Specificity of NP antibodies

We have previously generated and characterized two polyclonal anti-peptide antibodies that specifically recognize NLS1 and NLS2 on NP [7, 8]. In this study, we used these anti-NLS antibodies to analyze the exposure of these NLSs within cells infected with influenza A virus or transfected with the cDNA of NP. Total NP was detected by using a monoclonal antibody specific for NP. To ensure that all three of the NP monoclonal, anti-NLS1, and anti-NLS2 antibodies were specific for NP and not for components of the cell, we first compared the antibody labeling in infected cells with that in mock-infected cells. We found that each of the respective antibodies gave a strong signal in infected cells compared with mock-infected cells in which no virus was added (Fig. 1). A similar specificity of the anti-NP monoclonal, anti-NLS1, and anti-NLS2 antibodies was observed in cells transfected with the cDNA of NP compared with mock-transfected cells (results not shown).

Besides testing for the specificity of the anti-NP antibodies, the results from Fig. 1 also indicated that NLS1 was generally more exposed than NLS2, and exposed in a greater number of influenza A virus-infected cells. This is in agreement with our previous studies examining the immunogold labeling of purified vRNPs with the anti-NLS1 or anti-NLS2 antibodies [8], and with our conclusion that NLS1 is stronger that NLS2 in mediating the nuclear import of the influenza vRNPs [7].

Exposure of NLS1 and NLS2 in influenza-infected cells

We performed double-immunolabeling studies with the monoclonal NP antibody in conjunction with either the polyclonal NP anti-NLS1 or with the polyclonal NP anti-NLS2 antibody to analyze the exposure of the NLSs in cells infected with the influenza A virus. As illustrated in Fig. 2, the NP monoclonal antibody detected NP in both the nucleus and cytoplasm of infected cells (Fig. 2c–d), with 28% of the infected cells showing only nuclear staining (Fig. 3a). Similarly, the NLS1 epitope on NP was exposed in both the nucleus and cytoplasm (Fig. 2e). In contrast, the NLS2 epitope was only exposed in the nucleus of the infected cells (Fig. 2f). Quantitative analysis showed that 100% of the infected cells labeled with the anti-NLS2 antibody had only nuclear staining of anti-NLS2, while 35% of the infected cells labeled with the anti-NLS1 antibody had only nuclear staining of anti-NLS1 (Fig. 3a).

To distinguish between incoming vRNPs and newly synthesized NP and progeny vRNPs, we next performed a similar double-immunolabeling experiment with cells infected with influenza A virus in the presence of cycloheximide (a protein synthesis inhibitor). As illustrated in Fig. 4, there was no NP fluorescence signal in cells treated with cycloheximide. This indicates that the NP being labeled in the infected cells (Fig. 2) represents indeed newly-synthesized NP. Therefore, this limits the type of cytoplasmic NP detected in infected cells to be either newly-synthesized NP or newly-assembled vRNPs that have undergone nuclear export.

From the above results, it was unclear why these infected cells did not contain an exposed NLS2 in the cytoplasm even though the cells contained NP in the cytoplasm. The experiment with cycloheximide helped us to conclude that the cytoplasmic NP does not represent incoming vRNPs. To distinguish whether the cytoplasmic NP is newly-synthesized NP or nuclear-exported vRNPs, we used leptomycin B (LMB) to inhibit the nuclear export of vRNPs. These experiments with LMB detect newly synthesized vRNPs that is trapped in the nucleus. LMB has been successfully used in the past to inhibit the nuclear export of vRNPs in infected cells [11, 13]. We repeated these experiments in the presence of LMB, to block vRNP nuclear export and to determine whether the cytoplasmic NP in the infected cells represented newly-synthesized NP or nuclear-exported vRNPs. As documented in Fig. 2k–l, and Fig. 3a, we found that in the presence of LMB 78% of the infected cells showed only nuclear, and no cytoplasmic, NP. Quantitative analysis showed that 22% of the infected cells, however, also still showed cytoplasmic NP in addition to nuclear NP accumulation (Fig. 3b). Because we were inhibiting nuclear export, this cytoplasmic NP represents newly-synthesized NP that had not yet undergone nuclear import.

Consistent with the notion that there were two pools of cytoplasmic NP in infected cells untreated with LMB (newly-synthesized NP and newly-assembled vRNPs that have undergone nuclear export), the experiment in the presence of LMB yielded cells in which the fluorescence intensity of the cytoplasmic NP was less intense than from cells without LMB. Of particular note, this cytoplasmic NP contained an exposed NLS1 (Fig. 2m). In fact, quantitative analysis showed that 26% of infected cells in the presence of LMB still contained both cytoplasmic and nuclear immunostaining with the anti-NLS1 antibody (Fig. 3b). This indicates that newly-synthesized cytoplasmic NP that had not yet undergone nuclear import contains an exposed NLS1 epitope.

A longer time point in infected cells (30 hours instead of 17 hours) was also performed, and there was even less, but still a small amount of cytoplasmic NP staining from both the monoclonal and the anti-NLS1 antibodies (results not shown), indicating that more NP had undergone nuclear import. Taken together, these results indicate that NLS1 (but not NLS2) exposure is a prerequisite for successful nuclear import of newly-synthesized NP.

Exposure of NLS1 and NLS2 in NP-transfected cells

To distinguish any differences in the localization between NP only and NP as part of the vRNP complex, we repeated the immunolocalization experiments in cells transfected with NP cDNA. Similar to infected cells, 71% of the transfected cells showed NP in both the cytoplasm and nucleus, as represented by immmunolabeling with the monoclonal anti-NP antibody (Fig. 5c–d, and Fig. 6b). However, NP NLS1 and NLS2 were only exposed in the nucleus, and not cytoplasm, of transfected cells (Fig. 5e–f, and Fig. 6). This contrasted to infected cells, which yielded 65% of the cells with NP NLS1 exposed in the cytoplasm (Fig. 2e and Fig. 3b). According to our results above, this would indicate that the cytoplasmic NP in these transfected cells represented NP that had been nuclear exported, and not newly-synthesized NP, since NLS1 was not exposed in the cytoplasm of transfected cells (Fig. 5e and Fig. 6b) even though 71% of the transfected cells showed NP existing in the cytoplasm (Fig 5c–d, and Fig. 6b). To confirm this and distinguish between the two populations of cytoplasmic NP (nuclear exported or newly-synthesized), we blocked NP nuclear export with LMB. As expected, LMB completely inhibited NP nuclear export, with all the NP being retained in the nucleus of the transfected cells (Fig. 5k–l, and Fig. 6a). This indicates that all the cytoplasmic NP in transfected cells in the absence of LMB (Fig. 5c–d) indeed represented nuclear-exported NP. Since these cytoplasmic NP molecules did not show immunolabeling of NLS1 or NLS2 (Fig. 5e–f), nuclear exported-NP has its NLSs hidden or masked.

Exposure of NLS1 and NLS2 within the nucleolus

We also observed that in infected cells NP localized to distinct nuclear spots, which were reminiscent of nucleoli. To verify this we performed double immunolabeling with the anti-NLS antibodies and a monoclonal antibody against the nucleolar protein fibrillarin. As illustrated in Fig. 7a, we found that in influenza-infected cells, NLS1 was not exposed in the nucleolus. NLS2 was, however, exposed both in the nucleoplasm and the nucleolus. This is in contrast to NP-transfected cells, which have NLS1 and NLS2 exposed in the nucleoplasm, without any exposure in the nucleolus (Fig. 7b). This indicates that one or more components from the influenza virus play a role in allowing NLS2 to become exposed in the nucleolus of influenza A virus-infected cells.

Cells, viruses, antibodies

HeLa cells (American Type Culture Collection) were cultured in DMEM (HyClone) supplemented with 9% fetal bovine serum (FBS; Sigma) and maintained at 37°C in a humidified atmosphere with 5% CO₂. Influenza A (A/WSN/1933) NP cDNA in the pCAGGS vector was kindly provided by Dr. G. Whittaker (Cornell University). The affinity-purified rabbit polyclonal antibodies against the NLSs of NP (NLS1, ¹MASQGTKRSYEQM¹³ and NLS2, ¹⁹⁸KRGINDRNFWRGENGRKTR²¹⁶) were produced by Pacific Immunology, and have been characterized previously [7, 8]. The mouse monoclonal NP and fibrillarin antibodies were purchased from Acris and Abcam, respectively. Influenza A virus (A/Aichi/1968) was obtained from Charles River Laboratories.

Influenza infection

HeLa cells were plated at 30% confluency the day before infection in growth media containing 9% FBS onto 12-mm glass cover slips in 12-well plates. The next day, the cells were washed with phosphate buffered saline (PBS), and then 1 ml of growth media containing 0.2% FBS was applied to each well. 30 μl of the influenza A virus at 2 mg/ml (MOI of 1) were applied to the cells. The virus was allowed to adsorb to the surface of the cells for 40 minutes at room temperature, with gentle rocking every 10–15 minutes. The media containing the virus was then removed, and replaced with 1 ml of media containing 2% FBS. The cells were incubated for 17 or 30 hours in a 37°C incubator containing 5% CO₂. After these incubation times, the cells were prepared for immunofluorescence microscopy as described below.

For some experiments, the protein synthesis inhibitor cycloheximide (Sigma, St. Louis) at a final concentration of 1 mM was added to the 2% FBS medium. To inhibit nuclear export, leptomycin B (LMB; Sigma) was added to the cells 6 hours after replacing the media containing 2% FBS, and cells were incubated for a total of 17 or 30 hours at 37°C. LMB was used at a concentration of 11 nM, which is effective for the inhibition of the nuclear export of NP and vRNPs, as previously reported [6, 11].

Transfections

Transfection of HeLa cells with NP-pCAGGS was carried out on glass coverslips with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. For some experiments, cycloheximide (final concentration 1 mM) was added after media was changed, and the cells were incubated for 24 hours at 37°C. To inhibit nuclear export, LMB (final concentration 11 nM) was added 6 hours after the change in media, and the cells were incubated for a total of 30 hours at 37°C. After these incubation times, the cells were prepared for immunofluorescence microscopy as described below.

Immunofluorescence microscopy

After infection or transfection, cells were washed three times with PBS, fixed for 10 minutes with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (Sigma) in PBS containing 10% goat serum (Sigma), and labeled with the NP or the fibrillarin monoclonal antibody and either the NP polyclonal anti-NLS1 or the NP polyclonal anti-NLS2 antibody for 1 hour, followed by incubation with secondary antibodies (goat anti-mouse rhodamine and goat anti-rabbit fluorescein, both from Invitrogen). After washing, coverslips were mounted with Prolong Gold antifade containing DAPI (Invitrogen). Fluorescence microscopy was performed on a Zeiss Axioplan 2.