RNA. EMBO J 4, 243247.[Abstract]
Digard, P., Elton, D., Bishop, K., Medcalf, E., Weeds, A. & Pope, B. (1999). Modulation of nuclear localization of the influenza virus nucleoprotein through interaction with actin filaments. J Virol 73, 22222231.
Elton, D., Simpson-Holley, M., Archer, K., Medcalf, L., Hallam, R., McCauley, J. & Digard, P. (2001). Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway. J Virol 75, 408419.
Elton, D., Tiley, L. & Digard, P. (2002). Molecular mechanisms of influenza virus transcription. Recent Res Dev Virol 4, 121146.
Fearns, R., Peeples, M. E. & Collins, P. L. (1997). Increased expression of the N protein of respiratory syncytial virus stimulates minigenome replication but does not alter the balance between the synthesis of mRNA and antigenome. Virology 236, 188201.[CrossRef][Medline]
Fodor, E., Pritlove, D. C. & Brownlee, G. G. (1994). The influenza virus panhandle is involved in the initiation of transcription. J Virol 68, 40924096.[Abstract]
Fodor, E., Crow, M., Mingay, L. J., Deng, T., Sharps, J., Fechter, P. & Brownlee, G. G. (2002). A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J Virol 76, 89899001.
Hay, A. J., Abraham, G., Skehel, J. J., Smith, J. C. & Fellner, P. (1977a). Influenza virus messenger RNAs are incomplete transcripts of the genome RNAs. Nucleic Acids Res 4, 41974209.[Abstract]
Hay, A. J., Lomniczi, B., Bellamy, A. R. & Skehel, J. J. (1977b). Transcription of the influenza virus genome. Virology 83, 337355.[Medline]
Hay, A. J., Skehel, J. J. & McCauley, J. (1982). Characterization of influenza virus RNA complete transcripts. Virology 116, 517522.[Medline]
Herz, C., Stavnezer, E., Krug, R. M. & Gurney, T., Jr (1981). Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells. Cell 26, 391400.[Medline]
Hsu, M.-T., Parvin, J. D., Gupta, S., Krystal, M. & Palese, P. (1987). Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle. Proc Natl Acad Sci U S A 84, 81408144.[Abstract]
Huang, T.-S., Palese, P. & Krystal, M. (1990). Determination of influenza virus proteins required for genome replication. J Virol 64, 56695673.[Medline]
Klumpp, K., Ruigrok, R. W. H. & Baudin, F. (1997). Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO J 16, 12481257.
Lee, M. T. M., Bishop, K., Medcalf, L., Elton, D., Digard, P. & Tiley, L. (2002). Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase. Nucleic Acids Res 30, 429438.
Lee, M.-T. M., Klumpp, K., Digard, P. & Tiley, L. (2003). Activation of influenza virus RNA polymerase by the 5' and 3' terminal duplex of genomic RNA. Nucleic Acids Res 31, 16241632.
Leppert, M., Rittenhouse, L., Perrault, J., Summers, D. F. & Kolakofsky, D. (1979). Plus and minus strand leader RNAs in negative strand virus-infected cells. Cell 18, 735747.[CrossRef][Medline]
Mahy, B. W. J., Carroll, A. R., Brownson, J. M. T. & McGeoch, D. J. (1977). Block to influenza virus replication in cells preirradiated with ultraviolet light. Virology 83, 150162.[CrossRef]
Markushin, S. G. & Ghendon, Y. Z. (1984). Studies of fowl plague virus temperature-sensitive mutants with defects in synthesis of virion RNA. J Gen Virol 65, 559575.[Abstract]
Medcalf, L., Poole, E., Elton, D. & Digard, P. (1999). Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein. J Virol 73, 73497356.
Mena, I., Jambrina, E., Albo, C., Perales, B., Ortín, J., Arrese, M., Vallejo, D. & Portela, A. (1999). Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication. J Virol 73, 11861194.
Momose, F., Basler, C. F., O'Neill, R. E., Iwamatsu, A., Palese, P. & Nagata, K. (2001). Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis. J Virol 75, 18991908.
Momose, F., Naito, T., Yano, K., Sugimoto, S., Morikawa, Y. & Nagata, K. (2002). Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J Biol Chem 277, 4530645314.
Pinschewer, D. D., Perez, M. & de la Torre, J. C. (2003). Role of the virus nucleoprotein in the regulation of lymphocytic choriomeningitis virus transcription and RNA replication. J Virol 77, 38823887.
Pleschka, S., Jaskunas, S. R., Engelhardt, O. G., Zürcher, T., Palese, P. & García-Sastre, A. (1996). A plasmid-based reverse genetics system for influenza A virus. J Virol 70, 41884192.[Abstract]
Plotch, S. J. & Krug, R. M. (1977). Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA. J Virol 21, 2434.[Medline]
Plotch, S. J., Bouloy, M., Ulmanen, I. & Krug, R. M. (1981). A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, 847858.[Medline]
Pons, M. W. (1971). Isolation of influenza virus ribonucleoprotein from infected cells. Demonstration of the presence of negative-stranded RNA in viral RNP. Virology 46, 149160.[CrossRef][Medline]
Poole, E., Elton, D., Medcalf, L. & Digard, P. (2004). Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites. Virology 321, 120133.[CrossRef][Medline]
Poon, L. L. M., Pritlove, D. C., Sharps, J. & Brownlee, G. G. (1998). The RNA polymerase of influenza virus, bound to the 5' end of virion RNA, acts in cis to polyadenylate mRNA. J Virol 72, 82148219.
Portela, A. & Digard, P. (2002). The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83, 723734.
Robertson, J. S., Schubert, M. & Lazzarini, R. A. (1981). Polyadenylation sites for influenza virus mRNA. J Virol 38, 157163.[Medline]
Shapiro, G. I. & Krug, R. M. (1988). Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer. J Virol 62, 22852290.[Medline]
Shapiro, G. I., Gurney, T. & Krug, R. M. (1987). Influenza virus gene expression: control mechanisms at early and late times of infection and nuclear-cytoplasmic transport of virus-specific RNAs. J Virol 61, 764773.[Medline]
Skorko, R., Summers, D. F. & Galarza, J. M. (1991). Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis. Virology 180, 668677.[CrossRef][Medline]
Smith, G. L. & Hay, A. J. (1982). Replication of the influenza virus genome. Virology 118, 96108.[CrossRef][Medline]
Takeuchi, K., Nagata, K. & Ishihama, A. (1987). In vitro synthesis of influenza viral RNA: characterization of an isolated nuclear system that supports transcription of influenza viral RNA. J Biochem (Tokyo) 101, 837845.[Abstract]
Thierry, F. & Danos, O. (1982). Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitve mutants of HK/68 influenza virus. Nucleic Acids Res 10, 29252938.[Abstract]
Vreede, F. T., Jung, T. E. & Brownlee, G. G. (2004). Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates. J Virol 78, 95689572.
Young, J. F., Desselberger, U., Graves, P., Palese, P., Shatzman, A. & Rosenberg, M. (1983). Cloning and expression of influenza virus genes. In The Origin of Pandemic Influenza Viruses, pp. 129138. Edited by W. G. Laver. Amsterdam: Elsevier.
Received 11 August 2004;
accepted 15 September 2004.
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