(Received for publication, June 27, 1995; and in revised form, July 31, 1995)
From the
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 °C it docks at the nuclear envelope
only in the presence of the heterodimeric karyopherin (either
karyopherin 1
or karyopherin
2
), and docking is
competitively inhibited by an unlabeled NLS containing substrate. Like
other NLS-containing proteins, at 20 °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)()-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 (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, 17, 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. ()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) .
Using digitonin-permeabilized
mammalian cells and an NLS-containing protein, a number of transport
factors required for import into nuclei have been isolated from
cytosol. A heterodimeric protein complex, termed
karyopherin(22) , is required for targeting an NLS substrate to
NPCs, and two proteins, the GTPase Ran (23, 24) and
p10(25) , are required for transport into the nucleus. Either
karyopherin 1 (20) (corresponding to NPI-1/hSRP-1/importin
60(19, 26, 27) ) or karyopherin
2 (21) (corresponding to
NPI-3/Rch-1/hSRP
1(28, 29) ) serves as NLS binding
subunit (20, 21, 29) whereas karyopherin
(or importin 90(30) ) serves as an adapter subunit that
mediates binding to peptide repeat-containing
nucleoporins(21, 31) .
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 prepared 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.
Figure 1:
Docking
and import of NP requires transport factors. For docking,
digitonin-permeabilized BRL cells were incubated for 30 min on ice with
FITC-labeled NP (200 ng/assay) in transport buffer only (panela) or in the presence of either 100 nM karyopherin 1 + 100 nM karyopherin
(panelb) or 100 nM karyopherin
2
+ 100 nM karyopherin
(panelc).
For import, digitonin-permeabilized BRL cells were incubated for 30 min
at room temperature with FITC-labeled NP in transport buffer only (paneld) or in the presence of either 100 nM each of karyopherin
1 +
, 100 µg/ml Ran, and 3
µg/ml p10 (panele), or 100 nM each of
karyopherin
2 +
, 100 µg/ml Ran, and 3 µg/ml p10 (panelf). Bar, 10
µm.
Figure 2:
Docking of vRNA at the nuclear envelope is
mediated by NP and requires karyopherin 1
or
2
heterodimers. Digitonin-permeabilized cells were incubated for 30 min
on ice with either vRNA* (200 ng/assay) alone (panelsa and c) or vRNA* + NP (200 ng/assay each) (panelsb and d) in the presence of 100 nM each
of karyopherin
1 +
(panelsa and b) or 100 nM each of karyopherin
2 +
(panelsc and d). Bar, 10
µm.
The NP of influenza A/PR/8/34 virus was
subcloned between the EcoRI and XhoI restriction
sites of pET28a(+) (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 µg/ml) was performed at
room temperature in 0.1 M NaCO
, pH
9.0, 50 mM NaCl, and 100 µg/ml FITC. FITC-NP was dialyzed
against 20 mM Hepes, pH 7.3, 100 mM KOAc.
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.
Figure 3:
Import of vRNA is mediated by NP.
Digitonin-permeabilized BRL cells were incubated for 30 min at room
temperature with either vRNA* (200 ng/assay) alone (panela) or vRNA* + NP (panelb) at 200
ng/assay each in the presence of 100 nM each of karyopherin
1 +
, 100 µg/ml Ran, and 3 µg/ml p10. Bar, 10 µm.
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 digitonin-permeabilized cells in the presence of the karyopherin heterodimer, Ran and p10.
We investigated the time course of import of NP* (Fig. 4A) or vRNA* + NP (Fig. 4B). In both cases import proceeded with similar kinetics with maximal import at about 20 min.
Figure 4:
Time course of import of NP and viral
RNA*-NP into nuclei of digitonin-permeabilized cells. Import into
digitonin-permeabilized cells was done for the times indicated in the
presence of 100 nM each of karyopherin 1 +
,
100 µg/ml Ran, and 3 µg/ml p10. 200 ng/assay NP* (A)
or 200 ng/assay RNA* + 200 ng/assay NP (B) were used.
Quantification was as described(32) . 100% corresponds to the
maximum of the observed mean nuclear
fluorescence.
The concentrations of vRNA (200 ng/assay) and NP (200 ng/assay) that were used in the experiments shown in Fig. 2Fig. 3Fig. 4amounted 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 Fig. 2Fig. 3Fig. 4was 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 µg 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.
Figure 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.
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 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.