JOURNAL OF VIROLOGY, Nov. 2010, p. 1207512081 0022-538X/10/$12.00 doi:10.1128/JVI.00046-10 Copyright 2010, American Society for Microbiology.

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Vol. 84, No. 22

Generation of Replication-Competent Recombinant Inuenza A Viruses Carrying a Reporter Gene Harbored in the Neuraminidase Segment
Feng Li,1 Liqiang Feng,1 Weiqi Pan,1 Zhenyuan Dong,1 Chufang Li,1 Caijun Sun,1 and Ling Chen1,2*
State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Biomedicine and Health (GIBH), Chinese Academy of Sciences, Guangzhou 510530,1 and Department of Medical Biotechnology, University of Science & Technology of China, Hefei 230026,2 China
Received 10 January 2010/Accepted 13 August 2010

Replication-competent inuenza viruses carrying reporter genes are of great use for basic research, screening of antiviral drugs, and neutralizing of antibodies. In this study, two recombinant inuenza A viruses with a neuraminidase (NA) segment harboring enhanced green uorescent protein (EGFP) in the background of A/PR/8/34 (PR8) were generated. The viral RNA (vRNA)-specic packaging signals for NA were largely retained for efcient packaging. An autocleave 2A peptide sequence, which was inserted at the N terminus or the COOH terminus of NA to link with EGFP, enabled NA and EGFP to be expressed monocistronically. Further analysis demonstrated that both viruses, named rPR8-EGFP NA and rPR8-NA EGPF, although with some characteristic differences in growth and EGFP expression, could replicate in noncomplementary cells and propagate to large quantities while maintaining genome stability after multiple passages in embryonated eggs. These replication-competent inuenza viruses carrying reporter genes are a great addition to the tool set for developing antiviral therapeutics and vaccines and for in vivo studies of viral dissemination and pathogenicity.

The worldwide transmission of the high-pathogenicity avian inuenza virus (HPAI) H5N1 and its occasional jumping to humans have posed great public health challenges and caused signicant economic losses since 1996. In 2009, the swine H1N1 virus spread in 208 countries and claimed more than 12,220 lives (WHO, 27 December 2009). Live recombinant inuenza viruses containing reporter genes, such as those encoding enhanced green uorescent protein (EGFP) or luciferase, could be of great use in studying inuenza virus and developing vaccines and antiviral drugs. The inuenza A viral genome is constituted by 8 single-stranded, negative-sense RNA segments. Many efforts have been made to insert extra genes into its genome. Small peptides have been successfully inserted in-frame into the functional nonessential region of the hemagglutinin (HA) (8, 13) and neuraminidase (NA) (2, 3, 19) proteins, but the insertion of full-length proteins, such as the EGFP reporter, have been less successful. A ninth synthetic segment has been added to the viral genome (14, 15), but its lack of competitiveness in viral RNA (vRNA) packaging means that it can get lost eventually and therefore is infeasible for extensive propagation and large-scale usage. The G glycoprotein of the vesicular stomatitis virus (VSV-G) and the HAesterase fusion (HEF) glycoprotein of the inuenza C virus have been substituted for HA and NA in pseudoinuenza viruses (5, 9, 25). These viruses could not be considered exactly the same as inuenza A viruses, since HA was replaced by HEF of inuenza C virus or VSV-G. The establishment of cell

* Corresponding author. Mailing address: Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, 190 Kai Yuan Avenue, Science Park, Guangzhou 510530, China. Phone: 086 20 3229 0481. Fax: 086 20 3201 5343. E-mail: chen_ling@gibh.ac.cn. These authors contributed equally to this work. Published ahead of print on 8 September 2010.

lines stably expressing HA proteins provides an alternative means to produce pseudotyped and single-round-replication inuenza A virus for drug screening (12, 16, 17). However, these recombinant viruses can grow only in cells providing HA in trans and therefore cannot be produced in embryonated eggs, which makes them unsuitable for scale-up production. Attempts have been made to generate replication-competent inuenza viruses by directly incorporating green uorescent protein (GFP) or chloramphenicol acetyltransferase (CAT) genes as the dicistronic NA segment (24), but the incorporated GFP gene was unstable and often silenced. So far, replicationcompetent inuenza virus carrying a gene such as that encoding GFP has not been generated successfully on a large scale for practical use. Recently, the coding region in each vRNA has been found to play critical roles in the selective incorporation of vRNAs into mature progeny virions, as referenced (7, 18). These segmentspecic packaging sequences allow the creation of better inuenza vectors with high efciency of packaging of foreign gene-containing segments (5, 7, 21, 23, 24). One example was that during the revision of this article, a paper was published in which a recombinant inuenza A virus harboring a ninth segment was generated, containing a modied PB1 gene with its original packaging sequences exchanged for those of the NA segment (6). This report describes an improved strategy for generating replication-competent recombinant inuenza A viruses containing the EGFP gene within the NA vRNA segment. The NA vRNA packaging signals, including both the 3 noncoding region (NCR) and the adjacent 183 nucleotides (nt) of the coding region and the 5 NCR and the adjacent 157 nt of the coding region, were maintained for the efcient packaging of recombinant NA vRNAs. A short sequence that codes for a 2A autocleave peptide was inserted between the EGFP and NA

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FIG. 1. Generation of inuenza A viruses rPR8-EGFP NA and rPR8-NA EGFP. (A) Schematic representation of wild-type NA (upper), chimera EGFP NA (middle), and NA EGFP vRNA (lower). The 3 - and 5 -end noncoding regions (NCRs; gray), and 183 nt and 157 nt in the NA-coding regions (black) were responsible for the efcient incorporation of vRNA into virus particles. An autocleave 2A peptide-coding sequence (gray and white text) was inserted between the EGFP (green) and NA (blank) genes. Protein translation start points (3) and stop points () are shown. (B) Eight-plasmid virus rescue system. The bidirectional cytomegalovirus promoter (CMV)-bovine growth hormone poly(A) signal (pA) and Pol I-mTer transcription cassettes in the pM plasmid and the whole eight plasmids are shown. (C) RT-PCR detection of NA (upper), EGFP (middle), and NP (lower) from allantoic uids. (D) EGFP expression in MDCK cells infected with inuenza A virus rPR8-EGFP NA or rPR8-NA EGFP (scale bar 100 m). (E and F) Viral genome stability analysis. Recombinant inuenza A viruses were consecutively inoculated into 10-day-old embryonated eggs for 7 passages. NA and EGFP genes were detected using RT-PCR. pM-PR8-EGFP NA and pM-PR8NA EGFP plasmids were used as positive controls. DNA markers: for DL2000, 2,000, 1,000, 750, 500, 250, and 100 bp, and for DL15000, 15,000, 10,000, 7,500, 5,000, 2,500, 1,000, and 250 bp.

genes, allowing EGFP and NA to be expressed monocistronically (Fig. 1A). Two plasmids, named pM-PR8-NA(183)EGFP-2A-NA and pM-PR8-NA-2A-EGFP-NA(157), were constructed by inserting the EGFP gene into the 3 and 5 ends of NA vRNA in a bidirectional expression pM vector (4) for translation before and after the NA gene, respectively. Recombinant inuenza A viruses rPR8-EGFP NA and rPR8-NA EGFP were successfully rescued in the PR8 background by cotransfection with the other seven plasmids into 293T cells (Fig. 1B). After 48 h, culture supernatants were inoculated into 10-day-old embryonated chicken eggs. Two days later, progeny viruses were harvested from the allantoic uid. Both rPR8-EGFP NA and rPR8-NA EGFP viruses were consecutively propagated for seven passages in 10-dayold embryonated eggs at about 50 PFU per egg. The genomic vRNAs were extracted with Trizol reagent (Invitrogen). Chimeric NA-, EGFP-, and nucleoprotein (NP)-specic bands

were amplied by reverse transcription-PCR (RT-PCR) (Fig. 1C) using specic primers (10). Expression of EGFP mediated by inuenza viruses was conrmed with MDCK cells at 24 h postinfection (p.i.) (Fig. 1D). Specic EGFP and NA recombinant vRNAs could be readily detected from passages 1 to 7 for both rPR8-EGFP NA and rPR8-NA EGFP viruses (Fig. 1E and F). These results indicated that recombinant inuenza A viruses carrying EGFP in NA vRNA, as either rPR8EGFP NA or rPR8-NA EGFP viruses, could be successfully generated and were stable throughout the course of our study. Interestingly, expression of EGFP in MDCK cells infected with these two inuenza A viruses showed different patterns in cellular distribution. Confocal laser scanning analyses were carried out using cells stained with 4 ,6-diamidino-2-phenylindole (DAPI) uorescent nuclear dye and NP-specic antibody (1:500; Southern Biotech) at 36 h p.i. EGFP was conrmed mainly located in the membrane in rPR8-EGFP NA-infected

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FIG. 2. Expression pattern of EGFP in recombinant inuenza A virus-infected MDCK cells. (A) Confocal laser scanning analysis of EGFP localization. MDCK cells were infected with wtPR8, rPR8-EGFP NA, and rPR8-NA EGFP viruses. Cells transfected with the pEGFP plasmid were used as a reference control for original EGFP expression. Cells were xed with formalin and stained with 4 ,6-diamidino-2-phenylindole (DAPI) and NP-specic antibody (dilution 1:500) 36 h later. Scale bar 10 m. (B) Schematic presentation of 2A self-cleavage in protein translation. EGFP and NA fusion proteins are translated from monocistronic mRNA, and the 2A peptide self-cleaves the fusion proteins into individual ones. The translation start and stop points are shown as in Fig. 1. (C) EGFP localization in MDCK cells infected with inuenza viruses from passages 1, 4, and 7. Cells were xed and stained with DAPI at 24 h p.i. and subjected to confocal laser scanning analysis. Scale bar 10 m.

MDCK cells, while EGFP was evenly distributed in rPR8NA EGFP-infected MDCK cells, including nuclei (Fig. 2A). The difference in distribution could be attributed to the fact that in rPR8-EGFP NA virus, fusion of the rst 183 nt of NA mRNA (encoding 61 amino acids [aa], comprising the trans-

location signal and the transmembrane region) to the NH2 terminus of EGFP (Fig. 2B) could lead to the targeting of the EGFP protein to the cell membrane. These viruses obtained not only maintained their genomic stability (Fig. 1E and F) but also were consistent for the patterns of EGFP expression in

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MDCK cells infected with viruses from passages 1, 4, and 7 (Fig. 2C). Western blot analysis using an EGFP-specic antibody (diluted 1:1,000; Southern Biotech) showed specic EGFP protein bands in virus-infected MDCK cells, indicating that EGFP was efciently cleaved by the 2A peptide from the fusion with NA (Fig. 3A). As expected, EGFP expressed by rPR8EGFP NA virus was larger than that expressed by rPR8NA EGFP, as the result of both the fusion of an additional 61-aa peptide from NA to the NH2 terminus of EGFP and the fusion of the 2A peptide to the COOH terminus of EGFP (Fig. 2B). It was reported that the fusion of the transmembrane domain of NA to bioactive cytokines could lead to their incorporation into viral particles (26). We next carried out an experiment to assess the presence of EGFP in the virions. Recombinant rPR8-EGFP NA and rPR8-NA EGFP inuenza viruses were puried from allantoic uid through twostep glucose cushion ultraspeed centrifugation (Beckman SW28 rotor) (5). We were able to detect the presence of the EGFP protein in rPR8-EGFP NA viral particles but not in rPR8-NA EGFP viral particles (Fig. 3B), conrming that the EGFP protein was incorporated into virions. The NA enzymatic activity from puried virus was measured using an NA substrate, 2 -(4-methylumbelliferyl)- -D-acetylneuraminic acid (MUNANA) (Sigma), as described previously (1). The amounts of NA in rPR8-EGFP NA and rPR8-NA EGFP viruses were compared with those in wtPR8 virus under the same amount of virion (Fig. 3C). The rPR8-NA EGFP virion possessed nearly as much NA activity ( 90%) as the wtPR8 virus, while the rPR8-EGFP NA virion possessed much less NA activity ( 17%), which may be the result of EGFP interference and the truncation of the N-terminal region of the NA protein. We wondered if the insertion of the EGFP gene into NA vRNA may affect the efciency of packaging of NA vRNA into the virions. A SYBR green I-based quantitative PCR assay using segment-specic primers for NA, NP, and PB2 (16) was used to determine the efciency of packaging of the NA segment into the virions. Compared wtPR8, nearly 80% of NA EGFP vRNA segments were packaged into the rPR8NA EGFP virions, while fewer than 50% of EGFP NA vRNA segments were packaged into the rPR8-EGFP NA virions (Fig. 3D).

We next evaluated the growth characteristics of these recombinant viruses, as these viruses are different in fusion arrangement and in the amounts of NA vRNAs and NA proteins in their virions. The plaque-forming capabilities in infected MDCK cells were tested by immune staining with an anti-NP monoclonal antibody as described previously (5). The results revealed that the plaques formed by rPR8-EGFP NA were smaller and that the plaques formed by rPR8-NA EGFP were similar to wtPR8 (Fig. 3E), indicating a signicant attenuation of the rPR8-EGFP NA virus. We then compared the growth curves of the rPR8-EGFP NA, rPR8-NA EGFP, and wtPR8 viruses in embryonated eggs (Fig. 3F), in MDCK cells (Fig. 3G), and in A549 cells (Fig. 3H). Viral titers in allantoic uid samples and cell culture medium collected at various time intervals were measured using a plaque formation assay. The rPR8-EGFP NA virus was signicantly attenuated, and its virus yield was lower than that of wtPR8. The rPR8NA EGFP virus maintained a growth rate similar to that for wtPR8. The NA activities measured for cell culture medium from MDCK and A549 cells infected with respective viruses also correlated with the growth characteristics of these viruses, with the rPR8-EGFP NA virus having the lower NA activity level (Fig. 3I and J). Replication-competent recombinant inuenza A viruses expressing a reporter gene can be used in many applications for studying inuenza A virus and developing vaccines and antiviral therapeutics. These viruses should have advantages for in vitro and in vivo studies over the pseudotyped lentivirus (20) and pseudotyped inuenza reporter virus (17). We thus tested a mouse anti-PR8 serum by incubating a series of 5-fold diluted sera with the rPR8-NA EGFP virus for 2 h, followed by infection of MDCK cells. EGFP-expressing cells were observed at 24 h p.i. under a uorescence microscope (Fig. 4A), and mean uorescence intensity was measured by uorescence-activated cell sorting (FACS) (Fig. 4B). With increases in the titer of anti-PR8 serum, there were decreases in the number of EFGP-positive cells and the uorescence intensity of EGFP. This proof-of-principle experiment indicated that replication-competent inuenza A viruses with a reporter can be used as a rapid-readout system for screening neutralizing antibodies or antiviral drugs for inuenza A virus. In this study, we demonstrated that the combination of 183 and 157 nt in the NA-coding region anked by 3 and 5 NCRs and the use of a synthetic autocleave 2A peptide between

FIG. 3. Virological characteristics of inuenza A viruses rPR8-EGFP NA and rPR8-NA EGFP. (A) Western blot analysis of EGFP in virus-infected MDCK cells. (B) Western blot analysis of EGFP in puried viral particles. Viral NP and M1 proteins were utilized as a normalization control. Cells transfected with the pEGFP plasmid were used as a positive control (Con.) for original EGFP. (C) Measurement of NA enzymatic activities in the puried viral particles. The enzymatic activity of the NA protein was measured using the NA substrate 2 -(4-methylumbelliferyl)-D-acetylneuraminic acid (MUNANA) (Sigma) and normalized to the level for the parental wtPR8 virus by use of HA titers. (D) Efciencies of packaging of NP, PB2, and NA vRNA into viral particles. The abundances of the NP, PB2, and NA segments were qualied by SYBR green I-based quantitative PCR method with segment-specic primer pairs, and their relative abundances were normalized to the level for the wtPR8 virus. (E) Comparison of plaque sizes in MDCK cells infected with rPR8-EGFP NA, rPR8-NA EGFP and wtPR8 viruses. Cells were infected with diluted viruses and overlaid with 0.8% agarose gel. Plaques were immunostained with anti-NP monoclonal antibody (dilution 1:500) at 48 h p.i. (F) Curves for virus growth in embryonated eggs. Ten-day-old embryonated chicken eggs were inoculated with 100 PFU of rPR8-EGFP NA, rPR8-NA EGFP, and wtPR8 and incubated at 37C. The average titers of the virus from triplicate eggs at each time point are shown (mean standard deviation [SD]). Lg, log10. (G) Virus growth curves in MDCK cells. (H) Virus growth curves in A549 cells. (I) NA activities in MDCK cells. (J) NA activities in A549 cells. MDCK and A549 cells were infected at a multiplicity of infection (MOI) of 0.001. Cell culture media were collected at the different time points. The virus titers and NA activities (relative uorescence units [RFU]) were measured. The data shown are means SD for triplicate wells at each time point.

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FIG. 4. Application of inuenza A virus rPR8-NA EGFP virus for evaluating antiviral neutralizing antibody. The rPR8-NA EGFP virus was incubated in 5-fold-diluted mouse serum against the wtPR8 virus and negative-control serum (Con. Serum) and in 1 phosphate-buffered saline (PBS) buffer for 2 h before application to MDCK cells. At 24 h p.i., EGFP was observed under inverted uorescence microscopy (A), and its expression levels were analyzed with FACS from triplicate wells (mean SD) (B). MFI, mean uorescence intensity; mock, no virus infection control. Scale bar 100 m.

EGFP and NA resulted in a great outcome. Previous reports indicate that neither the insertion of the EGFP gene into the inuenza viral genome in the NS1- or NA-encoding segment nor the use of dicistronic expression cassettes was successful, as the EGFP expression was often unstable or the expression level was very low or even undetectable (11, 14, 24). An earlier report indicates that the insertion of the CAT gene with the 2A sequence into NA vRNA was exploited, but there were no follow-up reports for its application (22). We speculate that the disruption of NA vRNA packaging signals at the 3 end in that study may have resulted in signicant attenuation of the virus, which rendered scale-up production and application difcult. During the revision of this article, a newly published paper described the generation of a nine-segmented inuenza A virus in which the PB1 open reading frame was anked by the NA segment-specic packaging sequences, including the NCRs and the coding region packaging signals (6), indicating the importance of NAs coding region. Our strategy of including 183 and 157 nt in the NA-coding region improved chimera NA vRNA packaging efciency. Instead of using a different promoter (24) or internal ribosome entry site sequence, introduction of the 2A fusion peptide is the most simple and effective way to insert an exogenous gene into a vRNA segment. The presence of EGFP in the context of replication-competent inuenza virus has several advantages. (i) The recombinant inuenza viruses harboring the EGFP gene in the NA segment in both formats can be rescued and propagated to a large quantity in embryonated eggs and, importantly, are stable for multiple passages. (ii) The expression of EGFP allows direct measurement of viral infection and replication in target cells without the need for using chemical (e.g., NA enzyme assay), biological (e.g., plaque assay or hemagglutinin inhibition assay), and molecular (e.g., PCR) ap-

proaches which in general are more time-consuming and costly. (iii) For the rPR8-EGFP NA virus, the fusion of the NA-anchoring transmembrane region to EGFP results in the localization of the EGFP protein to the cell membrane, thus enabling live monitoring the inuenza virus in vivo. (iv) The rPR8-NA EGFP virus has biological characteristics similar to those of the parental virus and thus has great potential for use in evaluating anti-inuenza drugs and neutralizing antibodies. In summary, we demonstrated a new strategy for generating replication-competent recombinant inuenza A viruses containing a reporter gene, such as that encoding EGFP, in NA vRNA while maintaining all native viral genes. These reporter viruses can replicate and propagate without the need for complementary cells to provide NA proteins in trans and are stable for multiple passages of propagation in embryonated eggs. Future studies for incorporating luciferase into this format should enable more-powerful assessment of inuenza virus infection and migration in animal models.
This study was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (no. KSCX1-YW-10), the National Key Science & Technology Specic Projects of China (2008ZX10001011), the National Science Fund for Distinguished Young Scholar (no. 30688004), and the Guangdong Natural Science Fund (no. 06200872).
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