Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment

Influenza A viruses (IAV) pose a constant threat to the human population and therefore a better understanding of their fundamental biology and identification of novel therapeutics is of upmost importance. Various reporter-encoding IAV were generated to achieve these goals, however, one recurring difficulty was the genetic instability especially of larger reporter genes. We employed the viral NS segment coding for the non-structural protein 1 (NS1) and nuclear export protein (NEP) for stable expression of diverse reporter proteins. This was achieved by converting the NS segment into a single open reading frame (ORF) coding for NS1, the respective reporter and NEP. To allow expression of individual proteins, the reporter genes were flanked by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. The resulting viruses encoding luciferases, fluorescent proteins or a Cre recombinase are characterized by a high genetic stability in vitro and in mice and can be readily employed for antiviral compound screenings, visualization of infected cells or cells that survived acute infection.

The immunoblot (IB) was carried out with NS1-and NEP-specific antibodies. Detection of tubulin served as loading control. Proteins corresponding to the size of the detected bands are indicated impairment of viral replication in cell culture and mice 24 . Based on this favorable property of PTV-1 2A, we engineered influenza A viruses harboring an NS segment encoding reporter genes flanked by two genetically distinct PTV-1 2A-encoding sequences. These viruses are genetically stable in cell culture and mice and express a variety of luminescent and fluorescent reporters as well as a Cre recombinase.

PTV-1 2A-mediated co-translational separation of NS1 and NEP does not interfere with viral replication.
To confirm that the NS-segment of the mouse-adapted IAV A/SC35M (H7N7) 25 allows satisfying co-translational separation of NS1 and NEP, we generated a pHW2000 based 26 rescue plasmid (NS1_2A_NEP) that encodes NS1 and NEP in a single ORF (Fig. 1A). Here, the splice donor and acceptor sites of the NS1 gene were silenced by site-directed mutagenesis without affecting the amino acid sequence. A PTV-1 2A-coding sequence was introduced to mediate co-translational separation of NS1 and NEP. As expected from our previous work 24 , a recombinant virus carrying this modified NS-segment (SC35M NS1_2A_NEP ) replicates as efficiently as wild type SC35M (SC35M WT ) in mammalian MDCK-II cells (Fig. 1B). To analyze whether this modified NS-segment causes an altered NS1:NEP protein ratio during infection, we determined the levels of these proteins in the lysates of MDCK-II cells infected with SC35M WT or SC35M NS1_2A_NEP at a multiplicity of infection (MOI) of 0.1. As shown in Fig. 1C, NS1 (26 kDa) and NEP (14.5 kDa) protein levels were comparable between the two viruses 24 hours post infection (h.p.i.). However, we also detected a 40 kDa band corresponding to an NS1-2A-NEP polyprotein with both an NS1-and an NEP-specific antibody in the cell lysate of SC35M NS1_2A_NEP -infected cells, indicating that the 2A-mediated "stop-carry on" recoding is not completely efficient.

Fusion of a GFP to the nuclear export protein (NEP) results in compromised viral growth.
Based on recent observation that N-terminally GFP-tagged NEP (GFP-NEP) retains its nuclear export and polymerase co-factor function in vitro 27,28 , we reasoned that reporter genes might be introduced into the viral genome as NEP-fusion constructs. To demonstrate this, we generated a pHW2000-based NS segment rescue plasmid coding for NS1, 2A and a GFP-NEP fusion protein ( Fig. 2A). Using this plasmid, we could indeed successfully generate a recombinant GFP-encoding virus (SC35M NS1_2A_GFP-NEP ). SC35M NS1_2A_GFP-NEP is characterized by severe attenuation in MDCK-II cells, which was most obvious at 24 and 36 h.p.i., when viral titers in the cell supernatant were reduced by several log 10 relative to cells infected with SC35M WT (Fig. 2A). In contrast to SC35M NS1_2A_NEP (Fig. 1C), we observed highly elevated levels of NEP in lysates of cells infected with SC35M NS1_2A_GFP-NEP (Fig. 2B). This is consistent with earlier observations that fusion of GFP increases the stability of NEP 27 and that higher levels of NEP impair viral growth 23 . Although there is an increasing body of evidence that NEP is a multifunctional protein crucial for vRNP nuclear export, polymerase activity and viral budding 29,30 , little is known about the spatiotemporal regulation of these diverse functions. As we hardly detected any NS1-2A-GFP-NEP polyprotein in the lysate of SC35M NS1_2A_GFP-NEP -infected cells (Fig. 2B), this virus might serve as a valuable tool for the visualization of changes in the subcellular localization of NEP or its interaction with host factors in the course of an infection.
Introduction of a second PTV-1 2A peptide permits the generation of stable reporter viruses. The fusion of GFP to NEP substantially compromised viral growth ( Fig. 2A). Since this represents a major drawback for many experimental approaches, we set out to generate rescue plasmids allowing the expression of a gene of interest without its fusion to NS1 or NEP. To achieve this, we introduced a second PTV-1 2A-coding sequence between the transgene and NEP (Fig. 3A). This sequence was genetically modified to the highest possible extent to prevent homologous recombination with the non-modified PTV-1 2A nucleotide sequence (see Material and Methods). Using this approach, we were able to rescue viruses encoding three different fluorescent reporter genes: GFP (SC35M NS1_2A_GFP_2A_NEP ), its blue fluorescent derivate Azurite (SC35M NS1_2A_Azurite_2A_NEP ) 31 and the red fluorescent protein dsRed (SC35M NS1_2A_dsRed_2A_NEP ). All reporter viruses replicated to similar high viral titers of 10 8 PFU/ml 36 h.p.i., which represents an attenuation of approximately one log 10 relative to SC35M WT (Fig. 3A). Replication of SC35M NS1_2A_dsRed_2A_NEP was particularly delayed, as indicated by the low viral titer 24 hours post infection (Fig. 3A). As expected, infection of A549 cells with the reporter viruses resulted in well detectable fluorescence signals performing live cell imaging microscopy (Fig. 3B). To analyze the genetic stability of the modified NS segments, viruses were passaged four times in A549 cells and the proportion of reporter-expressing infectious viral particles was determined by plaque assay and subsequent fluorescent microscopy. As shown in table 1, all viruses retained their reporter gene after passaging in human cells, indicating a favorable stability of the reporter gene at least over 4 rounds of passaging. To analyze the recoding efficiency of the two PTV-1 2A peptides, NS1, GFP and NEP protein levels were determined in lysates of cells infected with SC35M NS1_2A_GFP_2A_NEP (Fig. 3C). Besides detection of the three individual proteins, we observed with both GFP-and NEP-specific antibodies a protein band of approximately 40 kDa corresponding to a GFP-2A-NEP fusion protein (Fig. 3C). A faint signal, which is visible above this GFP-2A-NEP band in the GFP-immunoblot might indicate the presence of low levels of an NS1-2A-GFP construct. Interestingly, we could not visualize a high molecular weight band corresponding to an NS1-2A-GFP-2A-NEP polyprotein. Consistently, we observed high levels of unprocessed reporter proteins fused to NEP in the lysates of cells infected with SC35M NS1_2A_dsRed_2A_NEP and SC35M NS1_2A_Azurite_2A_NEP (Fig. S1). To further characterize the fluorescent reporter viruses in vivo, BALB/c mice were infected intranasally with 1,000 PFU of the respective viruses and viral lung titers were determined 48 h.p.i. All reporter viruses replicated to significant titers between 5 × 10 5 and 5 × 10 6 PFU/lung (Fig. 3D). This also included SC35M NS1_2A_dsRed_2A_NEP , which showed delayed replication properties in cell culture compared to both SC35M NS1_2A_GFP_2A_NEP and SC35M NS1_2A_Azurite_2A_NEP (Fig. 3A). However, viral lung titers of all reporter viruses were substantially lower than the titers observed after infection of BALB/c mice with SC35M WT. Accordingly, the LD 50 of the fluorescent reporter viruses is increased compared to SC35M WT 32 ( Table 2). To monitor the genetic stability of the three reporter viruses in vivo, plaque assays were performed on the lung homogenates of the infected BALB/c mice and screened for reporter expression by fluorescent microscopy. This revealed that in all analyzed plaques (100/reporter virus) reporter expression was maintained, again demonstrating the high genetic stability of the here presented viruses (Table 1). To visualize spread of the reporter viruses in lungs of infected animals, BALB/c mice were infected intranasally with 10,000 PFU of SC35M NS1_2A_GFP_2A_NEP or SC35M NS1_2A_dsRed_2A_NEP . 48 hours post infection lungs were collected and subjected to microscopic analysis. Virus replication, as monitored by green or red fluorescence, could be observed in the epithelial layer of the bronchiolar tube (Fig. 3E, filled arrow heads) as well a in the distal lung tissues (Fig. 3E, transparent arrow heads). Taking together, these data indicate that our fluorescent reporter viruses are genetically stable and allow tracing of different virus-infected lung cells in mice.  Generation of luciferase-encoding viruses for screening approaches. Luciferases have been proven to be valuable tools in screening approaches for the identification of novel antiviral substances or host factors [33][34][35][36] . To generate a stable, luciferase-encoding virus, the gene for the Renilla luciferase (RenLuc) (~0.9 kB) was introduced into the NS-segment. MDCK-II cells infected with the resulting virus (SC35M NS1_2A_RenLuc_2A_NEP ) at an MOI of 0.001 released 10 8 PFU/ml into the cell supernatant after 48 hours (Fig. 4A). However, compared to SC35M WT -infected cultures, SC35M NS1_2A_RenLuc_2A_NEP revealed impaired viral growth. To prove stable expression of luciferase, SC35M NS1_2A_RenLuc_2A_NEP was passaged 4 times in human A549 cells. Reporter analysis of ten plaque-purified viruses revealed no loss of luciferase activity (Tab. 1), indicating stable expression of the RenLuc gene. To analyze whether SC35M NS1_2A_ RenLuc_2A_NEP could be employed for antiviral compound screenings, we treated infected MDCK-II cells with increasing concentrations of the viral polymerase inhibitor ribavirin and determined luciferase activity 24 hours post infection. As shown in Fig. 4B, RenLuc activity decreased in a dose-dependent manner reaching a baseline at approximately 100 μ M of ribavirin. The classic approach to analyze the antiviral activity in cell culture is the determination of viral titers. There, effects at an early stage within the first 6 hours of infection, prior to efficient particle formation, cannot be visualized. As the expression of virus-encoded luciferase is dependent on viral RNA synthesis and not particle release, we reasoned that SC35M NS1_2A_RenLuc_2A_NEP would allow quantification of antiviral activity of the viral polymerase inhibitor ribavirin at early time points post infection. To show this, SC35M NS1_2A_RenLuc_2A_NEP -infected MDCK-II cells were cultured in the presence of 0, 30 or 60 μ M of ribavirin and analyzed at different time points post infection for viral titers in the supernatant and luciferase activity in the cell lysate (Fig. 4C). Already at 3 h.p.i., we observed a substantial dose dependent difference in luciferase activity, whereas a first reduction of viral titers was detected at 12 hours post infection. Furthermore, we could not resolve differences in the antiviral efficacy between cells treated with 30 μ M or 60 μ M ribavirin by determination of viral titers, while this was possible as soon as 6 hours post infection by measuring luciferase activity.
The Gaussia luciferase (GLuc) represents an attractive luminescent reporter for many experimental approaches, not least due to its small size and its secretion from mammalian cells 37,38 . Indeed, insertion of the GLuc-coding sequence into the viral NS segment revealed a recombinant virus (SC35M NS1_2A_GLuc_2A_NEP ),    which was only partially attenuated but highly stable in cell culture (Fig. 4D, Tab.1). As expected, the supernatant of MDCK-II cells infected with this virus contained significant levels of secreted GLuc (Fig. 4E). To evaluate whether this extracellular reporter activity could be exploited as a measure for viral replication, we infected MDCK-II cells in the presence of ribavirin and determined luciferase activity and virus titers from the supernatant at different time points post infection. As anticipated, luciferase activity differed dependent on the concentration of ribavirin (Fig. 4F). However, especially at 24 hours post infection, differences in luciferase activity were not proportional to differences in virus titers determined from the same supernatants, most probably due to extracellular accumulation of the highly stable GLuc 38 .
As for the fluorescent reporter viruses, we detected high levels of unprocessed, luciferase-NEP fusion proteins in lysates of cells infected with SC35M NS1_2A_GLuc_2A_NEP and SC35M NS1_2A_RenLuc_2A_NEP (Fig. S1).
A Cre recombinase-encoding virus allows visualization of cells that are infected or survived acute infection. The Cre-Lox recombination is a widely used method to control gene expression in cell culture or animal models 39,40 . To generate a genetically stable virus that would allow activation or deactivation of genes specifically in infected cells, we introduced a Cre recombinase gene into the NS segment (SC35M NS1_2A_Cre_2A_NEP ). Infection of MDCK-II cells revealed a high viral titer of 10 8 PFU/ml cell culture supernatant 48 h post infection (Fig. 5A). However, SC35M NS1_2A_Cre_2A_NEP showed a delayed viral growth especially at 12 and 24 hours post infection compared to SC35M WT (Fig. 5A). To functionally monitor expression of Cre, we established a human airway derived Calu-3 cell line harboring a loxP-flanked dsRed gene followed by a silenced eGFP gene 41 (Fig. 5B). Upon Cre-mediated recombination, the dsRed gene is eliminated and cells express eGFP, resulting in a switch from red to green fluorescence and consequently constitutive GFP expression is inherited to progeny cells after proliferation. As IAV suppress host protein synthesis and induce apoptosis 42 (Fig. 6 A and B). Of note, all infectious viruses isolated from the lungs of infected animals by plaque purification 2 days post infection encoded a functional Cre recombinase as judged by GFP expression in the Calu-3 reporter cells, highlighting the genetic stability of SC35M NS1_2A_Cre_2A_NEP in mice (Table 1). Taken together, this reporter virus in combination with flow cytometric analysis represents a powerful tool to identify and quantify acutely infected lung cells as well as cells that survived acute infection.

Stable introduction of transgenes into the PB2-segment of SC35M is at the cost of viral replication capacity.
As for the NS-segment, it was shown that the PB2-segment tolerates the integration of foreign genes downstream of the PB2 ORF 11,12,46 . Since this approach represents an alternative to the NS segment as a vector for transgene expression, we generated pHW2000-based rescue plasmids encoding GFP or GLuc separated from the PB2 ORF by a PTV-1 2A-coding sequence (Fig. 7A). To guarantee packaging of the modified segment into viral particles, a terminal stretch of the segment was duplicated and fused downstream to the reporter gene as described by others 47 . Both viruses, encoding GFP (SC35M PB2_2A_GFP ) or GLuc (SC35M PB2_2A_GLuc ) could be successfully generated. Specific fluorescent signals were observed upon infection of A549 cells with SC35M PB2_2A_GFP by live cell imaging microscopy   (Fig. 7B). Analysis of the replication efficiency of these viruses in MDCK-II cells, revealed a substantial attenuation of SC35M PB2_2A_GLuc compared to SC35M (Fig. 7A). In sharp contrast, viral growth of SC35M PB2_2A_GFP was, to our surprise, as efficient as wild type virus (Fig. 7A). However, analysis of viral plaques obtained with SC35M PB2_2A_GFP revealed that only 1 out of 123 plaques was GFP positive (Tab. 1), indicating that the vast majority of these viruses lost the intact reporter gene after a single passage on human cells. Indeed, sequencing of the RNA extracted from six GFP-negative plaques revealed that the GFP gene was deleted, most likely by homologous recombination at the duplicated packaging signals. Interestingly, SC35M PB2_2A_GLuc did not lose its reporter activity after 4 passages (Tab. 1), suggesting that smaller genes might be tolerated on the expense of viral fitness.
To improve the genetic stability of the GFP-encoding PB2 segment, the highest possible number of silent mutations was introduced into the last 129 nucleotides of the PB2 ORF to prevent homologous recombination at the duplicated packaging signals (Fig. 7C). This modification resulted in a virus, designated SC35M PB2mod_2A_GFP , which was strongly attenuated in cell culture but genetically stable over four passages in A549 cells (Tab. 1). To rule out the possibility that this attenuation resulted from mutation of the PB2 ORF rather than from insertion of the transgene, we deleted the GFP-coding sequence from the PB2 segment (Fig. 7D). This virus (SC35M PB2mod ) replicated in cell culture almost as efficiently as SC35M WT . In summary, this suggests that stable integration of a transgene into the PB2-segment of SC35M is possible but associated with severe attenuation.

Discussion
Influenza A reporter viruses are important tools to study the biology of IAV and screening approaches. However, these viruses show in general impaired viral replication efficiencies, probably resulting in an unfavorable selective pressure towards loss of the reporter gene. Especially the latter may cause difficulties in the interpretation of the results observed with reporter viruses. In this study, we developed a strategy that allows the stable integration of reporter genes into the NS-segment of IAV. We achieved this by converting the NS segment into a single ORF encoding NS1, the respective gene of interest and NEP. The reporter gene was flanked by two sequences coding for the porcine Teschovirus-1 2A peptide (PTV-1 2A), thereby allowing co-translational separation of NS1, the reporter protein and NEP. The feasibility of this method was demonstrated by the successful generation of recombinant IAV, encoding a variety of fluorescent reporter proteins or catalytically active enzymes. Dependent on the gene inserted into the NS segment, we observed various degrees of attenuation. Most importantly, these genetically modified viruses displayed a high genetic stability over 4 passages in cell culture as well as over a single passage in mice and can be readily used for a broad range of in vitro and in vivo applications.
The levels of viral mRNA transcripts coding for NS1 or NEP in influenza A virus-infected cells is regulated by splicing, resulting in defined protein ratios of NEP and NS1. This is of special importance with respect to recent findings suggesting that the relative amount of NEP coordinates the intracellular timing of an infection and that an aberrant NS1 to NEP ratio results in inhibition of viral replication 23 . Intriguingly, the co-translational processing by PTV-1 2A seems rather inefficient, resulting in a substantial proportion of uncleaved polyprotein in cells infected with the recombinant viruses (Fig. 1C). As a consequence, the level of free NEP is comparable to that detected in cells infected with wild type virus preventing significant attenuation as exemplified best with the recombinant virus SC35M NS1_2A_NEP harboring no additional reporter gene (Fig. 1C). Interestingly, cells infected with SC35M NS1_2A_GFP_2A_NEP expressed high levels of a GFP-2A-NEP fusion protein, while NS1-2A-GFP was almost not detectable ( Fig. 3C and Fig. S1). This might be the result of context-specific differences in 2A-mediated recoding efficiency or could be explained by a higher stability of GFP-2A-NEP compared to NS1-2A-GFP.
Integration of foreign genes into the NS segment using the method presented in this study, can lead to impaired viral fitness. This might be a result of (I) low levels of NEP due to inefficient PTV-1 2A activity (Fig. 3C), (II) an intrinsic feature of the inserted reporter gene or (III) increased overall length of the NS segment. Indeed, integration of larger genes into the viral genome including firefly luciferase (~2 kb) or β -galactosidase (~3 kb) failed, suggesting a particular length restriction.
We could show for SC35M that insertion of the GFP gene into the PB2-segment results in the rapid loss of reporter activity due to homologous recombination of the duplicated packaging sequences (Fig. 7A and Tab.1). Loss of the reporter gene could be prevented by extensive modification of these sequences but was associated with significantly impaired viral growth (Fig. 7C and Tab.1). In contrast to the NS segment, introduction of reporter genes into the PB2 segment of SC35M did not result in viruses that are suited for further in vitro and in vivo studies. However, we cannot exclude the possibility that this might be different for other influenza A virus strains.
Importantly, the recombinant viruses described in this study harboring reporter genes in the NS segment are genetically stable in cell culture and mice. This includes fluorescent protein-encoding genes shown to be readily eliminated in cell culture or mice when present as NS1-fusion genes 15,16,20 . Genetically stable reporter viruses offer several advantages, including the reliable identification and determination of the relative abundance of virus-infected cells by microscopic tracing of these cells in a model organism. Because of their genetic stability, luciferase-encoding viruses might be robust tools for high throughput screening approaches. Here, SC35M NS1_2A_RenLuc_2A_NEP might be superior, as the activity of the virus-encoded RenLuc allows measurements already early upon infection and parallels with viral titers at later time points (Fig. 4C).
Sublethal infection of mice with influenza A virus is cleared by day 10-14 and no infectious particles can be isolated from the lung of these animals there upon 48 . Using the Cre recombinase-encoding virus, we could trace cells that survived viral infection in the lungs of infected rosa mT/mG mice 21 days after viral challenge. GFP-positive cells were found in various lung tissues including the epithelial layers lining the larger and also smaller airways (alveoli and bronchioles) of the respiratory tract. The fact that we could only observe the formation of GFP-positive cell clusters at later time points post infection (Fig. 5D) suggests that a proportion of these cells represents progeny of formerly infected and surviving cells, as GFP expression is inherited. Consistently, flow cytometry revealed the presence of GFP-positive cells among the stem and/or progenitor population 2, 7 and also 21 days post infection (Fig. 6B). Intriguingly, similar experiments were recently performed with an engineered H1N1 virus (A/Puerto Rico/8/1934) encoding a Cre recombinase within the PB2 segment 46 . In this case the authors could identify recombined cells exclusively in the larger airways of the respiratory tract. The reason for this discrepancy is unclear but could be related to differences in the segment used as vector for Cre recombinase-expression or might result from the different subtype of hemagglutinin (HA) of both mouse-adapted virus strains. While HA of PR/8 (H1N1) possesses a monobasic cleavage site, HA of SC35M (H7N7) harbors a multibasic cleavage site, which might facilitate infection of a broader spectrum of lung cells. Of note, influenza virus replication is known to be accompanied by the production of defective interfering particles (DIs) as well as semi-infectious particles (SIs) [49][50][51][52] . We cannot exclude the possibility that recombination events observed in cells of infected rosa mT/mG mice were caused by the incorporation of such particles, which might not have the capacity to induce cell death. Importantly, the in vivo prevalence of DIs and SIs remains to be elucidated 53 .
Taken together, we present a strategy that permits the introduction of foreign genes into the NS genome segment of Influenza A viruses. This was achieved by conversion of the intron-containing NS segment into a segment that allows the expression of reporter genes by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. Such engineered viruses were shown to be genetically stable over at least 4 passages in cell culture and a single passage in mice and thus represent attractive tools to study influenza A viruses in vitro and in vivo.

Methods
Plasmid construction. The Azurite gene was amplified from pGEMHE-X-Azurite, which was kindly provided by Max Ulbrich. The template used to amplify the Cre recombinase gene (pMIG-Cre) was a gift from Hassan Jumaa. The Gaussia Luciferase gene sequences derived from pT7-NYMVmg-Gluc 54 and the Renilla luciferase-coding sequence was obtained from pRL-SV40 (Promega). The eGFP gene was amplified from pCAGGS-GFP-P 27 . pHW2000-based 26 rescue plasmids to generate SC35M were described by others 32 . The NS1_2A_NEP plasmid was generated by deletion of the Flag-tag sequence of NS1_2A_Flag-NEP 24 by performing overlapping fusion PCR. The same plasmid served as template to create NS1_2A_GFP-NEP where the Flag-tag sequence was replaced by an eGFP gene. NS1_2A_ GFP_2A_NEP was obtained by insertion of a second genetically altered PTV-1 2A encoding sequence (GCCACAAATT TCTCTCTCCT CAAGCAAGCC GGGGACGTCG AGGAGAATCC CGGGCCC) between the genes for eGFP and NEP by overlapping fusion PCR. Furthermore, a SacII and a KpnI restriction site upstream and downstream of the eGFP gene respectively were introduced. NS1_2A_ Renilla_2A_NEP and NS1_2A_Cre_2A_NEP were generated by overlapping fusion PCR using NS1_2A_ GFP_2A_NEP as template. All other transgene-encoding NS-segment rescue plasmids were generated by PCR amplification of the transgene and SacII/KpnI-digestion-ligation into NS1_2A_GFP_2A_NEP. The PB2_2A_GFP and PB2_2A_GLuc plasmids were generated in two steps. First, a PB2_2A_GFP_2A_ NEP and a PB2_2A_GLuc_2A_NEP intermediate was generated by PCR amplification of PB2 and digestion-ligation into NS1_2A_GFP_2A_NEP and NS1_2A_GLuc_2A_NEP. In a second step, the 2A_ NEP sequence was replaced by 166 nucleotides of the 3' end of the PB2 segment by overlapping fusion PCR. To obtain PB2mod_2A_GFP, silent mutations within the last 129 nucleotides of the 3' end of the PB2 ORF (GCcAAaGGcG AaAAaGCcAA cGTcCTgATc GGcCAgGGcG AtGTcGTccT GGTcATGAAa aGaAaaGaGA tagctccATc CTgACcGAtt ccCAaACaGC cACaAAgAGg ATcaGaATGG CtATtAAc) were introduced into PB2_2A_GFP by annealing of two synthetic DNA oligonucleotides and subsequent overlapping fusion PCR. PB2mod was generated by removal of the 2A_GFP sequence from PB2mod_2A_ GFP via PCR amplification and digestion-ligation.

Virus rescue.
To generate recombinant influenza viruses, HEK293T cells were transfected in a 6 well format with 8 bidirectional pHW2000 rescue plasmids 26  Passaging and analysis of reporter stability. For each passage of fluorescent and luminescent reporter viruses, A549 cells were infected at an MOI of 0.01 and cultured in infection medium (see preceding chapter). 48 hours post infection, supernatant was collected and virus titer was determined by plaque assay. After 4 passages, plaques induced by fluorescent reporter viruses were analyzed and counted using a fluorescent microscope. To determine the reporter expression of passaged, luciferase-encoding viruses, 10 plaques were randomly picked and used for infection of MDCK-II cells and 24 hours post infection luciferase activity was measured. SC35M NS1_2A_Cre_2A_NEP was passaged on MDCK-II cells. After 4 passages and subsequent plaque assay, Calu-3 cells containing the loxp-dsRed-loxp-eGFP expression cassette were infected with plaque-purified viruses. After 3 hours, medium was replaced by Ribavirin-containing (100 μ M) medium. Recombination events resulting in a switch from red to green fluorescence were analyzed with a fluorescent microscope.
Determination of luciferase activity. Luciferase activity in whole cell lysates or in 5 μ l of supernatant of MDCK-cells cultured in 6-well plates was determined using a luciferase assay system (Promega) according to the manufacturer's instructions.
Immunoblot analysis. Virus infected cells were incubated with lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 1% protease inhibitor mix G [Serva, Heidelberg, Germany], 1 mM dithiothreitol [DTT]) for 15 min on ice. After centrifugation at 13,000 rpm at 4 °C, supernatants were complemented with SDS page sample buffer 55 and incubated at 95 °C. Proteins were separated in SDS-PAGE gels (15%), and transferred to nitrocellulose membranes. Antibodies for detection of NEP and NS1 were a gift from Thorsten Wolff and Christina Ehrhardt respectively. The commercial antibodies for detection of GFP and tubulin were purchased from Santa Cruz Biotechnology (GFP) and Sigma-Aldrich (tubulin).

Animal experiments.
All animal experiments were performed in accordance with the relevant guidelines (German animal protection law (TierSchG)) and approved by the welfare committees of the University of Freiburg, as well as the local authorities. Six-to-eight-week-old mice were anaesthetized with a mixture of ketamin (100 μ g per gram body weight) and xylazine (5 μ g per gram) administered intraperitoneally and inoculated intranasally with the indicated doses of viruses in 40 μ l PBS containing 0.3% bovine serum albumin (BSA). Animals were sacrificed, if severe symptoms developed, or body weight loss approached 25% of the initial value. Lung homogenates were prepared using the FastPrep24 system (MP Biomedicals). Briefly, after addition of 800 μ l of PBS containing 0.2% BSA, lungs were subjected to two rounds of mechanical treatment for 10 s each at 6.5 ms −1 . Tissue debris was removed by low-speed centrifugation. The LD 50 values were calculated based on the infectious dose (PFU). BALB/c mice were obtained from Janvier (Strasbourg). Rosa mT/mG mice (Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo) (Jackson laboratory) contain the two-color fluorescent rosa mT/mG allele from which the cell membrane localized red fluorescent tdTomato is expressed. Upon Cre-Lox recombination directed by lox-P sites flanking the tdTomato gene, eGFP expression is induced. Tissue histology. At appropriate time points, mice were sacrificed and lungs were transcardially perfused with 0.9% NaCl prior to fixation in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer at 4 °C overnight. After washing in ddH 2 O the lungs were first transferred into a 15% sucrose solution in 0.1 M phosphate buffer (w/v) for 4 h and thereafter in a 30% sucrose solution in 0.1 M phosphate buffer (w/v) overnight (4 °C). For cryosection, lungs were embedded within Tissue-Tek O.C.T. compound, solidified on dry ice and cut to 15 μ m thickness using a cryotome (Leica Microsystems, Germany). The sections were mounted onto gelatine-coated slides and dried at room temperature overnight in the dark. The slides were washed twice in phosphate buffer, DAPI (Invitrogen) stained at an end concentration of 300 nM in 0.1 M phosphate buffer for 7 min and again washed 3 times in 0.1 M phosphate buffer. Dried slides were embedded within IMMU-Mount ™ (ThermoShandon), coverslipped and stored in the dark at 4 °C until further use.

Isolation of murine distal lung cells.
Mouse lungs were perfused with HBSS (Gibco) followed by instillation of dispase (BD Biosciences) into the lung through the trachea and incubation in dispase for 40 minutes as previously described 56 . Trachea and large airways were dissected and the remaining distal lung tissue was homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01% DNase (Serva) and filtered through 100 μ m and 40 μ m nylon filters. Cell suspensions were incubated with biotinylated rat anti-mouse CD45, CD16/32 and CD31 mAb (BD Biosciences) for 30 minutes at 37 °C followed by incubation with biotin-binding magnetic beads and magnetic separation to deplete leukocytes and endothelial cells prior to flow cytometric analysis.
Fluorescence microscopy. Fluorescence images of cultured cells seeded in black, clear bottom 96 well microplates (Greiner) were acquired on a Zeiss Observer.Z1 inverted epifluorescence microscope (Carl Zeiss, Jena) equipped with an AxioCamMR3 camera using a 40x objective. Fluorescence microscopy of lung sections was performed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Jena) equipped with an ApoTome optical sectioning module using a 10x objective. Images were recorded with an AxioCamMR camera (Carl Zeiss, Jena).