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Nuclear functions of the influenza A and B viruses NS1 proteins: Do they play a role in viral mRNA export?
Vaccine 27 (2009) 6312–6316
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Nuclear functions of the influenza A and B viruses NS1 proteins: Do they play a role in viral mRNA export? Jana Schneider ∗ , Thorsten Wolff Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 28 November 2008 Accepted 7 January 2009
Keywords: Influenza virus NS1 protein mRNA export
a b s t r a c t Although it is known for decades that influenza viruses replicate and transcribe their genome in the nucleus of the host cell, there is little knowledge about the cellular and viral factors mediating the nuclear transport of viral mRNA transcripts to the cytoplasm. Efficient export of mature cellular mRNA is coupled to their synthesis by the RNA polymerase II and subsequent processing events such as splicing. This linkage necessitated influenza viruses to evolve a strategy to integrate their unspliced mRNAs generated by the viral polymerase into a cellular mRNA export pathway. Recent findings suggest that the major cellular mRNA export receptor Tap/NXF1 promotes the influenza virus mRNA export. Here, we review functions of the NS1 proteins of influenza A and B viruses and discuss the emerging evidence supporting a role of these viral factors in the export of viral mRNAs. © 2009 Elsevier Ltd. All rights reserved.
Influenza viruses belong to the few RNA viruses that replicate their genome in the nucleus of the host cell [1]. Therefore, bidirectional transport of viral nucleic acids and proteins across the nuclear membrane is a pivotal aspect of viral propagation. While the nuclear import of viral proteins and RNA has been extensively investigated, their export is less well understood and there is only little knowledge about how viral mRNAs are translocated into the cytoplasm [1,2]. Molecular cell biological research has led in the past few years to a detailed understanding of the complex process of cellular mRNA export, including the identification of multi-protein complexes, that function to connect the mRNA export to earlier steps in gene expression. One feature of most cellular transcripts in metazoans is the tight coupling of their export to the splicing process [3–5]. However, the genome of influenza viruses consists of eight single-stranded RNA segments from which only one (type B virus) or two (type A virus) of the eleven viral proteins are expressed from spliced transcripts [1]. Therefore, the majority of the viral mRNAs are per se not well recognized by the cellular export machinery, which forced these viruses to evolve an adaptive strategy to favor viral gene expression. Here, we discuss recent findings that give clues about how influenza viruses may access a nuclear export pathway in order to facilitate the expression of viral genes. The transport of RNA from the nucleus to the cytoplasm is mediated by export receptors that couple the cargo with components of the nuclear pore complex [6]. Currently, four receptor proteins have
∗ Corresponding author at: Robert Koch-Institute, P15, Nordufer 20, 13353 Berlin, Germany. Tel.: +49 30 187542280; fax: +49 30 187542328. E-mail address: [email protected] (J. Schneider). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.015
been described, which are specialized in mediating export of distinct classes of nuclear RNA: CRM1 mediates the export of small nuclear RNA and ribosomal RNA, Exportin-t is the export receptor for tRNA and Exportin-5 transports micro RNA to the cytoplasm [6]. The major export factor for cellular mRNA is Tap/NXF1 that forms a functional heterodimer with the p15 protein [7,8]. Most mammalian mRNAs carry large intron sequences that are removed before export by the cellular splicing machinery. Therefore, nuclear export is tightly coupled to the splicing process to ensure that only correctly processed mRNAs are recognized and transported through the nuclear pore complex [4]. The coupling of transcription and downstream processing events is achieved by the C-terminal domain (CTD) of the cellular polymerase II, that associates with proteins involved in 5 -capping, pre-mRNA splicing, 3 -end formation and nuclear export of the nascent RNA transcripts [4,9]. Binding of these various proteins is regulated by different phosphorylation states of the CTD [4]. Some of the CTD-associated factors are components of three major protein complexes that have been reported to mediate the recruitment of Tap/NXF1 to the processed transcript, including the cap binding complex (CBC), the exon-junction complex (EJC) and the transcription/export (TREX) complex (Fig. 1) [4,10,11]. Nascent transcripts first interact with the CBC and the EJC, a protein complex that is deposited on the transcript upstream of exon–exon junctions formed by RNA splicing (Fig. 1). The EJC consists of the four core proteins MLN51, Magoh, Y14 and eIF4AIII, which remain associated with the transcript throughout the export process. The EJC is thought to present a platform for transiently associating factors involved in the nonsense-mediated mRNA decay and mRNA export, including the export factors Aly/Ref and UAP56 (Fig. 1) [11]. Aly/Ref
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Fig. 1. Nuclear export of intron-containing cellular mRNAs in metazoans. (A) Upon splicing the multi-protein exon-junction complex (EJC) is loaded upstream of exon junctions on nascent mRNAs. The EJC is involved in mRNA quality control and export and is thought to serve as a binding platform for factors involved in these processes. The export factors UAP56 and Aly/Ref are transiently associated with the EJC. Together with the THO complex, a multi-protein complex that has various functions in co-transcriptional mRNP formation, UAP56 and Aly/Ref have also been shown to form a TREX complex, which is recruited to mRNAs in a transcription and 5 -cap-dependent manner. Upon binding of the export receptor Tap/NXF1 to Aly/Ref, UAP56 dissociates from the mRNP complex. Tap/NXF1 mediates translocation of the mRNP complex through the nuclear pore. The herpesvirus proteins ICP27 of HSV-1 and UL69 of HCMV and NS1 proteins of influenza A and B viruses have been shown to interact with components of the cellular export machinery, including Aly/Ref, UAP56, Tap/NXF1 and PABP2, as indicated in the scheme. (B) The herpesvirus proteins ICP27 protein and UL69 of HCMV recruit the mRNA export factors Aly/Ref and UAP56, respectively, to unspliced viral mRNAs to facilitate their export from the nucleus. The interaction of the A/NS1 and B/NS1 proteins with components of the cellular mRNA machinery might indicate a similar recruiting function thereby integrating viral transcripts into the nuclear mRNA export pathway.
is an adapter protein that binds to RNA and bridges the interaction of Tap/NXF1 with transcripts, a function that can also be mediated by other adapters, including several SR proteins [12,13]. The recruitment of Aly/Ref to mRNAs is facilitated by the RNA helicase UAP56 [14]. Both proteins assemble together with the THO complex into the TREX complex (Fig. 1) [4,5]. Loading of the human TREX complex onto transcripts depends on the 5 -cap and is coupled to the splicing process, as it is only recruited to processed but not to intronless transcripts [6,15,16]. Subsequently, Tap/NXF1 binds to Aly/Ref, thereby displacing UAP56 from the complex, and delivering the competent transcript to the nuclear pore complex to facilitate its export (Fig. 1). Although the majority of cellular transcripts contain intron sequences, there are also intronless transcripts that need to be transported into the cytoplasm. It has been shown that SR proteins, including SRp20 and 9G8 mediate the export of intronless mRNAs [17]. There is also evidence, that UAP56 and Aly/Ref are involved in the export of unspliced cellular mRNA, but the mechanistic aspects of this process remain to be elucidated [18,19]. The genes of many nuclear replicating viruses such as retroviruses and herpesviruses, are transcribed by the cellular RNA polymerase II. Many of those viral transcripts do not contain introns or need to remain either incompletely or fully unprocessed for expression, which necessitated these viruses to evolve a strategy
to integrate their transcripts into cellular export pathways. The human immunodeficiency virus (HIV) uses the CRM1-dependent export pathway to translocate intron-containing viral mRNAs into the cytoplasm [20]. HIV encodes for a regulator of expression of viral proteins, the Rev protein. Rev contains a leucine-rich nuclear export signal (NES), which is recognized by the nuclear export receptor CRM1 [20]. Rev binds to cis-acting rev responsive elements (RREs) within respective viral RNA transcripts and thus promotes their transport into the cytoplasm by the interaction with CRM1 [20]. The simple Mason-Pfizer monkey retrovirus does not encode for a Rev-like transactivating protein and has therefore evolved another strategy to favor export of its own intron-containing viral mRNAs. These transcripts contain an RNA sequence element, the constitutive transport element (CTE) that is recognized directly by the Tap/NXF1 protein [7]. The same export pathway is used by herpesviruses. Herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) express the multifunctional proteins ICP27 and UL69, respectively, that bind to unspliced viral mRNAs and promote their export by interacting with the mRNA export proteins Aly/Ref or UAP56. Thus, it is common that viral adapter proteins recruit these factors to viral transcripts, thereby introducing them into the cellular mRNA export pathway (Fig. 1) [21–23].
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Influenza viruses replicate explosively in a permissive host cell, which involves strong and apparently selective translation of viral transcripts. However, the current concept of how cellular RNAs gain access to the export machinery raises at least two major questions for our understanding of influenza virus mRNA export. First, influenza virus mRNAs are transcribed by the viral polymerase complex, and not by the cellular polymerase II [1]. So, how have these viruses adapted to force efficient export of viral mRNAs that are not produced by the cellular polymerase II? Second, similar to herpesvirus transcripts, six (type A) or seven (type B) influenza virus transcripts do not contain introns [1]. Hence, influenza viruses must have evolved a strategy to favor export of their transcripts, but little is currently known about how they achieve this. One strategy to cope with the first problem might be the binding of the viral polymerase complex with the polymerase II CTD. This interaction possibly provides viral transcripts with access to cellular activities mediated by the CTD, e.g. the loading of heterogenous nuclear RNPs onto viral transcripts [24,25]. In line with this, influenza virus mRNA export has been reported to depend on active polymerase II transcription, in addition to the supply with cellular 5 -cap-structures [25,26]. Moreover, considerable progress has also been made concerning the second open question, as recent findings suggest that the Tap/NXF1 export receptor mediates transport of viral mRNA into the cytoplasm. Thus, Tap/NXF1 co-precipitated with viral transcripts [27] and knockdown of Tap/NXF1 by siRNA transfection lead to strongly impaired influenza A virus replication [28]. The fact that viral mRNA export is not inhibited by the CRM1 inhibitor leptomycin B, excludes the possibility of a CRM1-dependent viral mRNA export, and supports a role of Tap/NXF1 in this process [25,27]. This distinguishes influenza virus mRNAs from the export of the viral genomic ribonucleoprotein that is facilitated by the CRM1-pathway [1]. How are mRNA transcripts of influenza viruses then integrated into the cellular mRNA export pathway? Clearly, expression of the first viral mRNAs produced during primary transcription in infected cells requires only the viral polymerase and nucleoprotein indicating that viral transcripts can be exported in the absence of additional viral proteins. However, evidence has emerged, which suggests that the multi-functional NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1, respectively) enhance viral mRNA export in later stages of infection (see below). The NS1 proteins are polypeptides of 26 kDa (A/NS1) and 32 kDa (B/NS1) that are known to bind to single- and double-stranded RNAs via their N-terminal domains ([29], Fig. 2). Early studies showed that ts-mutations in the viral NS1 gene strongly reduced late viral protein production (summarized in Refs. [30,31]), but more defined NS1 functions were not revealed before the mid 1990s. Thus, the A/NS1 and B/NS1 proteins were both shown to antagonize the type I interferon system and to block activation of the antiviral kinase PKR despite a low degree of sequence identity of <25% [32]. The A/NS1 protein was also reported to increase viral mRNA translation by interacting with the eukaryotic initiation factor eIF4GI and the cytoplasmic poly(A)-binding protein PABP1 [33–35]. Both NS1 proteins access the nucleus during infection, which is facilitated by nuclear localization signals that were mapped within the RNA binding domains (Fig. 2). However, the trafficking of the two NS1 proteins appears to be differentially regulated (Fig. 2). The A/NS1 protein is detected in the nucleus and the cytoplasm of the cell throughout infection, which is possibly also influenced by a second C-terminal NLS that is present in some virus strains (Fig. 2; [36,37]). Interestingly, a recent study revealed for the influenza B virus NS1 protein a more dynamic picture showing an accumulation in nuclear dot-like domains early in infection and a relocation to the cytoplasm at later time-points (Fig. 2; [38]). One intensively described nuclear function of the A/NS1 protein is the inhibition of the splicing and nucleo-cytoplasmic export of cellular mRNAs
[39,40]. The latter process was explained by an inhibition of 3 end formation via interactions of A/NS1 with the cleavage and polyadenylation specificity factor (CPSF)-30 kDa subunit and the nuclear poly(A)-binding protein PABP2 [41,42]. However, the A/NS1 protein also inhibits the splicing and, hence, export of its own collinear mRNA when expressed by the viral RNA polymerase [43]. This finding indicates that the protein can act on the processing of cellular and viral mRNA transcripts. Interestingly, this posttranscriptional activity appears not to be conserved in the B/NS1 protein [29] raising the question what function this protein might fulfill in the nucleus? One recent line of evidence showed that the influenza B virus NS1 protein interacts in a distinct nuclear compartment with RNA export factors. Double-staining experiments colocalized the B/NS1 protein with the mRNA export proteins Aly/Ref and UAP56 and the splicing factor SC35 in nuclear speckles at early time-points of infection [38,44]. In vitro binding assays also confirmed a physical association of the B/NS1 protein with UAP56, a component of the TREX complex [38]. Intriguingly, expression of the B/NS1 protein in infected or transfected cells lead to a rounded appearance of the normally irregularly shaped nuclear speckle domains, which is indicative for a disturbance of their normal function [38,44]. Speckles are nuclear non-membranous compartments, in which proteins involved in mRNA transcription, processing, nonsense-mediated decay and export accumulate [45]. Therefore, nuclear speckles seem to have an important function in RNA biogenesis, although it is unlikely that they are sites of active RNA processing [38,44]. Rather, the concept is that speckles serve as a spatial and temporal link for the maturation and export of cellular mRNA [45]. It therefore appears possible that the transient speckle association of the B/NS1 protein reflects an activity to recruit cellular factors such as UAP56 to viral RNAs. The cytoplasmic relocation of the B/NS1 protein late in infection also occurred when the CRM1-dependent pathway was blocked, suggesting that this process is mediated by a different receptor such as Tap/NXF1 [38]. The dynamic intracellular trafficking of the B/NS1 protein is strikingly similar to observations made with the ICP27 protein of HSV-1 suggesting functional analogies between the two proteins despite the absence of recognizable sequence homology. ICP27 transiently localized to nuclear speckle regions during infection [46] and was detected in the cytoplasm at later time-points [47]. The protein was also shown to bind to viral RNA transcripts as well as the Aly/REF adapter and the TAP export receptor proteins [21]. Recent gene array analysis of WT and mutant herpesviruses showed that ICP27 is in fact the major export adaptor for HSV-1 mRNA transcripts [47]. The parallels between the two herpes and influenza virus proteins encourage future studies to characterize in detail the kinetics and specificity of NS1 interactions with viral RNA transcripts, the effects of NS1 mutations on transcript localization, and the interactions with export adaptors. Additional recent reports revealed that also the influenza A viruses NS1 protein interacts with factors of the cellular mRNA export pathway. Satterly et al. [48] showed that the A/NS1 protein binds to Tap/NXF1, p15, Rae1, E1B-AP5, and the nucleoporin Nup98. These interactions were suggested to contribute to the known inhibition of cellular mRNA export, which, however, does not exclude a stimulatory activity on viral transcripts [48]. This hypothesis was in fact supported by a study from Wang et al. [27] that detected an association of the A/NS1 protein with several viral mRNA transcripts by RNA immunoprecipitation analysis. The A/NS1 amino acid sequence also contains a latent nuclear export signal in the C-terminal region [49]. However, it has not been tested yet whether this signal contributes to an RNA export activity. Viruses with a nuclear replication strategy have evolved mechanisms to ensure efficient transport of their transcripts to the
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Fig. 2. Cellular trafficking of the NS1 proteins of influenza A and B viruses during infection is very distinct. (A) Scheme of the A/NS1 and B/NS1 amino acid sequences. Both proteins contain a nuclear localization signal (NLS) in the N-terminal region. Some influenza A viruses contain a second NLS2 in the very C-terminal region. Further the A/NS1 protein contains a nuclear export signal (NES) in the C-terminal region. The N-terminal 90 amino acids are the minimal B/NS1 sequence that mediates speckle association of the protein. (B) A549 cell were infected with influenza A/PR/8/34 virus or B/Yam/1/73 virus at a multiplicity of infection of 1. At 4 h and 16 h (type A) or 8 h and 16 h (type B) post infection cells were fixed, permeabilized and stained for the NS1 proteins with A/NS1- or B/NS1-specific rabbit serum, respectively, followed by detection with secondary ␣-rabbit IgG-Alexa 488 antibodies. The settings for this experiment have been described elsewhere [38]. The cells were analyzed with confocal laser scanning microscopy using the 488 nm laser setting. Scale bar = 10 m.
cytoplasm. From studies of retro- and large DNA viruses the concept has emerged that this task is mediated by viral adaptors that bind viral transcripts and connect them to one of the cellular export pathways. However, for influenza viruses we are just in the beginning to understand this important aspect of the viral life-cycle. Recent evidence suggested that influenza virus mRNA export is facilitated by the Tap/NXF1 export receptor. For the viral transcripts that are generated by splicing, it is conceivable that they associate with TAP in a similar way as cellular mRNAs. However, several key issues remain to be addressed for a clear understanding of how the majority of intronless viral transcripts reach the cytoplasm. Here, we have summarized the indications suggesting that the NS1 proteins of influenza A and B viruses play a role in viral mRNA export, possibly by recruiting transport factors to viral transcripts [27,28,38,48]. However, to firmly establish this model will require more detailed investigations on the interactions of specific domains of the A/NS1 and B/NS1 proteins with cellular export factors and with viral mRNA transcripts in vitro and in vivo. Those analyses should also reveal
similarities and differences between the two NS1 proteins. Further, it was shown, that the A/NS1 protein co-precipitates mRNA of the viral NA, M1 and PB1 genes [27]. It will be important to determine whether additional viral transcripts are also recognized by NS1 and to characterize the sequence elements that facilitate these interactions. In this context, it is important to mention that segment-specific mRNA transport events were observed, which differed in their sensitivity towards RNA polymerase II inhibition [25]. Exploring the precise chain of events and the factors that mediate influenza virus mRNA export will not only lead to a more detailed understanding of the biology of these notorious pathogens, but may also reveal novel opportunities for antiviral interventions in the future. Acknowledgements The authors acknowledge support by grants of the Deutsche Forschungsgemeinschaft (Wo 554-3/2), the Influenza research pro-
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Nuclear functions of the influenza A and B viruses NS1 proteins: Do they play a role in viral mRNA export? Jana Schneider ∗ , Thorsten Wolff Robert Koch-Institute, Nordufer 20, 13353 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 28 November 2008 Accepted 7 January 2009
Keywords: Influenza virus NS1 protein mRNA export
a b s t r a c t Although it is known for decades that influenza viruses replicate and transcribe their genome in the nucleus of the host cell, there is little knowledge about the cellular and viral factors mediating the nuclear transport of viral mRNA transcripts to the cytoplasm. Efficient export of mature cellular mRNA is coupled to their synthesis by the RNA polymerase II and subsequent processing events such as splicing. This linkage necessitated influenza viruses to evolve a strategy to integrate their unspliced mRNAs generated by the viral polymerase into a cellular mRNA export pathway. Recent findings suggest that the major cellular mRNA export receptor Tap/NXF1 promotes the influenza virus mRNA export. Here, we review functions of the NS1 proteins of influenza A and B viruses and discuss the emerging evidence supporting a role of these viral factors in the export of viral mRNAs. © 2009 Elsevier Ltd. All rights reserved.
Influenza viruses belong to the few RNA viruses that replicate their genome in the nucleus of the host cell [1]. Therefore, bidirectional transport of viral nucleic acids and proteins across the nuclear membrane is a pivotal aspect of viral propagation. While the nuclear import of viral proteins and RNA has been extensively investigated, their export is less well understood and there is only little knowledge about how viral mRNAs are translocated into the cytoplasm [1,2]. Molecular cell biological research has led in the past few years to a detailed understanding of the complex process of cellular mRNA export, including the identification of multi-protein complexes, that function to connect the mRNA export to earlier steps in gene expression. One feature of most cellular transcripts in metazoans is the tight coupling of their export to the splicing process [3–5]. However, the genome of influenza viruses consists of eight single-stranded RNA segments from which only one (type B virus) or two (type A virus) of the eleven viral proteins are expressed from spliced transcripts [1]. Therefore, the majority of the viral mRNAs are per se not well recognized by the cellular export machinery, which forced these viruses to evolve an adaptive strategy to favor viral gene expression. Here, we discuss recent findings that give clues about how influenza viruses may access a nuclear export pathway in order to facilitate the expression of viral genes. The transport of RNA from the nucleus to the cytoplasm is mediated by export receptors that couple the cargo with components of the nuclear pore complex [6]. Currently, four receptor proteins have
∗ Corresponding author at: Robert Koch-Institute, P15, Nordufer 20, 13353 Berlin, Germany. Tel.: +49 30 187542280; fax: +49 30 187542328. E-mail address: [email protected] (J. Schneider). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.015
been described, which are specialized in mediating export of distinct classes of nuclear RNA: CRM1 mediates the export of small nuclear RNA and ribosomal RNA, Exportin-t is the export receptor for tRNA and Exportin-5 transports micro RNA to the cytoplasm [6]. The major export factor for cellular mRNA is Tap/NXF1 that forms a functional heterodimer with the p15 protein [7,8]. Most mammalian mRNAs carry large intron sequences that are removed before export by the cellular splicing machinery. Therefore, nuclear export is tightly coupled to the splicing process to ensure that only correctly processed mRNAs are recognized and transported through the nuclear pore complex [4]. The coupling of transcription and downstream processing events is achieved by the C-terminal domain (CTD) of the cellular polymerase II, that associates with proteins involved in 5 -capping, pre-mRNA splicing, 3 -end formation and nuclear export of the nascent RNA transcripts [4,9]. Binding of these various proteins is regulated by different phosphorylation states of the CTD [4]. Some of the CTD-associated factors are components of three major protein complexes that have been reported to mediate the recruitment of Tap/NXF1 to the processed transcript, including the cap binding complex (CBC), the exon-junction complex (EJC) and the transcription/export (TREX) complex (Fig. 1) [4,10,11]. Nascent transcripts first interact with the CBC and the EJC, a protein complex that is deposited on the transcript upstream of exon–exon junctions formed by RNA splicing (Fig. 1). The EJC consists of the four core proteins MLN51, Magoh, Y14 and eIF4AIII, which remain associated with the transcript throughout the export process. The EJC is thought to present a platform for transiently associating factors involved in the nonsense-mediated mRNA decay and mRNA export, including the export factors Aly/Ref and UAP56 (Fig. 1) [11]. Aly/Ref
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Fig. 1. Nuclear export of intron-containing cellular mRNAs in metazoans. (A) Upon splicing the multi-protein exon-junction complex (EJC) is loaded upstream of exon junctions on nascent mRNAs. The EJC is involved in mRNA quality control and export and is thought to serve as a binding platform for factors involved in these processes. The export factors UAP56 and Aly/Ref are transiently associated with the EJC. Together with the THO complex, a multi-protein complex that has various functions in co-transcriptional mRNP formation, UAP56 and Aly/Ref have also been shown to form a TREX complex, which is recruited to mRNAs in a transcription and 5 -cap-dependent manner. Upon binding of the export receptor Tap/NXF1 to Aly/Ref, UAP56 dissociates from the mRNP complex. Tap/NXF1 mediates translocation of the mRNP complex through the nuclear pore. The herpesvirus proteins ICP27 of HSV-1 and UL69 of HCMV and NS1 proteins of influenza A and B viruses have been shown to interact with components of the cellular export machinery, including Aly/Ref, UAP56, Tap/NXF1 and PABP2, as indicated in the scheme. (B) The herpesvirus proteins ICP27 protein and UL69 of HCMV recruit the mRNA export factors Aly/Ref and UAP56, respectively, to unspliced viral mRNAs to facilitate their export from the nucleus. The interaction of the A/NS1 and B/NS1 proteins with components of the cellular mRNA machinery might indicate a similar recruiting function thereby integrating viral transcripts into the nuclear mRNA export pathway.
is an adapter protein that binds to RNA and bridges the interaction of Tap/NXF1 with transcripts, a function that can also be mediated by other adapters, including several SR proteins [12,13]. The recruitment of Aly/Ref to mRNAs is facilitated by the RNA helicase UAP56 [14]. Both proteins assemble together with the THO complex into the TREX complex (Fig. 1) [4,5]. Loading of the human TREX complex onto transcripts depends on the 5 -cap and is coupled to the splicing process, as it is only recruited to processed but not to intronless transcripts [6,15,16]. Subsequently, Tap/NXF1 binds to Aly/Ref, thereby displacing UAP56 from the complex, and delivering the competent transcript to the nuclear pore complex to facilitate its export (Fig. 1). Although the majority of cellular transcripts contain intron sequences, there are also intronless transcripts that need to be transported into the cytoplasm. It has been shown that SR proteins, including SRp20 and 9G8 mediate the export of intronless mRNAs [17]. There is also evidence, that UAP56 and Aly/Ref are involved in the export of unspliced cellular mRNA, but the mechanistic aspects of this process remain to be elucidated [18,19]. The genes of many nuclear replicating viruses such as retroviruses and herpesviruses, are transcribed by the cellular RNA polymerase II. Many of those viral transcripts do not contain introns or need to remain either incompletely or fully unprocessed for expression, which necessitated these viruses to evolve a strategy
to integrate their transcripts into cellular export pathways. The human immunodeficiency virus (HIV) uses the CRM1-dependent export pathway to translocate intron-containing viral mRNAs into the cytoplasm [20]. HIV encodes for a regulator of expression of viral proteins, the Rev protein. Rev contains a leucine-rich nuclear export signal (NES), which is recognized by the nuclear export receptor CRM1 [20]. Rev binds to cis-acting rev responsive elements (RREs) within respective viral RNA transcripts and thus promotes their transport into the cytoplasm by the interaction with CRM1 [20]. The simple Mason-Pfizer monkey retrovirus does not encode for a Rev-like transactivating protein and has therefore evolved another strategy to favor export of its own intron-containing viral mRNAs. These transcripts contain an RNA sequence element, the constitutive transport element (CTE) that is recognized directly by the Tap/NXF1 protein [7]. The same export pathway is used by herpesviruses. Herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) express the multifunctional proteins ICP27 and UL69, respectively, that bind to unspliced viral mRNAs and promote their export by interacting with the mRNA export proteins Aly/Ref or UAP56. Thus, it is common that viral adapter proteins recruit these factors to viral transcripts, thereby introducing them into the cellular mRNA export pathway (Fig. 1) [21–23].
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Influenza viruses replicate explosively in a permissive host cell, which involves strong and apparently selective translation of viral transcripts. However, the current concept of how cellular RNAs gain access to the export machinery raises at least two major questions for our understanding of influenza virus mRNA export. First, influenza virus mRNAs are transcribed by the viral polymerase complex, and not by the cellular polymerase II [1]. So, how have these viruses adapted to force efficient export of viral mRNAs that are not produced by the cellular polymerase II? Second, similar to herpesvirus transcripts, six (type A) or seven (type B) influenza virus transcripts do not contain introns [1]. Hence, influenza viruses must have evolved a strategy to favor export of their transcripts, but little is currently known about how they achieve this. One strategy to cope with the first problem might be the binding of the viral polymerase complex with the polymerase II CTD. This interaction possibly provides viral transcripts with access to cellular activities mediated by the CTD, e.g. the loading of heterogenous nuclear RNPs onto viral transcripts [24,25]. In line with this, influenza virus mRNA export has been reported to depend on active polymerase II transcription, in addition to the supply with cellular 5 -cap-structures [25,26]. Moreover, considerable progress has also been made concerning the second open question, as recent findings suggest that the Tap/NXF1 export receptor mediates transport of viral mRNA into the cytoplasm. Thus, Tap/NXF1 co-precipitated with viral transcripts [27] and knockdown of Tap/NXF1 by siRNA transfection lead to strongly impaired influenza A virus replication [28]. The fact that viral mRNA export is not inhibited by the CRM1 inhibitor leptomycin B, excludes the possibility of a CRM1-dependent viral mRNA export, and supports a role of Tap/NXF1 in this process [25,27]. This distinguishes influenza virus mRNAs from the export of the viral genomic ribonucleoprotein that is facilitated by the CRM1-pathway [1]. How are mRNA transcripts of influenza viruses then integrated into the cellular mRNA export pathway? Clearly, expression of the first viral mRNAs produced during primary transcription in infected cells requires only the viral polymerase and nucleoprotein indicating that viral transcripts can be exported in the absence of additional viral proteins. However, evidence has emerged, which suggests that the multi-functional NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1, respectively) enhance viral mRNA export in later stages of infection (see below). The NS1 proteins are polypeptides of 26 kDa (A/NS1) and 32 kDa (B/NS1) that are known to bind to single- and double-stranded RNAs via their N-terminal domains ([29], Fig. 2). Early studies showed that ts-mutations in the viral NS1 gene strongly reduced late viral protein production (summarized in Refs. [30,31]), but more defined NS1 functions were not revealed before the mid 1990s. Thus, the A/NS1 and B/NS1 proteins were both shown to antagonize the type I interferon system and to block activation of the antiviral kinase PKR despite a low degree of sequence identity of <25% [32]. The A/NS1 protein was also reported to increase viral mRNA translation by interacting with the eukaryotic initiation factor eIF4GI and the cytoplasmic poly(A)-binding protein PABP1 [33–35]. Both NS1 proteins access the nucleus during infection, which is facilitated by nuclear localization signals that were mapped within the RNA binding domains (Fig. 2). However, the trafficking of the two NS1 proteins appears to be differentially regulated (Fig. 2). The A/NS1 protein is detected in the nucleus and the cytoplasm of the cell throughout infection, which is possibly also influenced by a second C-terminal NLS that is present in some virus strains (Fig. 2; [36,37]). Interestingly, a recent study revealed for the influenza B virus NS1 protein a more dynamic picture showing an accumulation in nuclear dot-like domains early in infection and a relocation to the cytoplasm at later time-points (Fig. 2; [38]). One intensively described nuclear function of the A/NS1 protein is the inhibition of the splicing and nucleo-cytoplasmic export of cellular mRNAs
[39,40]. The latter process was explained by an inhibition of 3 end formation via interactions of A/NS1 with the cleavage and polyadenylation specificity factor (CPSF)-30 kDa subunit and the nuclear poly(A)-binding protein PABP2 [41,42]. However, the A/NS1 protein also inhibits the splicing and, hence, export of its own collinear mRNA when expressed by the viral RNA polymerase [43]. This finding indicates that the protein can act on the processing of cellular and viral mRNA transcripts. Interestingly, this posttranscriptional activity appears not to be conserved in the B/NS1 protein [29] raising the question what function this protein might fulfill in the nucleus? One recent line of evidence showed that the influenza B virus NS1 protein interacts in a distinct nuclear compartment with RNA export factors. Double-staining experiments colocalized the B/NS1 protein with the mRNA export proteins Aly/Ref and UAP56 and the splicing factor SC35 in nuclear speckles at early time-points of infection [38,44]. In vitro binding assays also confirmed a physical association of the B/NS1 protein with UAP56, a component of the TREX complex [38]. Intriguingly, expression of the B/NS1 protein in infected or transfected cells lead to a rounded appearance of the normally irregularly shaped nuclear speckle domains, which is indicative for a disturbance of their normal function [38,44]. Speckles are nuclear non-membranous compartments, in which proteins involved in mRNA transcription, processing, nonsense-mediated decay and export accumulate [45]. Therefore, nuclear speckles seem to have an important function in RNA biogenesis, although it is unlikely that they are sites of active RNA processing [38,44]. Rather, the concept is that speckles serve as a spatial and temporal link for the maturation and export of cellular mRNA [45]. It therefore appears possible that the transient speckle association of the B/NS1 protein reflects an activity to recruit cellular factors such as UAP56 to viral RNAs. The cytoplasmic relocation of the B/NS1 protein late in infection also occurred when the CRM1-dependent pathway was blocked, suggesting that this process is mediated by a different receptor such as Tap/NXF1 [38]. The dynamic intracellular trafficking of the B/NS1 protein is strikingly similar to observations made with the ICP27 protein of HSV-1 suggesting functional analogies between the two proteins despite the absence of recognizable sequence homology. ICP27 transiently localized to nuclear speckle regions during infection [46] and was detected in the cytoplasm at later time-points [47]. The protein was also shown to bind to viral RNA transcripts as well as the Aly/REF adapter and the TAP export receptor proteins [21]. Recent gene array analysis of WT and mutant herpesviruses showed that ICP27 is in fact the major export adaptor for HSV-1 mRNA transcripts [47]. The parallels between the two herpes and influenza virus proteins encourage future studies to characterize in detail the kinetics and specificity of NS1 interactions with viral RNA transcripts, the effects of NS1 mutations on transcript localization, and the interactions with export adaptors. Additional recent reports revealed that also the influenza A viruses NS1 protein interacts with factors of the cellular mRNA export pathway. Satterly et al. [48] showed that the A/NS1 protein binds to Tap/NXF1, p15, Rae1, E1B-AP5, and the nucleoporin Nup98. These interactions were suggested to contribute to the known inhibition of cellular mRNA export, which, however, does not exclude a stimulatory activity on viral transcripts [48]. This hypothesis was in fact supported by a study from Wang et al. [27] that detected an association of the A/NS1 protein with several viral mRNA transcripts by RNA immunoprecipitation analysis. The A/NS1 amino acid sequence also contains a latent nuclear export signal in the C-terminal region [49]. However, it has not been tested yet whether this signal contributes to an RNA export activity. Viruses with a nuclear replication strategy have evolved mechanisms to ensure efficient transport of their transcripts to the
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Fig. 2. Cellular trafficking of the NS1 proteins of influenza A and B viruses during infection is very distinct. (A) Scheme of the A/NS1 and B/NS1 amino acid sequences. Both proteins contain a nuclear localization signal (NLS) in the N-terminal region. Some influenza A viruses contain a second NLS2 in the very C-terminal region. Further the A/NS1 protein contains a nuclear export signal (NES) in the C-terminal region. The N-terminal 90 amino acids are the minimal B/NS1 sequence that mediates speckle association of the protein. (B) A549 cell were infected with influenza A/PR/8/34 virus or B/Yam/1/73 virus at a multiplicity of infection of 1. At 4 h and 16 h (type A) or 8 h and 16 h (type B) post infection cells were fixed, permeabilized and stained for the NS1 proteins with A/NS1- or B/NS1-specific rabbit serum, respectively, followed by detection with secondary ␣-rabbit IgG-Alexa 488 antibodies. The settings for this experiment have been described elsewhere [38]. The cells were analyzed with confocal laser scanning microscopy using the 488 nm laser setting. Scale bar = 10 m.
cytoplasm. From studies of retro- and large DNA viruses the concept has emerged that this task is mediated by viral adaptors that bind viral transcripts and connect them to one of the cellular export pathways. However, for influenza viruses we are just in the beginning to understand this important aspect of the viral life-cycle. Recent evidence suggested that influenza virus mRNA export is facilitated by the Tap/NXF1 export receptor. For the viral transcripts that are generated by splicing, it is conceivable that they associate with TAP in a similar way as cellular mRNAs. However, several key issues remain to be addressed for a clear understanding of how the majority of intronless viral transcripts reach the cytoplasm. Here, we have summarized the indications suggesting that the NS1 proteins of influenza A and B viruses play a role in viral mRNA export, possibly by recruiting transport factors to viral transcripts [27,28,38,48]. However, to firmly establish this model will require more detailed investigations on the interactions of specific domains of the A/NS1 and B/NS1 proteins with cellular export factors and with viral mRNA transcripts in vitro and in vivo. Those analyses should also reveal
similarities and differences between the two NS1 proteins. Further, it was shown, that the A/NS1 protein co-precipitates mRNA of the viral NA, M1 and PB1 genes [27]. It will be important to determine whether additional viral transcripts are also recognized by NS1 and to characterize the sequence elements that facilitate these interactions. In this context, it is important to mention that segment-specific mRNA transport events were observed, which differed in their sensitivity towards RNA polymerase II inhibition [25]. Exploring the precise chain of events and the factors that mediate influenza virus mRNA export will not only lead to a more detailed understanding of the biology of these notorious pathogens, but may also reveal novel opportunities for antiviral interventions in the future. Acknowledgements The authors acknowledge support by grants of the Deutsche Forschungsgemeinschaft (Wo 554-3/2), the Influenza research pro-
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