- Short report
- Open Access
Heterologous influenza vRNA segments with identical non-coding sequences stimulate viral RNA replication in trans
https://doi.org/10.1186/1743-422X-5-2
© Ng et al; licensee BioMed Central Ltd. 2008
- Received: 10 October 2007
- Accepted: 11 January 2008
- Published: 11 January 2008
Abstract
The initiation of transcription and replication of influenza A virus requires the 5' and 3' ends of vRNA. Here, the role of segment-specific non-coding sequences of influenza A virus on viral RNA synthesis was studied. Recombinant viruses, with the nonstructural protein (NS) segment-specific non-coding sequences replaced by the corresponding sequences of the neuraminidase (NA) segment, were characterized. The NS and NA vRNA levels in cells infected with these mutants were much higher than those of the wild type, whereas the NS and NA mRNA levels of the mutants were comparable to the wild-type levels. By contrast, the PB2 vRNA and mRNA levels of all the tested viruses were similar, indicating that vRNA with heterologous segment-specific non-coding sequences was not affected by the mutations. The observations suggested that, with the cooperation between the homologous 5' and 3'segment-specific sequences, the introduced mutations could specifically enhance the replication of NA and NS vRNA.
Keywords
- Viral mRNA
- Viral Transcription
- Viral Polymerase
- vRNA Template
- vRNA Level
Background
The genome of influenza A virus contains 8 RNA segments of negative polarity [1]. Each virion RNA (vRNA) can be used as a template for transcription and replication to generate viral mRNA and complementary RNA (cRNA), respectively. cRNA is a faithful complementary copy of vRNA and is used as a template for vRNA synthesis. By contrast, the transcription of the viral mRNA is terminated at a track of uridines (U) which is about 17 nucleotides away from the 5' end of the vRNA template [2, 3] and the polymerase then starts to polyadenlyate the mRNA by reiteratively copying of the U-track [4, 5]. It is generally believed that there is a control mechanism to regulate the polymerase's transcriptase and replicase activities [6]. However, recent studies have suggested an alternative hypothesis that such switching mechanism might not exist [7–10].
Sequence analyses of all the vRNA segments revealed that the first 12 and 13 nucleotides at their 3' and 5' ends are highly conserved [11]. Extensive studies on these sequences indicated that these regions are the promoter for transcription and replication. These sequences were shown to be involved in the viral polymerase binding [12–14], cap-snatching [14, 15], and transcription initiation [16, 17]. The 5' and 3' ends of each vRNA are partially inverted complementary and can form a corkscrew structure that is known to be critical for the above biological processes [6]. Within these conserved sequences, there is a single natural variation (U or C) at the 4th residue of the 3' end [11]. Of all the vRNA segments, the polymerase segments (PB2, PB1 and PA) invariably carry a C residue at this position (C4), whereas most of the other segments contain a U residue at this position (U4). Mutagenic studies of this polymorphic site suggested that this nucleotide variation might modulate viral transcription and replication [18, 19]. Adjacent to the universally conserved regions, each vRNA segment contains additional non-coding sequences at its 5' and 3' regions. The lengths and sequences of these non-coding sequences are segment specific. Growing evidences have supported the hypothesis that these sequences are parts of the viral RNA packaging signals [20–24]. In addition, disrupting the NA segment-specific sequences were shown to have effects on viral RNA synthesis [25–27], indicating these segment specific sequences might modulate viral RNA synthesis.
Findings
Growth properties of the wild type and NSNA mutants in MDCK cells. (A) Quantitation of infectious progeny viral particles generated from infected cells by standard plaque assays. (B) Plaque morphologies of the wild type (WT) and NSNA mutant.
Quantitation of PB2, NA and NS vRNA (A) and mRNA (B) in cells infected with the wild-type (WT) or NSNA virus at different postinfection time points. Uni-12 primer (0.2 ng/μl) [35] was used for the cDNA synthesis of vRNA, whereas oligo dT20 (25 μM) was used to generated cDNA of viral mRNA. In a typical reverse transcription reaction, 0.5 μg of DNase-treated RNA sample was mixed with 1 μl of the corresponding primer, 4 μl of 5x first stand buffer, 2 μl of 0.1M dithiothreitol, and 1 μl of 10 mM deoxyribonucleoside triphosphates (Strategene), 150 U of SuperScript II reverse transcriptase in a 20 μl reaction. For detecting NA and NS RNA species, RNase-treated cDNA was examined by 5'-nuclease-based assays in a 7300 Sequence Detection System (Applied Biosystems). Briefly, 5 μl of the corresponding diluted cDNA samples were mixed with 12.5 μl superMix-UDG (Invitrogen), 0.5 μl of Rox reference dye, 1 μl of 10 mM forward primer, 1 μl of 10 mM reverse primers, 1 μl of 10 mM probe and 4 μl of water. Reactions were first incubated at 50°C for 2 min, followed by 95°C for 10 min. Reactions were then thermal-cycled for 45 cycles (95°C for 15 sec, 56°C for 1 min). Primers used in the NA detection assay were 5'-ACCGACCATGGGTGTCCTT-3' (corresponds to nt 870–888 of the NA cRNA) and 5'-GAAAATCCCTTTACTCCGTTTGC-3' (complementary to nt 998–1020 of the NA cRNA). Primer used in the NS detection assay were 5'-TACCTGCATCGCGCTACCTA-3' (corresponds to nt 277–296 of the NS cRNA) and 5'-ATGATCGCCTGGTCCATTCT-3' (complementary to nt 378–397 of the NS cRNA) were used. The probes used in the NA and NS assays were 5'-FAM-CGTCCCAAAGATGGA-NFQ-3' (corresponds to nt 950–964 of the NA cRNA; FAM, 6-carboxyfluorescein; NFQ, nonfluorescent quencher) and 5'-VIC-CACTGGTTCATGCTCA-NFQ-3' (corresponds to nt 327–342 of the NA cRNA; VIC, a proprietary dye), respectively. For the quantitation of PB2 RNA species, cDNA samples were amplified by using FastStart DNA Master SYBR Green I kit (Roche) in a LightCycler platform (Roche). In a typical reaction, 5 μl of RNase-treated cDNA was mixed with 2 μl master mixtures, 1.6 μl of MgCl2, 1 μl of forward primer (5'-CCGCAGTTCTGAGAGGATTC-3', corresponds to nt 2090–2109 of PB2 cRNA), 1 μl of reverse primer (5'-TCCGTTTCCGTTTCATTACC-3', complementary to nt 2226–2245 of the PB2 cRNA) and 1.6 μl of water. Reactions were first incubated at 95°C for 10 min, followed by a thermal-cycling (95°C for 10 sec, 58°C for 5 sec, 72°C for 15 sec; 40 cycles). The specificities of the amplified products were all confirmed by melting curve analysis. In all the PCR assays, serially diluted plasmids containing the corresponding sequences were used as standard controls. All the data were derived from three independent assays. The levels of mRNA and vRNA from the studied mutants were analyzed by two-tails paired t-test.
Quantitation of PB2, NA and NS RNA species in cells infected with the All-U or NSNA-U mutants at various postinfection time points. (A) PB2, NA and NS vRNA levels as indicated. (B) PB2, NA and NS mRNA levels as indicated. All the data were derived from three independent assays.
One of the possible mechanisms account for the elevation of NS and NA vRNA levels is that the 5' and 3' segment-specific regions would facilitate the initiation of vRNA replication. This stimulating effect, however, might require the presence of the 5' and 3' segment-specific regions from homologous segments. As the NA and NS vRNA segments in the NSNA and NSNA-U mutants had the identical non-coding sequences, the availability of compatible 5' ends for initiating NS and NA vRNA replications would be increased. This hypothesis is supported by two of our observations. First, our data demonstrated that the mutations had no effect on vRNA which has heterologous segment-specific sequences (i.e. PB2). In addition, our data showed that the transcription of the NA and NS segments were not up-regulated. These agreed with previous findings that the viral polymerase has to bind to the 5' and 3' ends of the same vRNA template for mRNA synthesis [12, 29]. Thus, the increases of compatible ends' populations would not expected to have stimulating effects on the NS and NA mRNA expressions. Interestingly, the NS mRNA levels from the NSNA and NSNA-U mutants in this study seemed to be less than that of the corresponding controls (Figs. 2B and 3B, right panels). It is possible that, due to the increase of the number of these compatible ends in infected cells, the polymerase might have less chance to bind to the ends of the same vRNA template for transcription initiation.
Detection of NA vRNA, cRNA and mRNA by primer extension assays. Total RNA from infected cells were harvested at 8 and 24 hr postinfection. The reaction conditions were identical to previously described assays [31], except fluorescent vRNA-specific primer (5'-Cy3-TGGACTAGTGGGAGCATCAT-3') and cRNA/mRNA-specific primer (5'-Cy5-TCCAGTATGGTTTTGATTTCCG-3') were used in the assays. The fluorescent products were resolved in 10% denaturing polyacrylamide gels and the images were analyzed by an imaging analyzer (Typhoon 8600 variable mode imager, Amersham Biosciences). Signals for the vRNA, cRNA and mRNA are shown as indicated. cRNA and vRNA signals of the NSNA-U were consistently higher than those of the All-U in independent attempts (Trials 1 and 2).
In the early phase of viral infections, vRNP predominantly synthesizes mRNA for viral protein synthesis [30]. This is followed by an active phase of viral RNA replication. It was previously proposed that the nascent NP expressed in infected cells might stimulate viral RNA replication [31, 32]. Recent evidences have provided an alternative hypothesis to explain this observation. Rather than stimulating the viral RNA replication, free NP and viral polymerase are proposed to protect nascent cRNA from degradation by binding to these newly synthesized cRNA transcripts [7, 9, 33]. The results from our current study might also help to explain the dramatic increase of cRNA levels in the late phase of viral infection. In the early phase of infection, the amount of vRNA is low and the viral polymerase is more likely to bind to the ends of the same vRNA template for transcription (i.e. activate in cis). Messenger RNA generated from this cis-acting transcription mode would be transported to cytosol for protein expression. Due to the lack of newly synthesized NP and viral polymerase, nascent cRNA generated from this cis-acting mode might be rapidly degraded at the early time point [7, 9, 33]. By contrast, during the mid- to late phase of infection, the accumulations of cRNP and vRNP make the viral polymerase complex has less chance to bind to the ends from the same vRNA or cRNA template. At this stage, the viral RNA polymerase is prone to utilize the vRNA/cRNA ends derived from different templates from transcription initiation (i.e. trans-activation mode). As the polyadenylation of viral mRNA requires the viral polymerase bind to the same viral template [12, 29], transcription initiated by the trans-activation mode would favor viral RNA replication and further increase the vRNA and cRNA levels. In our study, the mutated NS segment could specifically enhance the NA vRNA and cRNA levels, suggesting the trans-activation mode might require the 5' and 3' vRNA ends derived from homologous RNA segments.
In conclusion, our result demonstrated that the segment specific regions have roles in controlling viral transcription and replication. Viral RNA with compatible segment-specific sequences might facilitate viral replication in trans. Given the fact that different viral RNA segments might have subtle sequence requirements for viral RNA synthesis [34], further studies on the segment-specific non-coding regions in other viral segments are needed.
Declarations
Acknowledgements
This project is supported by National Institutes of Health (NIAID contract HHSN266200700005C), Research Grant Council of Hong Kong (HKU 7356/03M to LLMP) and Area of Excellence Scheme of the University Grants Committee (Grant AoE/M-12/06). We thank RG Webster (St. Jude Children's Research Hospital, Memphis, USA) for plasmids.
Authors’ Affiliations
References
- Lamb RF, Krug RM: Orthomyxoviridae: The viruses and their replication. In Fields virology. 3rd edition. Edited by: Knipe DM, Howley PH. Philadelphia: Lippincott-Raven Publishers; 1996:353-1395.Google Scholar
- Li X, Palese P: Characterization of the polyadenylation signal of influenza virus RNA. J Virol 1994, 68: 1245-1249.PubMed CentralPubMedGoogle Scholar
- Robertson JS, Schubert M, Lazzarini RA: Polyadenylation sites for influenza virus mRNA. J Virol 1981, 38: 157-163.PubMed CentralPubMedGoogle Scholar
- Poon LL, Pritlove DC, Fodor E, Brownlee GG: Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template. J Virol 1999, 73: 3473-3476.PubMed CentralPubMedGoogle Scholar
- Zheng H, Lee HA, Palese P, Garcia-Sastre A: Influenza A virus RNA polymerase has the ability to stutter at the polyadenylation site of a viral RNA template during RNA replication. J Virol 1999, 73: 5240-5243.PubMed CentralPubMedGoogle Scholar
- Neumann G, Brownlee GG, Fodor E, Kawaoka Y: Orthomyxovirus replication, transcription, and polyadenylation. Curr Top Microbiol Immunol 2004, 283: 121-143.PubMedGoogle Scholar
- Deng T, Vreede FT, Brownlee GG: Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication. J Virol 2006, 80: 2337-2348. 10.1128/JVI.80.5.2337-2348.2006PubMed CentralView ArticlePubMedGoogle Scholar
- Mullin AE, Dalton RM, Amorim MJ, Elton D, Digard P: Increased amounts of the influenza virus nucleoprotein do not promote higher levels of viral genome replication. J Gen Virol 2004, 85: 3689-3698. 10.1099/vir.0.80518-0View ArticlePubMedGoogle Scholar
- Vreede FT, Brownlee GG: Influenza virion-derived viral ribonucleoproteins synthesize both mRNA and cRNA in vitro. J Virol 2007, 81: 2196-2204. 10.1128/JVI.02187-06PubMed CentralView ArticlePubMedGoogle Scholar
- Vreede FT, Jung TE, Brownlee GG: Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates. J Virol 2004, 78: 9568-9572. 10.1128/JVI.78.17.9568-9572.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Desselberger U, Racaniello VR, Zazra JJ, Palese P: The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity. Gene 1980, 8: 315-328. 10.1016/0378-1119(80)90007-4View ArticlePubMedGoogle Scholar
- Fodor E, Pritlove DC, Brownlee GG: The influenza virus panhandle is involved in the initiation of transcription. J Virol 1994, 68: 4092-4096.PubMed CentralPubMedGoogle Scholar
- Tiley LS, Hagen M, Matthews JT, Krystal M: Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs. J Virol 1994, 68: 5108-5116.PubMed CentralPubMedGoogle Scholar
- Hagen M, Chung TD, Butcher JA, Krystal M: Recombinant influenza virus polymerase: requirement of both 5' and 3' viral ends for endonuclease activity. J Virol 1994, 68: 1509-1515.PubMed CentralPubMedGoogle Scholar
- Lee MT, Klumpp K, Digard P, Tiley L: Activation of influenza virus RNA polymerase by the 5' and 3' terminal duplex of genomic RNA. Nucleic Acids Res 2003, 31: 1624-1632. 10.1093/nar/gkg253PubMed CentralView ArticlePubMedGoogle Scholar
- Flick R, Neumann G, Hoffmann E, Neumeier E, Hobom G: Promoter elements in the influenza vRNA terminal structure. RNA 1996, 2: 1046-1057.PubMed CentralPubMedGoogle Scholar
- Fodor E, Pritlove DC, Brownlee GG: Characterization of the RNA-fork model of virion RNA in the initiation of transcription in influenza A virus. J Virol 1995, 69: 4012-4019.PubMed CentralPubMedGoogle Scholar
- de Wit E, Spronken MI, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, Fouchier RA: Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Res 2004, 103: 155-161. 10.1016/j.virusres.2004.02.028View ArticlePubMedGoogle Scholar
- Lee KH, Seong BL: The position 4 nucleotide at the 3' end of the influenza virus neuraminidase vRNA is involved in temporal regulation of transcription and replication of neuraminidase RNAs and affects the repertoire of influenza virus surface antigens. J Gen Virol 1998, 79: 1923-1934.View ArticlePubMedGoogle Scholar
- Dos Santos Afonso E, Escriou N, Leclercq I, van der Werf S, Naffakh N: The generation of recombinant influenza A viruses expressing a PB2 fusion protein requires the conservation of a packaging signal overlapping the coding and noncoding regions at the 5' end of the PB2 segment. Virology 2005, 341: 34-46. 10.1016/j.virol.2005.06.040View ArticlePubMedGoogle Scholar
- Fujii K, Fujii Y, Noda T, Muramoto Y, Watanabe T, Takada A, Goto H, Horimoto T, Kawaoka Y: Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions. J Virol 2005, 79: 3766-3774. 10.1128/JVI.79.6.3766-3774.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Fujii Y, Goto H, Watanabe T, Yoshida T, Kawaoka Y: Selective incorporation of influenza virus RNA segments into virions. Proc Natl Acad Sci USA 2003, 100: 2002-2007. 10.1073/pnas.0437772100PubMed CentralView ArticlePubMedGoogle Scholar
- Liang Y, Hong Y, Parslow TG: cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments. J Virol 2005, 79: 10348-10355. 10.1128/JVI.79.16.10348-10355.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Watanabe T, Watanabe S, Noda T, Fujii Y, Kawaoka Y: Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes. J Virol 2003, 77: 10575-10583. 10.1128/JVI.77.19.10575-10583.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Bergmann M, Muster T: Mutations in the nonconserved noncoding sequences of the influenza A virus segments affect viral vRNA formation. Virus Res 1996, 44: 23-31. 10.1016/0168-1702(96)01335-4View ArticlePubMedGoogle Scholar
- Muster T, Subbarao EK, Enami M, Murphy BR, Palese P: An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice. Proc Natl Acad Sci USA 1991, 88: 5177-5181. 10.1073/pnas.88.12.5177PubMed CentralView ArticlePubMedGoogle Scholar
- Zheng H, Palese P, Garcia-Sastre A: Nonconserved nucleotides at the 3' and 5' ends of an influenza A virus RNA play an important role in viral RNA replication. Virology 1996, 217: 242-251. 10.1006/viro.1996.0111View ArticlePubMedGoogle Scholar
- Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG: A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 2000, 97: 6108-6113. 10.1073/pnas.100133697PubMed CentralView ArticlePubMedGoogle Scholar
- Poon LL, Pritlove DC, Sharps J, Brownlee GG: The RNA polymerase of influenza virus, bound to the 5' end of virion RNA, acts in cis to polyadenylate mRNA. J Virol 1998, 72: 8214-8219.PubMed CentralPubMedGoogle Scholar
- Hay AJ, Lomniczi B, Bellamy AR, Skehel JJ: Transcription of the influenza virus genome. Virology 1977, 83: 337-355. 10.1016/0042-6822(77)90179-9View ArticlePubMedGoogle Scholar
- Mena I, Jambrina E, Albo C, Perales B, Ortin J, Arrese M, Vallejo D, Portela A: Mutational analysis of influenza A virus nucleoprotein: identification of mutations that affect RNA replication. J Virol 1999, 73: 1186-1194.PubMed CentralPubMedGoogle Scholar
- Shih SR, Krug RM: Surprising function of the three influenza viral polymerase proteins: selective protection of viral mRNAs against the cap-snatching reaction catalyzed by the same polymerase proteins. Virology 1996, 226: 430-435. 10.1006/viro.1996.0673View ArticlePubMedGoogle Scholar
- Dalton RM, Mullin AE, Amorim MJ, Medcalf E, Tiley LS, Digard P: Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes. Virol J 2006, 3: 58. 10.1186/1743-422X-3-58PubMed CentralView ArticlePubMedGoogle Scholar
- Catchpole AP, Mingay LJ, Fodor E, Brownlee GG: Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene. J Gen Virol 2003, 84: 507-515. 10.1099/vir.0.18795-0View ArticlePubMedGoogle Scholar
- Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR: Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 2001, 146: 2275-2289. 10.1007/s007050170002View ArticlePubMedGoogle Scholar
Copyright
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.