Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import.

We have fluorescently labeled one of the eight genomic segments of influenza virus RNA and a recombinant influenza viral protein, the nucleoprotein (NP), to investigate the requirement for their uptake into nuclei of digitonin-permeabilized cells. We found that the influenza viral NP behaves like a nuclear localization sequence (NLS) containing protein. Thus, at 0 degrees C it docks at the nuclear envelope only in the presence of the heterodimeric karyopherin (either karyopherin alpha 1 beta or karyopherin alpha 2 beta), and docking is competitively inhibited by an unlabeled NLS containing substrate. Like other NLS-containing proteins, at 20 degrees C NP is imported into the nucleus after further addition of the GTPase Ran and of p10. In contrast, the fluorescently labeled, 890-nucleotide-long viral RNA segment does not dock to the nuclear envelope or enter the nucleus either in the presence of exogenous cytosol or of karyopherin heterodimer, Ran, and p10. However, in the presence of NP the RNA is able to dock and enter the nucleus with transport requirements indistinguishable from those for docking and entry of NP. These data indicate that uptake of the influenza virus RNA segment is not via a signal in the RNA but via an NLS of a viral protein such as NP.

The mechanism of entry of ribonucleoproteins into the nucleus is not known. Microinjection studies have suggested that import of some of the U small nuclear RNAs occurs by two biochemically distinct pathways that do not compete with the nuclear localization sequence (NLS) 1 -mediated route for nuclear import of proteins (1). However, all three pathways use the nuclear pore complex (NPC) as antibodies to some nucleoporins (a collective term for NPC proteins) or wheat germ agglutinin (some nucleoporins contain N-acetylglucosamine residues) block import of proteins as well as of RNPs (1) (for review see Ref. 2).
The genome of influenza virus is transcribed and replicated in the nucleus (3,4). The entry of isolated influenza viral RNPs into the nucleus has been explored by microinjection and also been shown to proceed via the NPC (5,6). However, it is not known by which pathway the viral RNP enters.
The influenza virus particle contains eight different negative strand RNAs varying in length from approximately 900 to 2,500 nucleotides (7). Each of the RNA molecules is coated by multiple copies of viral nucleoprotein (NP) at one NP per 20 nucleotides (8). A heterotrimeric RNA polymerase complex is located at one end of each of the viral RNAs (9). The viral matrix protein, M1, forms an inner shell around the RNPs and is bounded by a membrane containing the integral membrane proteins M2, hemagglutinin, and neuraminidase (10,11). Virus entry into the cell is via hemagglutinin-mediated membrane fusion in a late endosomal compartment (12,13). The M2 functions as an acid-activated channel for monovalent cations (14,15) and appears to be required to divest the multimeric RNP complex of its M1, yielding individual viral RNPs. It is these RNPs that enter the nucleus both after infection (5) and after microinjection (6). Nuclear uptake is an ATP-and temperature-sensitive process (5,6).
As each of the four viral RNA-associated proteins in the microinjected RNPs has been reported to contain an NLS (16 -18), nuclear import might proceed via an NLS-mediated pathway. Consistent with this idea is the finding that in a yeast two-hybrid system, NP interacted with two proteins, termed NPI-1 (19) and NPI-3. 2 These two proteins were subsequently shown to be the transport factors karyopherin ␣1 and karyopherin ␣2, respectively, that have been demonstrated to bind to NLS-containing proteins (20,21).
To address the question of whether influenza viral RNA can enter the nucleus by itself or whether its entry is mediated by an associated NLS-containing protein, such as NP, we pre-* This work was made possible in part by funds granted through a fellowship program sponsored by the Charles H. Revson Foundation (to R. E. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  pared unlabeled or fluorescently labeled components, either RNA (NS segment of influenza A/WSN/33 virus) or NP. These components were then incubated with digitonin-permeabilized cells in reactions assaying docking to the nuclear envelope and uptake into the nucleus using all recombinant transport factors. We found that NP by itself could be docked and imported into the nucleus in the presence of transport factors but that RNA by itself could not. However, RNA could be docked and imported in the presence of NP, demonstrating that viral RNA import into nuclei is by an NLS-mediated pathway.

EXPERIMENTAL PROCEDURES
Assays for Nuclear Docking and Import-Bufallo rat liver (BRL) cells were grown on coverslips and were extracted with digitonin (50 g/ml for 5 min at room temperature) as described previously (20,21). Docking of various fluorescently labeled substrates to the nuclear envelope of the digitonin-permeabilized cells was assayed by incubation at 0°C in the presence of components indicated in the legends to Figs. 1 and 2 and was monitored by fluorescence microscopy (32). In contrast, import was assayed at room temperature (22°C) and the fluorescent signal quantitated as described previously (32).
The NP of influenza A/PR/8/34 virus was subcloned between the EcoRI and XhoI restriction sites of pET28a(ϩ) (Novagen, Inc.) for expression of a His 6-tagged recombinant protein. Bacterially expressed NP (containing 37 additional N-terminal residues and 6 N-terminal His) was purified by nickel-nitrilotriacetic acid affinity chromatography. Fluorescein isothiocyanate (FITC) labeling of NP (260 g/ml) was performed at room temperature in 0.1 M Na 2 CO 3 , pH 9.0, 50 mM NaCl, and 100 g/ml FITC. FITC-NP was dialyzed against 20 mM Hepes, pH 7.3, 100 mM KOAc.

Import of Recombinant Protein NP of Influenza Virus into Nuclei of Digitonin-permeabilized Cells Requires Transport
Factors-It was reported that influenza virus NP could enter into and accumulate in nuclei after microinjection into oocytes of NP itself or of the corresponding mRNA or cDNA (17). The sequence of NP that functions as an NLS to allow entry into the nucleus has not yet been mapped although a region encoding amino acids 327-345 has been reported to function in nuclear retention (17). To determine whether NP behaves like an NLScontaining substrate and is transported into nuclei of digitoninpermeabilized cells in a transport factor-dependent fashion, we prepared recombinant fluorescently labeled NP (NP*). As previously reported for a model import substrate (NLS conjugated to human serum albumin (NLS-HSA)) there was docking of NP* to the nuclear envelope only in the presence of karyo-pherin ␣1 and ␤ (Fig. 1b) or karyopherin ␣2 and ␤ (Fig. 1c). Neither buffer alone (Fig. 1a) nor karyopherin ␣ alone (not shown) nor karyopherin ␤ alone (not shown) yielded docking. Docking could be competitively inhibited by NLS-HSA (not shown), indicating that recombinant NP* contains a functional NLS that can be recognized and docked by either karyopherin heterodimer, ␣1␤ or ␣2␤. Moreover, as previously reported for NLS-HSA (20,21), NP* can be imported into the nucleus in the presence of either one of the two karyopherin heterodimers and of Ran and p10 (Fig. 1, e and f).
Nuclear Import of a Fluorescently Labeled Influenza Virus RNA Occurs in the Presence of Viral NP-We fluorescently labeled an 890-nucleotide negative-sense RNA of the NS segment of influenza virus (see "Experimental Procedures") and incubated the labeled viral RNA (vRNA*) with digitonin-permeabilized cells in the presence of Xenopus cytosol or of Xenopus cytosol fraction A (32) (not shown) or of karyopherin ␣1 and ␤ (Fig. 2a) or karyopherin ␣2 and ␤ (Fig. 2c). Neither of these incubations yielded docking of the vRNA* at the nuclear envelope. However, when NP (not fluorescently labeled) was included in the docking reaction there was docking of the vRNA* at the nuclear envelope with either karyopherin ␣1␤ (Fig. 2b) or karyopherin ␣2␤ (Fig. 2d) and no docking when either ␣1 and ␣2 or ␤ were omitted (not shown). These results indicated that viral RNA by itself was unable to dock at the nuclear envelope and that docking occurred only in the presence of NP in an NLS-and karyopherin-mediated fashion.
Likewise, there was no import of the vRNA* alone in the presence of the karyopherin heterodimer, Ran and p10 (Fig.  3a). However, there was import when NP was present in the reaction (Fig. 3b). Import was competitively inhibited by NLS-HSA (data not shown). Thus, we conclude that import of vRNA* into nuclei of digitonin-permeabilized cells could be mediated by NP in an NLS-dependent pathway using all the transport factors that are required for import of an NLS-containing protein.
We have also tested whether influenza virus NP can mediate nuclear import of non-viral RNA. A 790-nucleotide-long, fluorescently labeled NPI-3 RNA by itself was not transported into the nucleus, but it was when NP was included into the assay (data not shown). Thus, NP can also bind to a non-virus-derived RNA and mediate its transport into the nucleus of digitoninpermeabilized cells in the presence of the karyopherin Nuclear Import of Viral RNA 22702 heterodimer, Ran and p10.
We investigated the time course of import of NP* (Fig. 4A) or vRNA* ϩ NP (Fig. 4B). In both cases import proceeded with similar kinetics with maximal import at about 20 min.
The concentrations of vRNA (200 ng/assay) and NP (200 ng/assay) that were used in the experiments shown in Figs. 2-4 amounted to a molar ratio of 4.8 NP/vRNA, i.e. far below the molar ratio of 45 NP/vRNA if the vRNA were fully coated by NP (8). To determine the effect of NP concentrations on the rate of vRNA* import, we varied the concentration of NP at a constant concentration of vRNA* (200 ng/assay). We found maximal import at NP concentrations of 100 -200 ng/assay (Fig. 5), i.e. the import of vRNA* in Figs. 2-4 was indeed at maximal rates. However, we do not know the total amount of vRNA*-NP that was imported nor what the actual molar NP/vRNA* ratio of the imported vRNA*-NP was. Interestingly, at very high molar NP/vRNA* ratios there was inhibition of vRNA*-NP import. For example at 10 g of NP and 200 ng of vRNA*/assay amounting to an NP/vRNA* ratio of 240 the rate of import of vRNA*-NP dropped to 70% of maximum (data not shown), probably as a result of competition between NP and vRNA*-NP. DISCUSSION Our data here suggest that influenza virus RNA does not contain its own ("cis-acting") signal for nuclear import but that import is via RNA-associated protein(s) containing an NLS and therefore requires all the transport factors (karyopherin, Ran, and p10) obligatory for uptake of NLS-containing proteins into nuclei of digitonin-permeabilized cells. There are potentially four such NLS-containing proteins associated with the genomic viral RNA: the heterotrimeric RNA polymerase complex and the NP (see the Introduction). It is therefore conceivable that the heterotrimeric RNA polymerase complex might mediate entry of the influenza virus RNA as well.
Unlike specific viral RNA binding proteins (35)(36)(37) NP can bind to any RNA provided that it is longer than 15 nucleotides (38). NP binding is to the sugar phosphate backbone leaving the bases exposed and melting the secondary structure of the RNA (39). These binding properties explain NP's ability to mediate import into the nuclei of digitonin-permeabilized cells even of a non-viral RNA. Whether these promiscuous binding properties of NP could affect synthesis, transport, function, and metabolism of endogenous RNAs of virally infected cells is not known. It should be kept in mind, however, that there are numerous cellular RNA-binding proteins with which NP would have to compete in the infected cell.
The binding site of NP for RNA has been mapped to amino acid residues 91-188 (40). The recombinant NP that we tested Import into digitonin-permeabilized cells was done for the times indicated in the presence of 100 nM each of karyopherin ␣1 ϩ ␤, 100 g/ml Ran, and 3 g/ml p10. 200 ng/assay NP* (A) or 200 ng/assay RNA* ϩ 200 ng/assay NP (B) were used. Quantification was as described (32). 100% corresponds to the maximum of the observed mean nuclear fluorescence.
FIG. 5. Import of RNA* in the presence of increasing concentrations of NP. Digitonin-permeabilized cells were incubated for 30 min at room temperature with 200 ng of RNA/assay in the presence of increasing concentrations of viral NP. Quantification was as described (32). 100% corresponds to the maximum of the observed mean nuclear fluorescence.
Nuclear Import of Viral RNA 22703 here contained an additional 37 N-terminal residues and a C-terminal His tag. Neither of these two NP modifications seemed to interfere with nuclear import of NP* or with the association of NP with RNA as RNA* could be imported in an NP-dependent fashion. It remains to be seen whether nuclear entry of nucleic acids via specifically or nonspecifically associated proteins and an NLS pathway is unique to genomic influenza viral RNAs or whether it is common to other viral or cellular nucleic acids. DNA viruses such as herpesvirus or adenovirus have been reported to dock at the NPC and are thought to inject their DNA across the NPC into the nucleus (41)(42)(43). Based on our studies it is more likely that associated, NLS-containing proteins mediate nuclear entry of the viral genomes of the DNA viruses as well. Lentiviruses, of which HIV-1 is a member, are unique among retroviruses in that they can infect non-dividing cells. The HIV-1 matrix protein contains an NLS that is required for this function (44,45). It is likely that the matrix protein functions in importing the HIV-1 preintegration complex into the nucleus.