Intracellular localization of influenza virus NP
Budding of influenza virus requires the assembly of all virion components at the apical cell surface. Although the viral membrane proteins contain targeting signals that direct them to the apical plasma membrane, it is unclear how the internal virion components reach this surface at late time points during infection. With respect to RNPs, an immunoelectron microscopy study showed a general distribution of NP throughout the cytoplasm and a low accumulation of it at the apical plasma membrane (31). In contrast, a recent study employed confocal microscopy to show NP apparently localised specifically at the apical plasma membrane (27). As an initial experiment therefore, we examined the distribution of NP and HA in virus A/PR/8/34 (PR8)-infected cells at late time points, using Madin–Darby canine kidney (MDCK) cells as a model polarised epithelial cell, baby hamster kidney 21 (BHK) cells as a source of nonpolarised fibroblasts, and human embyronic kidney cells (293-T) as a cell type with an intermediate epithelioid phenotype. All three cell types are fully permissive for influenza virus replication and produce infectious progeny (e.g. 13,22,32).
Immunofluorescent staining of HA on infected MDCK cells fixed at 8 h p.i. showed the expected pattern of distribution. In z-axis cross-sections taken through the depth of the cells, HA was present only on apical and not on basolateral surfaces (Figure 1a, panel iii). Single optical sections taken in the xy-plane through small clumps of cells showed predominant labelling of the exterior (i.e. apical) surface of the cell clumps, with weaker labelling of internal lateral membranes (Figure 1a, panel iii). This latter localization pattern might be due to some missorting of HA to lateral membranes, or possibly to visualization of HA at areas of apical membrane above tight junctions. The staining pattern observed for NP was similar to that of HA in that, in xy-sections, the protein was found predominantly at the exterior of the cell clump, while z-sections showed its association with the apical surface (Figure 1a, panel i). In contrast to HA, no NP was seen to be associated with lateral membranes. Cells similarly infected and double-stained for NP and DNA (to delineate the nucleus) confirmed that the majority of NP had exited the nucleus and, furthermore, clearly showed that NP did not localise to all areas of the cytoplasm but instead was specifically found in areas adjacent to the apical plasma membrane (Figure 1a, panel iv). This pattern was maintained when cells were grown to confluency on porous filters to promote full differentiation of apical and basolateral surfaces (Figure 1d, panel i). Identical results were obtained with the human polarised epithelial CACO-2 cell line, either as confluent monolayers on permeable filters or as subconfluent cells on solid supports (data not shown). Examination of infected 293-T cells at 8 h p.i. showed a very similar localization pattern for NP in which although the protein had largely exited the nucleus, it showed a marked accumulation at apical cell surfaces rather than being distributed throughout the cytoplasm, either in subconfluent (Figure 1b, panels i and iv) or confluent cells (Figure 1d, panel ii). However, in 293-T cells, HA did not show a marked preference for the apical membrane but instead was found on all surfaces of the PM (Figure 1b, panel iii). Similarly, when infected BHK fibroblasts were tested, HA distributed all over the PM (Figure 1c, panel iii). In many cells, NP was distributed diffusely throughout the cytoplasm (data not shown). However, in approximately 50% of cells, NP associated preferentially to areas of cytoplasm adjacent to the plasma membrane (Figure 1c, panel i). Furthermore, this juxta-membrane positioning of NP was polar, with little NP associated with the basal plasma membrane (Figure 1c, panel i). Thus at late times postinfection, NP localises specifically to regions of the cytoplasm adjacent to apical (or active) membrane surfaces in several different cell types. Furthermore, in confirmation of the results of Mora et al. (27), this behaviour of NP does not obligatorily correlate with HA localization. This apical localization pattern is unlikely to be a staining artifact resulting from incomplete permeabilization of the cells because it was also seen with methanol permeabilised cells and with the histological technique of applying antibody to cut sections of fixed cells (data not shown).
Figure 1. Cellular distribution of NP and HA at late times postinfection. Virus-infected MDCK (a, d, i), 293-T (b, d, ii) or BHK (c) cells were fixed at 8 h p.i. and double-stained for NP and HA, NP and DNA (with propidium iodide) as labelled, or (d), NP (green) and LAP-2 (red). Cells in (d) were grown to confluency on (i) porous supports until tight junctions had formed as assessed by measurement of the trans-epithelial electrical resistance or (ii) glass coverslips. Single optical sections in the xy plane (upper panels) and xz planes (lower panels) are shown for individual or merged fluorophores as labelled. Scale bar = 5 μm.
Since NP localises to the apical plasma membrane of cells apparently independently of HA, we went on to examine the localization of other internal virion components known to be involved in RNP trafficking. Experiments were carried out on subconfluent clumps of cells because this permitted visualization of apical targeting in the xy plane, where the microscope has greater resolving power. In addition, it permitted direct comparison between infection and transfection (see later) as the latter technique requires subconfluent cells for reasonable efficiency. In MDCK cells at 8 h p.i., the M1 polypeptide was found primarily at apical plasma membrane surfaces in a very similar localization pattern to that of NP and the two antigens showed strong colocalization (Figure 2a). Similar results were obtained when infected 293-T cells were examined except that a slightly larger proportion of M1 was detectable in the nucleus (data not shown). However, in BHK fibroblasts, M1 showed little tendency to accumulate at the plasma membrane and was generally found throughout the cytoplasm even in cells where NP had localised preferentially to the upper surface of the cell membrane (Figure 2b). When MDCK cells were stained for NS2/NEP the protein was found throughout both nucleus and cytoplasm, showing no preference for any membrane surface or any polarity in z-axis projections (Figure 2c, panel iii). This behaviour was in contrast to NP, which as before localised preferentially at the apical plasma membrane, where it colocalised with a minor fraction of NS2/NEP (Figure 2c, panels i and ii). A similar pattern was found in infected 293-T cells (Figure 2d) and BHK cells (data not shown) in which NS2/NEP localised throughout the cell only showing limited colocalization with NP present at the apical plasma membrane. Therefore the marked accumulation of NP at the apical surface is not matched by similar specific trafficking of the NS2/NEP polypeptide, or, in BHK cells, the M1 protein.
Figure 2. Cellular distribution of NP, M1 and NEP at late times postinfection. Virus infected MDCK (a c), 293-T (d) or BHK (b) cells were fixed at 8 h p.i. and double-stained for NP and M1 or NP and NEP as labelled. Single optical sections in the xy plane (upper panels) and xz planes (lower panels) are shown for individual or merged fluorophores as labelled. Scale bar = 5 μm.
Nevertheless, a proportion of the M1 and NS2/NEP polypeptides were found to colocalise with NP at the apical PM (Figure 2). Potentially this could reflect a ternary complex which, once assembled, contains the necessary signals for apical targeting. Alternatively, it is possible that NP itself contains a signal that directs its specific accumulation at this surface independently of other viral polypeptides. To test this latter hypothesis, we examined the intracellular localization of exogenously expressed NP. When expressed in the absence of other viral proteins, NP shuttles between nucleus and cytoplasm and depending on expression levels and the cellular environment can appear resident in either compartment when examined by immunofluorescence (22,33,34). Accordingly, 293-T cells were transfected with a high dose of a plasmid (pCDNA-NP) containing the NP gene under the control of an RNA polII promoter and incubated for 48 h before fixation and immunofluorescent analysis. As expected (22,34) under these conditions of high-level NP expression the majority of transfected cells contained cytoplasmic NP. The high transfection efficiency obtainable with 293-T cells resulted in the formation of many contiguous clumps of NP-expressing cells. Similar to the pattern observed in virus-infected cells, NP in these transfected cells localised predominantly to areas of cytoplasm adjacent to the edges of the cell sheet (Figure 3a). Z-axis reconstructions confirmed that this did indeed result from preferential localization of NP to the apical surface of the cells (Figure 3a). The poor transfection efficiency of MDCK cells meant that large clumps of NP-expressing cells could not be obtained. Nevertheless, when individual or pairs of NP expressing cells were examined, the protein showed preferential accumulation at the apical surface (data not shown). When a similar experiment was carried out in BHK fibroblasts, again the majority of transfected cells contained cytoplasmic NP. In many cases, this NP localised diffusely throughout the cytoplasm (data not shown). However, in approximately half the cells, single optical sections taken through the body of the cell showed marked peripheral accumulation of NP with clear regions of unstained cytoplasm between them and the nucleus (Figure 3b, panels i and ii). Z-axis reconstructions generated from several such optical planes of focus confirmed that this resulted from close association of NP with the upper but not basal membrane surfaces of the cell (Figure 3b, panel iii). Overall, therefore, NP localises to the apical cell periphery in the absence of other influenza virus proteins.
Figure 3. Cellular distribution of NP in transfected and infected cells.a) 293-T or b) BHK cells were transfected with plasmid pCDNA-NP and subsequently fixed and stained for NP (green) and (a) DNA with propidium iodide or (b) Nup 62 (red). c) Virus infected MDCK cells were fixed at 8 h p.i. and double- stained for NP and the PB2 subunit of the viral polymerase. Single optical sections in the xy plane (large panels) and xz planes (small panels) are shown for individual or merged fluorophores as labelled. Scale bar = 5 μm.
Although NP is generally considered to be a useful marker for RNP localization (17,18), the finding that NP can traffick to the apical PM in the absence of other viral components raised the possibility that the NP seen there in virus-infected cells might not necessarily be in the form of RNPs. To test whether the apical NP staining did represent RNPs, we stained infected MDCK cells at 8 h p.i. for the PB2 component of the viral RNA-dependent RNA polymerase. The majority of PB2 remained in the nucleus (consistent with the existence of a pool of non-RNP-associated polymerase ), but significant antibody reactivity was also visible at the apical PM (Figure 3c, panel iii). Furthermore, this subpopulation of PB2 colocalised with NP also present at the membrane (Figure 3c, panels i, ii). Identical results were obtained when infected MDCK cells were double-stained for PB1 and NP and when the same experiments were performed in 293-T cells (data not shown). This suggests that the NP seen at the apical plasma membrane late in virus infection is at least in part in the form of RNP particles.
Membrane association of NP
Immunofluorescence analysis showed the specific accumulation of NP at the apical plasma membrane both during virus infection and when expressed alone. To provide a biochemical test of the association of NP with the plasma membrane, these findings were further investigated by sucrose gradient flotation analysis of cellular membrane fractions. Infected 293-T cells were separated into nuclear and cytoplasmic fractions at 8 h p.i. Cytoplasmic membrane-associated proteins were then separated by sucrose gradient flotation assay and analysed by Western blotting for viral polypeptides. The proportion of membrane-associated protein (defined here as the top four fractions) was quantified by densitometry of the Western blots. These analyses revealed the presence of HA, M1 and NP in low buoyant density membrane-associated fractions (Figure 4a,b). Densitometric quantification of the amount of HA present in the top four and bottom six fractions showed that around half of the total protein was present in the form of low buoyant density material (Figure 4e). This was in agreement with earlier studies, which also observed the presence of this protein in both nonbuoyant and membrane associated fractions by flotation analysis (36,37). Likewise, a substantial fraction of M1 (50%) was found in membrane-associated fractions, although in a slightly lower proportion to that reported previously (∼ 70%) (37,38). Most NP protein remained at the bottom of the gradients, but a small but reproducible proportion (14 ± 0%) was found in membrane-associated fractions (Figure 4a,e). This finding suggests the association of some NP with membranes and is in agreement with earlier studies that also detected NP in low buoyant density fractions after flotation assay of virus-infected cells (36,37). Virus specific polypeptides were not detected when extracts from mock-infected cells were subjected to similar analysis (data not shown). This observed association of NP with membranes might be due its interaction with any of the influenza virus membrane-associated proteins, such as HA, NA, or M1. Therefore, to determine whether NP associates with membranes in the absence of other influenza virus proteins, 293-T cells were transfected or mock-transfected with plasmid pCDNA-NP and analysed by flotation assay at 48 h post transfection. No NP was detected in gradient fractions from untransfected cells (Figure 4d). Analysis of transfected cells, however, again showed the presence of NP in membrane-associated fractions (Figure 4c). Although the majority of NP was found at the bottom of the sucrose gradients, a similar proportion to that found in infected cells (12 ± 4%) floated to the top fractions (Figures 4c,e), indicating that NP is able to associate with membranes even in the absence of other viral proteins.
Figure 4. Membrane flotation analysis of infected and transfected cells. Postnuclear supernatants prepared from 293-T cells (a b) infected with virus for 8 h (c) transfected with plasmid pCDNA-NP or (d) mock transfected were separated by sucrose gradient flotation and fractions analysed by SDS-PAGE and Western blotting with anti-PR8 virus (a, b) or anti-RNP serum (c, d). Arrows mark the indicated polypeptides. e) The proportions of HA and NP from infected or transfected (tNP) cells present in low density fractions (top four) from untreated cells (– TX), cells treated with TX-100 (+ TX) or TX-100 and the indicated concentrations (mm) of MBCD were determined by densitometry of the Western blots and plotted as a percentage of the total protein. The mean and range of duplicate experiments is shown except for the + TX samples, where the mean and standard error from five experiments is shown.
Several studies have shown that the influenza virus glycoproteins associate with lipid raft domains in the plasma membrane and that these domains are incorporated into budding virus particles (9–14,16,39). We therefore tested whether NP also associated with detergent-resistant membrane domains. First, 293-T cells were infected with virus and at 8 h p.i., treated with the nonionic detergent Triton X-100 (TX-100) on ice. Proteins associated with TX-100-insoluble lipid rafts were isolated by sucrose gradient centrifugation and analysed by Western blotting as before. The proportion of each protein present in low buoyant density fractions (again defined as the top four fractions) was quantified by densitometry (Figure 4e). HA and M1 were recovered in both low buoyant density (raft) and nonraft fractions (Figure 5a, panels i and iii), consistent with previously published results (11–14,37). When the proportion of each polypeptide that partitioned into low buoyant density fractions was quantified, one third of the HA was found to be raft associated (Figure 4e). This is comparable to but slightly lower than previously published values (43–49% [12,13]). In the case of M1, 13 ± 5% (n = 5) was found to be raft associated. This is substantially lower than the 60% value found by Zhang et al. (12). However, the study of Ali et al. (37) showed that the fraction of M1 associated with detergent-insoluble lipid domains during flotation analysis is sensitive to small changes in TX-100 concentration, providing a possible methodological explanation for our findings. When NP was examined, the majority of it remained at the bottom of the gradient, but a reproducible and meaningful proportion (on average, 16%) floated with the detergent-insoluble membrane fractions similarly to HA and M1 (Figure 5a, panel ii; quantification data in Figure 4e). However, this behaviour was not a universal feature of influenza virus polypeptides as the NS1 polypeptide showed no tendency to associate with low buoyant density material (Figure 5a, panel iv). Thus, consistent with previous observations (12,14,37) we found that some NP associates with lipid rafts in the context of virus infection. We therefore went on to test whether this was an intrinsic property of the polypeptide that did not require other viral components. 293-T cells were transfected with plasmid pCDNA-NP, treated on ice with TX-100 at 48 h post transfection, and analysed as above. As with previous flotation experiments, a high proportion of NP remained in the high density fractions at the bottom of the gradient, but the same amount as in infected cells (16%) floated to the low buoyant density fractions (Figure 5b, panel ii; quantification data in Figure 4e). This could not be ascribed to a failure of the lipid raft extraction procedure as the cellular clathrin heavy chain protein, a membrane-associated polypeptide that does not interact with lipid rafts (40,41), remained wholly in the detergent-soluble fractions (Figure 5b, panel i). In addition, flotation of NP was abolished if cells were detergent extracted at room temperature (data not shown), a condition expected to solubilise lipid rafts (42). This suggests that NP associates with lipid rafts in the absence of other viral polypeptides.
Figure 5. Lipid raft flotation analysis of infected and tran- sfected cells. Infected (a) or pCDNA-NP transfected (b) cells were lysed on ice with TX-100, separated by sucrose gradient flotation and the resulting fractions analysed by SDS-PAGE and Western blotting with anti-sera to the indicated poly-peptides. Where indicated, the cells were first depleted of cholesterol by treatment with MBCD as described in the text.
Effect of cholesterol depletion on NP trafficking
Cholesterol is a known structural component of lipid rafts. If it is removed, disorganization of these microdomains and dissociation of proteins bound to the rafts ensues (9,43,44). Therefore, the association of NP with lipid rafts was evaluated after treatment with cholesterol depleting agents. For this, 293-T cells were grown in the presence of mevastatin and mevalonic acid lactone (to inhibit the cholesterol biosynthesis pathway) and treated with either low or high doses of the cholesterol-extracting agent methyl-β-cyclodextrin (MBCD) for long or short times, respectively (45). In addition, the cells were either infected with virus or transfected with plasmid pCDNA-NP before lipid rafts and associated proteins were isolated by gradient centrifugation, analysed by Western blotting and quantified by densitometry as described above. When cells were treated with 3.5 mm MBCD from 1 h p.i and harvested at 8 h p.i. the proportion of HA associated with low buoyant density material decreased by approximately two-thirds (Figure 4e). Similarly, when cells were treated with 30 mm MBCD for 40 min prior to harvest, less than 10% of the HA remained raft associated (Figure 4e). Metabolic labelling experiments showed that the drug treatments did not significantly affect synthesis of the NP, M1/NS1 and NEP polypeptides, although HA synthesis was reduced (Figure 6a). Western blotting experiments confirmed normal accumulation of NP (Figure 6b) and M1 after cholesterol depletion (data not shown). However, when the cells were fractionated into soluble cytoplasmic and insoluble/nuclear fractions, the amount of soluble NP was significantly reduced by both drug treatment regimens (Figure 6b, compare lanes 2,4 and 6). Furthermore, when this soluble NP was subjected to flotation analysis, a much lower proportion (2% with both high and low MBCD concentrations) was associated with low buoyant density fractions (Figure 5a, panels v,vi; quantification data in Figure 4e) compared to that from untreated cells. When the parallel experiment was carried out on cells transfected with an NP-expressing plasmid, a similar decrease in the levels of soluble NP was seen (data not shown). Again, the proportion of low buoyant density NP in the soluble fraction decreased to 3% in cells treated with 3.5 mm MBCD for 7 h prior to harvesting (Figures 4e and 5b, panel iii), and to undetectable levels in cells treated with 30 mm MBCD for 40 min (Figures 4e and 5b, panel iv). Thus NP shows a cholesterol-dependent association with detergent-resistant membrane fractions that does not require the presence of other influenza virus proteins.
Figure 6. Analysis of viral protein synthesis in infected cells with and without cholesterol depletion.a) Lysates from 293-T cells infected with PR8 or mock (M)-infected and pulse labelled with 35S-methionine for 2-h periods ending at the indicated times p.i. were separated by SDS-PAGE and detected by autoradiography. Arrows denote abundant viral polypeptides. b) Aliquots of infected 293-T cells taken at 8 h p.i. and treated with TX-100 and the indicated concentrations of MBCD were analysed by SDS-PAGE and Western blotting with anti-NP either before (T) or after subcellular fractionation into soluble cytoplasmic (S) fractions.
In addition to the effect of cholesterol depletion on the solubility of raft-associated proteins, previous studies have shown that extraction of cholesterol causes missorting of apically targeted membrane proteins, including the influenza virus HA, possibly due to the disruption of lipid rafts (45). We therefore determined the effect of cholesterol-depleting agents on the cellular distribution of NP and HA. 293-T cells were grown in the presence of mevastatin and mevalonic acid lactone, infected with influenza virus and treated with MBCD as above. Untreated cells were used as a control. Cells were fixed at 8 h p.i., double-stained for NP and HA, and analysed by confocal microscopy. In the absence of drug treatment, HA localised to all plasma membrane surfaces, while NP was found concentrated at the apical surface (Figure 7a, panel i). Treatment with MBCD from 1 h p.i. resulted in the failure of HA to reach the plasma membrane in detectable amounts, instead localising to foci near the nuclei (Figure 7a, panel ii), presumably representing its retention in the Golgi apparatus (45). Furthermore, NP was no longer found only at the apical plasma membrane. Although it still showed a bias towards the apical sides of cells, its overall staining pattern was less polarised and more diffuse, including areas of cytoplasm not directly opposed to the apical membrane (Figure 7 a, panel ii, arrows). Extraction of cells with a higher dose of MBCD for 40 min prior to cell fixation had less effect on HA distribution with some cell surface staining still visible (Figure 7 a, panel iii). However, the tight association of NP with the apical plasma membrane was lost, with the protein again showing an increased tendency to localise throughout the cell, including areas of cytoplasm away from the apical cell surface (Figure 7a, panel iii, arrows). To provide a semiquantitative measure of NP distribution in treated and untreated cells, the fluorescence intensity across the width of several similarly sized clumps of cells was measured and the average plotted. The profile derived from untreated cells confirmed the predominant localization of NP towards the apical surface, with around 10-fold higher fluorescence intensities found at the exterior of the cell sheet than the interior (Figure 8). However, after cholesterol depletion (with either high or low concentrations of MBCD), markedly higher fluorescence intensities were observed in the interior of the cell sheet, and although a bias towards peripheral staining was still evident, the difference in average intensity between interior and exterior was now only around twofold (Figure 8). In part, the more diffuse localization of NP also appeared to result from increased nuclear staining. To test this, cells were double-stained for NP and nuclear lamin-associated polypeptide 2 (LAP-2) in order to visualise the nuclear envelope. At 7 h 20 min p.i., in cells treated only with mevastatin and mevalonic acid lactone, the nuclei were largely devoid of NP staining (Figure 7b, panel i). The identical pattern was seen in infected cells not treated with any drug (data not shown). However, after treatment with 3.5 mm MBCD for the preceding 7 h, a higher proportion of NP remained inside the nuclei at 8 h p.i. (Figure 7b, panel ii). Similarly, when cells were treated with 30 mm MBCD for 40 min at 7 h 20 min p.i., a substantial proportion of NP was found within the nuclei (Figure 7b, panel iii). Thus cholesterol depletion decreases the association of RNPs with the apical PM at late times postinfection and also results in their nuclear retention or possibly re-import.
Figure 7. Effect of cholesterol depletion on NP distribution in infected cells. PR8-infected 293-T cells were treated with MBCD as indicated, fixed and stained for NP and HA (a) or LAP-2 (b) as labelled. Single optical sections in the xy plane (upper panels) and xz planes (lower panels) are shown for individual or merged fluorophores as labelled. Arrows indicate areas of cytoplasmic NP staining not immediately adjacent to the apical PM. Scale bar = 5 μm.
Figure 8. Semiquantitative analysis of NP localization in infected cells. The average fluorescence intensity in the xy plane across four similarly sized clumps of cells (including those shown in Figure 7) treated with the indicated concentrations of MBCD is plotted.