After infection by many eukaryotic viruses, there is often an inhibition of cellular protein synthesis at times when viral proteins are maximally synthesized(1, 2, 3, 4) . The best understood viral translational strategies are those utilized by adenovirus and poliovirus. In both of these systems, the selective translation of viral mRNAs is assured by invoking cap-independent mechanisms for translational initiation(5, 6, 7) . The inhibition of cellular protein synthesis in poliovirus-infected cells correlates with the degradation of P220, a component of the cap-binding protein complex, termed eukaryotic initiation factor 4F, eIF4F()(8) . In the absence of functional P220, cellular mRNAs, which initiate translation in a cap-dependent manner, cannot be translated while poliovirus mRNAs, which initiate cap-independently and internally, continue to be translated(9) . Selective translation in the adenovirus system appears to be dependent on the dephosphorylation of eIF4E, which is also a component of eIF4F(5, 10) . This reduction in phosphorylation levels results in functional limitations of the initiation factor and the subsequent translation of mRNAs that have a reduced requirement for eIF4E, such as those adenovirus mRNAs containing the tripartite leader(11) .

Like adenovirus and poliovirus, influenza virus establishes translation controls that regulate viral and cellular protein synthesis. However, unlike these viruses, influenza virus mRNA translation occurs in a cap-dependent manner similar to cellular mRNAs. Indeed influenza virus even ``steals'' the cap and 5` end of cellular mRNAs as part of its transcriptional strategy(12) . Influenza virus has therefore evolved more subtle strategies to ensure the selective and efficient translation of its mRNAs(2, 13) . These include, but are not limited to, (i) degrading newly synthesized cellular mRNAs in the nucleus of infected cells(14) ; (ii) inhibiting pre-existing cellular mRNA translation at both the initiation and elongation stages(15) ; and (iii) encoding complex tactics to down-regulate the double-stranded RNA-activated protein kinase(4, 16) . Accumulating evidence now suggests that the structure of influenza viral mRNAs is critical for selective viral protein synthesis(17, 18) . This was most convincingly demonstrated by the development of a transfection/infection assay in which representative viral and cellular cDNAs were introduced into COS cells that were subsequently infected with influenza virus(17) . Using cDNA chimeras containing the noncoding and coding regions of cellular and viral mRNAs, it was subsequently demonstrated that this selective translation was mediated by sequences present within the 5`-untranslated region (UTR) of the viral mRNAs(18) . These data have been corroborated by others who have shown that sequences contained within the viral 5`-UTR may be important for efficient viral mRNA translation(19, 20, 21, 22) .

The current study was undertaken to further define the molecular mechanisms mediating viral mRNA 5`-UTR-driven selective mRNA translation. Using gel mobility shift and UV cross-linking analysis, we identified critical cis-acting sequences within the viral mRNA 5`-UTR and trans-acting proteins that interacted with this region. We were particularly focused on identifying novel cellular proteins which regulate influenza virus gene expression, since virtually nothing is known of the role of such proteins. We succeeded in identifying several cellular proteins which specifically interacted with select regions of viral 5`-UTRs, but not a cellular 5`-UTR. Finally, we found that the influenza virus NS1 protein also bound to several viral mRNA 5`-UTRs.

MATERIALS AND METHODS

Preparation of Cell Extracts

In Vitro Transcription

Gel Mobility Shift Analysis

Gel Mobility Supershift Assay

UV-induced Cross-linking Analysis

Northwestern Analysis

Preparation of GST-NS1 Fusion Proteins

RESULTS

Analysis of Cellular Proteins That Interact with the 5`-UTRs of NP and NS mRNAs

Figure 1: Schematic diagram of in vitro transcribed RNA transcripts used as probes in the gel mobility shift and UV cross-linking assays. The RNA probes were transcribed in vitro as described under ``Materials and Methods.'' Underlined sequences represent the conserved 12-nucleotide sequences found on all influenza virus type A mRNAs. A, the 5`-UTR of NP mRNA was divided into four regions (regions A, B, C, and D). The sequences of in vitro transcripts representing the 5`-UTR of nucleocapsid protein (NP) mRNA and its deletion mutants (NP-A, NP-B, NP-C, and NP-D) are depicted on top of the panel. Region A contained the conserved 12-nucleotide sequences. Region C contained the inverted repeat sequences ( ). The NS 5`-UTR is shown along with sequences of the two NS substitution mutants, NS-B and NS-B2 (mutated bases are shown in lowercase letters). Below are shown the sequences of the SEAP 5`-UTR along with the SEAP 5`-UTR appended to region A (SEAP + A) and region B (SEAP + B). B, sequence comparisons between the NP, NS, and mRNAs 5`-UTRs. Homologous regions within region B are shown in bold letters.

Figure 2: Gel mobility shift assays utilizing NP, NS, and SEAP 5`-UTRs. Uninfected HeLa cell cytoplasmic extracts (2 µg) were incubated in the absence or presence of the nonspecific competitor, heparin (0.125 mg/ml), or poly(I-C) (1.25 mg/ml) in the presence of P-labeled 5`-UTR RNAs (250,000 cpm/ng) as indicated on top of the figure and described under ``Materials and Methods.'' Roman numerals I-V (on the left) refer to the major shifted bands. Predominant bands II and III are indicated by arrows on the right. Position of the free RNA probe is indicated.

To more directly examine the specificity of observed RNA-protein complexes, a competition experiment was performed. The 5`-UTR of NP was radiolabeled and reacted with HeLa cell extracts in the presence of a 25 , 50 , or 100 molar excess of either the homologous H-labeled NP 5`-UTR or the heterologous H-labeled cellular SEAP 5`-UTR competitor (Fig. 3A). Only bands II and III showed greater decreases with specific competitor (NP) than with nonspecific competitor (SEAP). In contrast, the intensity of band I decreased in the presence of excess homologous and heterologous competitor, while bands IV and V did not decrease in the presence of either competitor. These data together argue that the interactions represented by bands II and III were the most specific.

Figure 3: Gel mobility shift and supershift assays. A, competition experiments. Gel mobility shift assays were performed as described under ``Materials and Methods'' in the presence of P-labeled NP 5`-UTR alone (lanes 1 and 5) or a 25-, 50-, or 100-fold molar excess of H-labeled NP 5`-UTR (lanes 2-4) or SEAP 5`-UTR (lanes 6-8). B, gel mobility supershift assay. A gel mobility supershift experiment was performed as described under ``Materials and Methods'' with the P-labeled NP 5`-UTR in the absence (lane 1) or presence (lane 2) of La monoclonal antibody. The arrow on left indicates the ``supershifted'' band. C, supershift competition assay. P-Labeled NP 5`-UTR was reacted alone (lanes 1 and 3) or with a 100-fold molar excess of H-labeled NP 5`-UTR (lane 2) or SEAP 5`-UTR (lane 4) as competitor (COMP) in the presence of the La monoclonal antibody as described under ``Materials and Methods.''

During titrations of the cellular extracts, we consistently observed that band III was the most intense, suggesting the concentrations of the band III reactive protein(s) were in excess relative to the concentration e.g. of the band II proteins. This might also explain why the band III complex could not be as effectively competed as band II in the above described experiment. Because the La autoantigen is known to be present in high amounts in HeLa cell extracts(26) , and because La was recently found to react both with the poliovirus and HIV mRNA 5`-UTRs(26, 27, 28) , we tested whether La was reacting with the NP 5`-UTR, thus representing at least one protein component of shifted band III. This was initially tested utilizing a monoclonal antibody to the La protein (24) in a gel mobility supershift assay (Fig. 3B). In the presence of the monoclonal antibody, a supershifted band appears (see arrow on left) along with a concomitant decrease in the intensity of shifted band III. No such shifted band was observed using control monoclonal antibody (data not shown). It is important to note that band III was not completely eliminated in the presence of monoclonal antibody, raising the possibility that additional proteins were represented in this shifted band. We then performed another competition experiment, this time in the presence of the La monoclonal antibody (Fig. 3C). Under these conditions, the intensity of band III was diminished, but only in the presence of H-labeled NP RNA, presumably since La has been removed and other protein(s) were more visibly out-competed with the homologous probe. Interestingly, the supershifted band containing the La protein was reduced, only in the presence of homologous competitor. Utilizing purified protein (kindly provided by Yuri Svitkin and Nahum Sonenberg), we have confirmed the interaction of La with the influenza virus mRNA 5`-UTR (data not shown). As of yet, however, it is premature to assign a biological significance to these observations until a functional role can be attributed to the La-NP 5`-UTR interaction. To gain more information about the nature of the proteins interacting with the viral 5`-UTRs, we performed gel mobility shift analysis with both rabbit reticulocye and wheat germ extracts. Compared with the HeLa cell extracts, there were only minor complexes formed using the wheat germ extracts. In contrast, there were multiple shifted bands using the reticulocyte extracts which closely resembled the HeLa cell extract pattern (data not shown).

Sequences within the NP and NS mRNA 5`-UTRs That Are Critical for the Binding of Specific Cellular Trans-acting Factors

Figure 4: Gel mobility shift and UV cross-linking analysis. A, gel shift assays. Wild-type and mutant P-labeled 5`-UTRs (described on top of the panel) were subjected to gel mobility shift analysis as described under ``Materials and Methods.'' B, in situ UV cross-linking. In situ UV-induced cross-linking assays were performed on shifted band II or III from the gel shift experiment described in A. The complexes were subsequently excised from the gel, treated as described under ``Materials and Methods,'' and analyzed by 10% SDS-PAGE. The P-labeled 5`-UTR RNAs used in the individual experiments are indicated on top of each lane. The position of the predominant cross-linked proteins are indicated by the arrows on the left, and the migration positions of molecular weight markers ( 10) are shown on the right.

We took advantage of this mutant analysis to investigate the molecular weight of the cellular proteins that were interacting with the influenza virus mRNA 5`-UTRs and giving rise to shifted bands II and III in particular. We also wanted to ascertain whether both the NS and NP 5`-UTRs were interacting with similar proteins. Bands II and III, shown in Fig. 4A, were therefore subjected to in situ UV cross-linking and then excised from the gel. After ribonuclease treatment, the radiolabeled proteins were subjected to SDS-PAGE (Fig. 4B). Band III is comprised of at least two proteins, approximately 43-46 kDa. In contrast, band II consists of one major protein, approximately 50 kDa. Unexpectedly, the shifted band in the SEAP + A reaction appeared to give rise to a single protein which comigrated with the faster migrating polypeptide contained in the doublet from band III (see ``Discussion'').

For a more complete picture of the proteins which interacted with the viral 5`-UTRs, we performed an in solution UV cross-linking assay utilizing the wild-type and mutant mRNA 5`-UTRs. We first examined the reactivity of the wild-type SEAP, NP, and NS 5` UTRs in the absence and presence of heparin (Fig. 5A). Several proteins cross-linked to both the NP and NS 5`-UTRs, while no detectable proteins were found to react with the SEAP 5`-UTR. While the higher molecular mass proteins (80-100 kDa; upper arrows on the left) were partially competed out in the presence of heparin, possibly indicating a lower affinity of these proteins for the UTRs, the lower molecular mass proteins (43-55 kDa; lower arrows on left) actually increased in response to the nonspecific competitor. This latter class of proteins corresponded well in size to the polypeptides detected in bands II and III by in situ UV cross-linking analysis (see Fig. 4B). We next proceeded to perform in solution UV cross-linking with a selection of mutant RNAs. In these experiments we radiolabeled the RNAs with both [P]ATP and [P]UTP (Fig. 5B) in contrast to the previously described experiment in which the RNAs were labeled only with [P]UTP (Fig. 5A). This was done because there are no uridine residues present within the conserved region A of the influenza virus 5`-UTR (see Fig. 1). By labeling with both UTP and ATP, it was possible to get a fuller representation of proteins interacting with all regions of the viral 5`-UTR. Indeed, if one now compares the proteins cross-linking to the NP 5`-UTR which was dually labeled (Fig. 5B, lane 4) to the NP RNA labeled with only UTP (Fig. 5A, lane 6), additional proteins were now detected. In addition to the predominant proteins in the 43-50-kDa range and the less predominant proteins around 80-100 kDa, there were at least two additional major polypeptides of approximately 60 and 70 kDa which interacted with the NP 5`-UTR. This indirectly suggested that these proteins interacted, at least in part, with the A region of the 5`-UTR. Mutant analysis supported this conclusion, in that the NP-A mutant RNA failed to react with the 60- and 70-kDa proteins. In addition the NP-B RNA failed to react with these proteins, suggesting that this region was also important, possibly because of necessary secondary structure which formed involving regions A and B. Both NP-B and NS-B also failed to react with the 50-kDa protein in accordance with the previous in situ data. Binding to the 80-100-kDa proteins was similarly reduced with these mutants. To test if regions A or B were by themselves sufficient to induce interaction with any of the described proteins, in solution cross-linking was performed with radiolabeled SEAP + A or SEAP + B 5`-UTRs. Whereas SEAP + B failed to interact with any of the proteins, SEAP + A interacted with the 60- and 70-kDa proteins, providing further evidence that these proteins interact with the conserved A region. In addition SEAP + A interacted with a smaller 43-kDa protein, which likely was identical to the protein detected by in situ cross-linking as described above. Preliminary experiments utilizing purified protein preparations suggested that this may represent the La autoantigen (data not shown). As demonstrated previously, deleting regions C or D of the 5`-UTR had minor effects on RNA-protein interactions.

Figure 5: In solution UV cross-linking assays. A, in solution UV cross-linking was performed as described under ``Material and Methods'' utilizing SEAP, NS1, or NP mRNA 5`-UTRs labeled with [-P]UTP in the absence (indicated as -) and presence (+) of heparin. Samples were analyzed by SDS-PAGE followed by autoradiography. Major cross-linked proteins are indicated on the left, and molecular mass markers on the right. B, UV cross-linking was performed with wild-type and mutant 5`-UTRs (indicated on top of the panel), radiolabeled with [-P]UTP/ATP in the presence of heparin. Samples were analyzed by 10% SDS-PAGE.

More recently we have performed gel shift and UV cross-linking analysis on another cellular UTR, which is present at the 5` end of the interleukin 2 mRNA. This was selected since our earlier work found that mRNAs containing this UTR were not translated in an influenza virus-infected cell(17, 18) . Similar to the SEAP 5`-UTR, the interleukin 2 5`-UTR interacted at best, weakly with cellular proteins present in the uninfected cell cytoplasmic extracts utilizing both assays (data not shown). Importantly we have now confirmed that the predominant proteins that interacted with the viral 5`-UTRs do not interact with the interleukin 2 5`-UTR just as they failed to react with the SEAP 5`-UTR.

Influenza Viral NS1 Protein Interacts with the Viral NP and NS 5`-UTRs

Figure 6: Northwestern analysis. Mock-infected extracts (M) and extracts from cells infected with influenza virus for 4, 9, 23, and 32 h (F4, F9, F23, F32) were subjected to 12% SDS-PAGE, followed by blotting of the gels onto nitrocellulose filters and probing with the P-labeled 5`-UTR (2 10 cpm/ml) representing SEAP (A), NP (B), or NP-B (C) as described under ``Materials and Methods.''

To directly analyze whether NS1 bound to influenza virus 5`-UTRs, we prepared a recombinant purified GST-NS1 fusion protein and reacted the protein with both wild-type and mutant RNAs in our gel shift assay in the presence of heparin (Fig. 7A). The recombinant protein bound to the NP, NS, and M 5`-UTRs, although reactivity with the homologous NS mRNA was clearly weaker (lanes 1, 6, and 9, respectively). In contrast NS1 did not bind to the SEAP 5`-UTR (lane 10) nor did it bind to NP-A, NP-B, NS-B, or NS-B2 5`-UTRs. The NS-B2 RNA contained only a 3-nucleotide change compared with the wild-type NS mRNA 5` UTR (Fig. 1), yet still did not bind the NS 1 protein. Despite the apparent dependence of regions A and B for NS1 protein binding, the protein failed to bind to either SEAP + A or SEAP + B strongly, suggesting that these regions were necessary but not sufficient for NS1 protein binding. It should be mentioned that control preparations, expressing only the GST tag or an unrelated GST fusion protein, failed to bind any of these RNAs (data not shown), demonstrating that binding was due to the NS1 protein itself. Competition experiments were then performed with homologous and heterologous competitor. The reactivity between the NS1 protein and the NP 5`-UTR could be successfully competed out by the homologous NP competitor but not by the heterologous SEAP RNA 5`-UTR (Fig. 7B), again suggesting that NS1 binding may be specific.

Figure 7: Gel mobility shift analysis with recombinant GST-NS1 protein. A, gel mobility shift analysis was performed with purified recombinant NS1 (200 ng) and the radiolabeled mutant and wild-type 5`-UTRs (10,000 cpm) indicated on top of the panel in the presence of heparin as described under ``Materials and Methods.'' The arrow indicates the major shifted band. B, a competition assay was performed with purified recombinant GST-NS1 (200 ng) and P-labeled NP 5`-UTR in the absence (lanes 1 and 3) or presence of 100-fold molar excess of H-labeled NP 5`-UTR (lane 2) or H-labeled SEAP 5`-UTR (lane 4).

DISCUSSION

In the current report we have demonstrated the interaction of trans-acting factors with influenza virus mRNA 5`-UTRs. We acknowledge that additional studies are required to assign a functional translational regulatory role to these proteins. Moreover, it is certainly possible that one or more of these cellular proteins play a role in other aspects of viral replication, since similar sequences are present on the influenza virus cRNA, the template for virion RNA synthesis(29) . It is relevant to cite a recent report identifying a cellular protein which interacted with the influenza virus NP protein and which therefore may play a role in viral gene expression(30) . In addition there has been indirect evidence that cellular proteins play a role in the regulation of influenza virus replication (reviewed in (29) ). To our knowledge, however, this is the first study which has examined the interaction of cellular proteins with influenza virus RNAs of any kind and thus represents a starting point to explore novel regulatory pathways.

Several cellular proteins were identified that bind mainly to the conserved A and the immediately downstream B regions of the influenza virus 5`-UTRs. The 60- and 70-kDa proteins bind predominantly to the A region based on radiolabeling and NP mutant analysis. Furthermore the A region is both necessary and sufficient for the binding of these two proteins, since SEAP + A alone interacted with the the 60- and 70-kDa proteins (Fig. 5B). In contrast, region B is necessary, but not sufficient, to bind the 50-kDa protein as revealed by both in situ and solution UV cross-linking assays, suggesting that other sequences and/or structures are important. Region B also appears to be the binding site of the larger 80-100-kDa proteins ( Fig. 5and Fig. 6). It is more problematic to assign a region of binding for the two closely migrating proteins around 43-46 kDa. The SEAP + A RNA binds to the faster migrating protein of the doublet which likely represents the La protein based on UV cross-linking studies with the purified recombinant protein (data not shown). Surprisingly, however, the NP-A RNA construct bound both this protein and the other component of the doublet, while the NS-B RNA failed to interact with either of these proteins (Fig. 5B). Based on this evidence it is likely that these proteins bind to multiple sites within the 5`-UTRs, and the interactions may be dictated by a specific secondary structure.

We found that NS1 bound to the 5`-UTRs of M and NP mRNAs and to a lesser extent to the NS mRNA 5`-UTR. Furthermore, the NS1 binding site mapped to regions A and B (Fig. 7). NS1 failed to bind to NP-A, NP-B, NS-B, and to NS-B2, which contains only a 3-nucleotide change in region B as compared with the wild-type. These data are consistent with two recent reports, suggesting a role for NS1 in regulating viral mRNA translation. Enami et al.(19) , using a ribonucleoprotein transfection system, showed that NS1-stimulated translation of the M-CAT mRNA (and NP-CAT) but not the NS-CAT mRNA. They also preliminarily attributed the stimulation to the GGUAGAUA sequences which are present at the very end of region A and the beginning of region B of the influenza virus 5`-UTR (see Fig. 1). As mentioned we detected weaker binding of the NS1 protein to the NS mRNA and also found the B region critical for binding. It is noteworthy that both NP and M have sequence identity to each other in this region, while there are minor differences with the same region present in the NS 5`-UTR (Fig. 1B). In a related recent report, Ortin and colleagues(22) , using cotransfection analysis, found that NS1 specifically enhanced the translation of M and NP mRNAs and mapped the effects to the influenza virus mRNA 5`-UTR. These data, together with our observations, are all consistent with a role of NS1 in the translational enhancement of viral mRNAs. NS1 has even been reported to be associated with ribosomes, although not as a structural protein(31) . Despite this evidence, caution is needed, particularly because of the multiple roles already assigned to NS1 in the literature. For example, NS1 has been described as a protein which binds both double-stranded RNA (32) or influenza virus minus sense RNA in vitro(33) . In addition NS1 has been reported to bind only the poly(A) region of influenza viral mRNAs and thus inhibit nuclear export of mRNAs containing poly(A)(34) . There is also evidence that the NS1 protein regulates the transport of spliced NS2 mRNA (35) and even may inhibit pre-mRNA splicing(36) . Clearly NS1 can perform more than one function. It is equally evident, however, that more direct evidence is needed to confirm the translational regulatory role of this interesting protein, preferably utilizing an in vitro system that can discriminate between cellular and viral mRNA translation.

There is ample precedence in the literature for a role of cellular and viral proteins acting as positive regulators of viral mRNA translation. There are several cellular proteins that bind to picornaviral 5`-UTRs and potentially up-regulate translation. The best characterized is the La autoantigen which Sonenberg and colleagues (27) have reported not only bound the poliovirus 5` UTR, but also stimulated the translation of the poliovirus P1 capsid precursor protein and eliminated the synthesis of aberrant polypeptides. Other proteins implicated in picornaviral translational control include p97 and p57 which may be identical to the polypyrimidine tract-binding protein (37) . The Sonenberg laboratory also demonstrated that the La autoantigen bound the HIV-1 mRNA trans-activation response element and alleviated the translational repression imparted on HIV-1 mRNAs due to the presence of the trans-activation response element(28) . While we have demonstrated that La also binds influenza virus mRNAs, we have not yet shown that the autoantigen can regulate selective influenza virus mRNA translation. Other examples of positive acting cellular factors which may regulate translation are those reported to bind to rubella virus and hepatitis C virus RNAs(38, 39) . Finally, there are also reports of viral proteins acting as positive regulators of mRNA translation, such as the adenovirus L4 100-kDa protein(40) .

In summary, influenza virus has developed multiple strategies to ensure the specific and efficient translation of its mRNAs. We propose that the 5`-UTR sequences, especially the nucleotides present in regions A and B, play a major role in directing selective viral protein synthesis. It is also likely that a subset of the proteins identified in this report, possibly including the La and NS1 proteins, participate in these regulatory events. Potentially relevant to this story is the observation that eIF4E is modestly dephosphorylated in influenza virus-infected cells(41) . Dephosphorylation leads to a functional decrease of this factor which is already present in rate-limiting amounts in eukaryotic cells(10) . It is tempting to speculate that the structure (or lack thereof) of the influenza virus 5`-UTR dictates a higher affinity or a reduced requirement for eIF4E, thus favoring translation of viral over the more structured cellular mRNAs. It remains to be determined whether any of our observed cellular RNA binding proteins are eIF-4E or any other protein synthesis initiation factors. An alternative hypothesis suggests that influenza virus infection results in a modification (e.g. phosphorylation or glycosylation) of certain cellular proteins (possibly including known initiation factors) which help stimulate translation. It can be argued that these modified factors would then have a higher affinity for viral over cellular mRNAs.

FOOTNOTES

*

This work was supported by United States Public Health Service Grants AI-22646 and RR 00166 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 206-543-8837; Fax: 206-685-0305; honey@u.washington.edu.

()

The abbreviations used are: eIF, eukaryotic initiation factor; UTR, untranslated region; PAGE, polyacrylamide gel electrophoresis; SEAP, secreted embryonic alkaline phosphatase; NP, nucleocapsid protein; NS, nonstructural protein; M, matrix protein.

ACKNOWLEDGEMENTS

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