División de Productos Biológicos y Biotecnología, Agencia Española del Medicamento, Majadahonda 28220, Madrid, Spain1
Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK2
Author for correspondence: Paul Digard. Fax +44 1223 336 926. e-mail pd1{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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General properties and replication cycle of influenza virus |
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Structure and activities of influenza virus NP |
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Homo-oligomerization
The existence of NPNP interactions that play a major role in maintaining RNP structure became evident from early work examining the physical characteristics of RNPs after the removal of RNA (Kingsbury & Webster, 1969 ; Pons et al., 1969
). The ability of purified RNA-free NP to form structures resembling authentic RNPs has been demonstrated subsequently by EM analyses (Ruigrok & Baudin, 1995
). NPNP interactions have been estimated to have a Kd of
200 nM in a binding assay, which made use of recombinant immobilized fusion proteins and in vitro-translated NP (Elton et al., 1999a
). This system was used further to identify NP sequences important for self-association. Deletion mutagenesis identified two independent regions, NP-1 and NP-2 (Fig. 3
), capable of forming NPNP contacts. Interestingly, the C-terminal 23 residues of NP were found to inhibit oligomerization. Point mutations within each of these areas with matching negative and positive effects on oligomerization were also identified (Elton et al., 1999a
).
NPpolymerase interactions
In recent years, it has been shown that NP interacts directly with PB1 and PB2 but not with PA, both in virus-infected cells and recombinant systems (Biswas et al., 1998 ; Medcalf et al., 1999
). Consistent with this, a recent EM reconstruction of an RNP clearly shows two regions of contact between the polymerase complex and separate NP monomers (Fig. 1b
) (Martín-Benito et al., 2001
). In addition, there is genetic evidence for a linkage between NP and PB2. A revertant of a temperature-sensitive (ts) virus with a lesion in NP was found to carry an extragenic suppressor mutation, most likely located in the PB2 gene (Mandler et al., 1991
). Similarly, an analysis of the compatibility between RNP polypeptides from human and avian strains of influenza virus found a consistent linkage between NP and PB2 (Naffakh et al., 2000
). Biswas et al. (1998)
identified three NP fragments (Fig. 3
), each of which could interact independently with PB2. They also observed that removal of the last 33 amino acids of NP increased the strength and stability of the NPPB2 interaction: a finding that is in agreement with the results described for NPNP oligomerization above.
NPM1 interactions
Initial studies of disrupted virions indicated an association between M1 and RNPs (Zvonarjev & Ghendon, 1980 ; Rees & Dimmock, 1981
) but, since M1 is an RNA-binding protein (Wakefield & Brownlee, 1989
), it was not known if this reflected a direct proteinprotein interaction between M1 and NP. However, a recent study that employed mutant M1 molecules unable to bind RNA and RNA-depleted RNPs indicated that M1 does in fact bind to NP directly, although M1RNA contacts also play a role (Ye et al., 1999
). However, the M1-binding site on NP has not been identified yet.
NPimportin interactions
Given our current understanding of the cellular nuclear transport machinery, it now seems self-evident that NP would interact with importin because of its nuclear localization signal (NLS). However, elucidation of this has not been straightforward. Soon after NP was shown to contain an NLS (Lin & Lai, 1983
), amino acids 327345 (Fig. 3
) were proposed to contain a nuclear accumulation signal, based on the behaviour of NP deletion mutants expressed in Xenopus oocytes (Davey et al., 1985
). A decade later, yeast two-hybrid experiments indicated that NP bound members of a family of cellular polypeptides of then uncertain function but which were almost simultaneously identified as components of the nuclear import machinery (ONeill & Palese, 1995
; Gorlich et al., 1994
; Moroianu et al., 1995
). However, mutational analysis of NP did not implicate the oocyte NLS' as being responsible for this interaction but, instead, identified a short sequence at the N terminus of the protein (Fig. 3
, NLS I) which also functioned as a transferable NLS in mammalian cells (Wang et al., 1997
). Mutation of this NLS in the context of full-length NP did not prevent nuclear import, indicating the presence of other signal(s) in the polypeptide (Wang et al., 1997
; Neumann et al., 1997
). Consistent with this, a sequence matching a canonical bipartite cellular NLS has been identified (Fig. 3
, NLS II) and shown to be active in the absence of NLS I (Weber et al., 1998
). However, no evidence has been found to suggest that the oocyte NLS functions as an NLS in mammalian cells (Wang et al., 1997
; Neumann et al., 1997
) and, in fact, it has been shown to act in opposition to cause cytoplasmic accumulation of the protein [Fig. 3
, cytoplasmic accumulation signal (CAS)] (Weber et al., 1998
; Digard et al., 1999
). In addition, systematic deletion analysis of NP suggests the presence of another potential NLS located between amino acids 320 and 400 (Bullido et al., 2000
).
NPF-actin interactions
Cell fractionation and co-localization studies suggested that, late in infection, cytoplasmic NP is associated with the cytoskeleton (Avalos et al., 1997 ; Husain & Gupta, 1997
). In confirmation of this, purified NP has been shown to bind F-actin in vitro, with a Kd of 1 µM and a stoichiometry of 1 NP per actin subunit, and, furthermore, co-localization of NP and
-actin has been detected in cells expressing recombinant NP (Digard et al., 1999
). A discrete sequence in NP capable of binding F-actin has not been identified, but a cluster of point mutations that disrupts the interaction has been characterized (Digard et al., 1999
). Curiously, these mutations lie within or close to the region identified originally as a nuclear accumulation signal in Xenopus oocytes but which has been shown subsequently to promote cytoplasmic accumulation of NP in mammalian cells (Fig. 3
, CAS/actin). Accordingly, it has been proposed that the CAS functions as a cytoplasmic retention signal for RNPs by tethering NP to the actin cytoskeleton (Digard et al., 1999
).
NPCRM1 interactions
In recent years, it has become evident that, in addition to multiple NLSs, NP also contains signals that work in opposition to cause cytoplasmic accumulation of the polypeptide. Exogenously expressed NP is not static in one cellular compartment but, instead, shuttles between the cytoplasm and the nucleus (Whittaker et al., 1996 ; Neumann et al., 1997
), suggesting that it contains a nuclear export signal (NES). Even in the absence of other influenza virus proteins, the balance between the import and export signals is not fixed, as, depending on the circumstances, static immunofluorescent snapshots' of NP distribution can show the bulk of it in either the nucleus or the cytoplasm, or distributed evenly between the two compartments (Neumann et al., 1997
; Digard et al., 1999
). Treatment of NP-expressing cells with the drug leptomycin B (LMB), which specifically inactivates CRM1/exportin-1, a cellular NES receptor (Fornerod et al., 1997
; Kudo et al., 1998
), biases NP towards a more nuclear distribution (Elton et al., 2001
). This suggests that NP contains an NES recognized by CRM1 and, in support of this hypothesis, overexpression of CRM1 biases transfected NP towards cytoplasmic accumulation and the two proteins interact in in vitro-binding assays (Elton et al., 2001
). The identity of the NP NES(s) remains uncertain. Residues 138 have been proposed to contain an NES (Neumann et al., 1997
) but this hypothesis awaits further testing. In addition, the CAS sequence is an alternative or additional candidate for an NES, as the techniques used so far to examine the behaviour of NP molecules bearing lesions within this region do not formally distinguish between cytoplasmic retention and nuclear export.
NPBAT1/UAP56 interactions
A cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases, BAT1/UAP56, was identified as an NP-interacting polypeptide by a convincing congruence of different techniques in independent laboratories. On one hand, it was identified by fractionation of nuclear extracts from uninfected cells in a search for a stimulatory factor for influenza virus RNA synthesis; on the other hand, by a yeast two-hybrid screen for NP-interacting polypeptides (Momose et al., 2001 ). Deletion analysis of the NPBAT1 interaction in vitro and in yeast led to the identification of a BAT1-binding site in the N-terminal 20 amino acids of NP (Fig. 3
) (Momose et al., 2001
).
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Roles of NP during the virus life cycle |
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Viral RNA synthesis
NP has long been known to be the major protein component of influenza virus RNPs (Pons et al., 1969 ) and, as these particles were quickly identified as transcriptase complexes (Bishop et al., 1971
), a role for NP in vRNA synthesis seemed likely. The most obvious role is a structural one of maintaining the RNA template in an ordered conformation suitable for transcription by the polymerase and/or packaging into virions. EM visualization of RNPs reveals rod-like particles, often with loops at one end; these structures have been interpreted as strands of NPRNA complexes bent into a hairpin and twisted into a helical structure (Fig. 1a
) (Jennings et al., 1983
). This model has been elegantly confirmed by the recent EM reconstruction of a recombinant mini-RNP (Martín-Benito et al., 2001
) in which the barrel' of the RNP is lost because of the artificial shortness of the vRNA molecule, leaving only the terminal loop (Fig. 1b
). Formation of these structures depends on a combination of NPRNA and NPNP interactions, as discussed in the preceding section. However, in the absence of an identified sequence-specific interaction between NP and the genome segments, it is unclear what directs the specific encapsidation of v- and cRNA but not mRNA (Hay et al., 1977
). One interesting possibility is that the sequence-specific interaction of the polymerase complex with the 5' end of vRNAs (Tiley et al., 1994
) functions as a specific encapsidation signal via ensuing proteinprotein interactions between NP and PB1 and/or PB2. Also, host cell proteins may influence RNP formation, as the cellular polypeptide BAT1/UAP56 has been proposed to act as a chaperone for non-RNA-bound NP (Momose et al., 2001
). NP may also act as a processivity factor for the polymerase, as RNPs which have been stripped of NP by treatment with high concentrations of CsCl retain apparently normal transcription initiation activities but are unable to synthesize long RNA products (Honda et al., 1988
). This could potentially reflect NPP protein interactions.
The function of NP during RNA synthesis which has received the most attention is its potential role in the switch from mRNA transcription to genome replication. The form of RNP packaged into virions only synthesizes mRNA in vitro (Skorko et al., 1991 ; Seong et al., 1992
) and, although input vRNA templates are transcribed into cRNA after infection of cells, an initial round of mRNA transcription and subsequent protein expression is essential (Hay et al., 1977
). While it is possible that host cell polypeptides are necessary, and some evidence has been presented to support this hypothesis (Shimizu et al., 1994
; Momose et al., 1996
), multiple lines of genetic and biochemical evidence implicate NP as a major factor. Several NP ts mutants have been isolated that are defective for replicative transcription at the non-permissive temperature (Krug et al., 1975
; Scholtissek, 1978
; Mahy et al., 1981
; Thierry & Danos, 1982
; Markushin & Ghendon, 1984
). Moreover, infected cell extracts that synthesize cRNA in vitro depend on a supply of non-RNP-associated NP for readthrough of the polyadenylation signal to produce a full-length copy of the vRNA template (Beaton & Krug, 1986
; Shapiro & Krug, 1988
). Similarly, in vitro synthesis of full-length vRNA was found to be dependent on a pool of soluble NP (Shapiro & Krug, 1988
). Moreover, nuclear extracts from cells infected with the NP mutant A/WSN/33 ts56 virus synthesized m-, c-and vRNA in vitro at the permissive temperature but only mRNA at the non-permissive temperature (Shapiro & Krug, 1988
).
Thus, NP is evidently essential for replicative transcription. However, its mode of action and the precise steps during genome replication which require it remain uncertain. Although the nuclear extract system developed by the Krug laboratory demonstrated the necessity of NP for readthrough of the polyadenylation signal, technical limitations imposed by the nature of the extracts prevented examination of the role of NP in transcription initiation. However, the mode of initiation and termination of positive-sense RNA synthesis is probably coupled in vivo, as Hay et al. (1982) observed that most full-length transcripts of the viral RNA templates were uncapped, while Shaw & Lamb (1984)
found that most polyadenylated vRNAs have host sequences at their 5' ends. In support of this, transcripts initiated in vitro with a capped primer are also polyadenylated, even in the presence of non-nucleocapsid NP (Beaton & Krug, 1986
). Accordingly, several hypotheses have been proposed for the role of NP in the switch between mRNA and cRNA synthesis (Fig. 2b
). The encapsidation hypothesis proposes that NP does not have a regulatory function as such but is merely an essential co-factor. In this hypothesis, other factors alter polymerase activity to change the modes of transcription initiation and termination and NP is required to co-transcriptionally coat the nascent cRNA segments (Shapiro & Krug, 1988
). This hypothesis perhaps has a precedent in that the analogous process of genome replication in the non-segmented negative-sense RNA viruses is known to depend on co-transcriptional encapsidation of the nascent replicative intermediate by the N protein (Wagner & Rose, 1995
). Alternatively, the template modification hypothesis holds that the interaction of soluble (i.e. not already present in the RNP structure) NP with the template RNA alters its structure and, therefore, the modes of transcription initiation and termination (Hsu et al., 1987
; Fodor et al., 1994
; Klumpp et al., 1997
). This is plausible, since the terminal sequences of the vRNA template are partially base-paired to form a panhandle structure (Hsu et al., 1987
; Baudin et al., 1994
) and recognition of this structure by the polymerase is intimately connected with the mechanisms of mRNA transcription initiation (Tiley et al., 1994
; Hagen et al., 1994
; Cianci et al., 1995
) and polyadenylation (Pritlove et al., 1998
; Poon et al., 1998
). A third more recent hypothesis concerns the ability of NP to bind directly to PB1 and PB2: in this model NP alters the transcriptional function of the polymerase through direct proteinprotein contacts (Biswas et al., 1998
; Mena et al., 1999
). These hypotheses await definitive testing but circumstantial evidence supports two of the three proposed mechanisms. It has been shown that the ts lesions of two NP mutants defective for replicative transcription result in ts RNA-binding activity without apparent effects on NP oligomerization or interactions with the P proteins (Medcalf et al., 1999
). This indicates that an NPRNA interaction is necessary to support genome replication and is consistent with both the encapsidation and template modification hypotheses.
The regulation of vRNA synthesis may well differ from that of cRNA, as genetic evidence indicates that the roles of NP in c- and vRNA synthesis are mutationally separable (Thierry & Danos, 1982 ; Markushin & Ghendon, 1984
; Mena et al., 1999
). The role of NP in vRNA synthesis is perhaps simpler, as cRNA templates do not support cap-primed transcription or contain a polyadenylation signal (Cianci et al., 1995
). Certainly, the premature termination of in vitro vRNA transcription in the absence of soluble NP is consistent with the necessity of NP for co-transcriptional encapsidation of the nascent segment (Shapiro & Krug, 1988
).
RNP trafficking
Assembled RNPs must be transported in both directions across the nuclear envelope: incoming RNPs from the uncoating virion are imported at the start of the infectious cycle, while later, newly assembled RNPs are exported to be packaged into progeny virions (Fig. 4a). This temporal regulation of RNP trafficking is reflected in the distribution of their major protein component, NP. At early times post-infection, NP is found predominantly in the nucleus of infected cells but, at later times post-infection, substantial amounts accumulate in the cytoplasm (Breitenfeld & Schafer, 1957
; Maeno & Kilbourne, 1970
). Depending on cell type and, possibly, virus strain, this can take the form of an almost complete reversal of NP distribution, with the nucleus apparently emptying (Fig. 4b
). The dramatic change in NP localization must reflect a regulated process and much evidence exists to suggest the involvement of several viral and host polypeptides. Nuclear import of NP and NPRNA complexes results from the interaction of NP with host cell importin
(ONeill et al., 1995
; Wang et al., 1997
). This trafficking event certainly operates during the early period of the infectious cycle to direct nuclear import of the infecting RNPs (Fig. 4a
, i) and of newly synthesized NP (Fig. 4a
, ii) to support the process of genome replication. However, later in infection, RNP export becomes dominant, although it is not clear whether nuclear import of NP is turned off or just overridden by the nuclear export process. In support of the former hypothesis, exported RNPs do not apparently shuttle back into the nucleus, perhaps because of interactions with M1 (Martin & Helenius, 1991
; Whittaker et al., 1996
).
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Future prospects |
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Acknowledgments |
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References |
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