Generation of recombinant A/PR/8/34 viruses carrying a ninth GFP segment.
At restrictive temperature, a temperature-sensitive influenza A virus has been shown capable of containing two sets of nonstructural protein (NS) segment-specific packaging signals located in two different segments: one set was derived from an NS segment that has a temperature-sensitive defect in the NS1 gene, and a second set was from the segment that carries a wild-type NS1 gene (
2). In the present study, to determine whether influenza A virus was able to incorporate two copies of NA segment-specific packaging sequences, the packaging signals of the PB1 segment were switched to those from the NA segment (Fig.
1A, left), while the original NA segment was unchanged. To accomplish this, the A/PR/8/34 PB1 ORF that carried serial synonymous mutations at the two ends, named PB1mut (Fig.
1A, left), was flanked by the NA segment-specific packaging sequences (including the 3′ and 5′ NCRs, as well as the terminal coding sequence of the NA ORF), thus generating the NA-PB1mut-NA segment (Fig.
1A, left). Based on our previous findings that the partial packaging signals in the HA or NS ORF region can affect viral RNA incorporation (
7), we decided to silently mutate the two ends of the PB1 ORF. The synonymous mutations in the PB1mut ORF region include 24-nucleotide (nt) and 17-nt changes in the 3′- and 5′-proximal regions, respectively. The ATGs in the 3′-proximal NA region of the chimeric NA-PB1mut-NA segment were all mutated by site-directed mutagenesis so that translation would be initiated at the PB1mut gene start codon. Based on our previous findings for the HA and NS segments (
7) and data from other studies (
4,
8,
9,
11-
13), we surmised that this chimeric NA-PB1mut-NA construct in Fig.
1A would most likely utilize the flanking NA packaging signals due to the absence of proper PB1-specific packaging sequences.
Using reverse genetics, a −PB1(ps) virus that carries seven wild-type A/PR/8/34 RNA segments (PB2, PA, HA, NP, NA, M, and NS) and one chimeric NA-PB1mut-NA segment was successfully rescued (Fig.
1B). The −PB1(ps) virus was attenuated compared with wild-type A/PR/8/34 virus, with lower titers in eggs and smaller plaques in MDCK cells (Fig.
1E and F). To determine whether the −PB1(ps) virus was able to incorporate a ninth segment that had PB1 segment-specific packaging signals, we generated a PB1-GFP-PB1 construct that carried 153 nt of PB1 packaging sequences in the 3′ end and 159 nt in the 5′ end (Fig.
1A, right). These 153-nt and 159-nt sequences consisted of both NCRs and terminal coding region packaging sequences and the six ATGs located in the 3′ 153-nt PB1 packaging sequences were all mutated by site-directed mutagenesis. We then successfully generated the −PB1(ps)+GFP virus that had all eight segments of the −PB1(ps) virus and a ninth GFP segment with PB1 segment-specific packaging signals (Fig.
1B). −PB1(ps)+GFP virus exhibited similar growth characteristics to the −PB1(ps) virus, with similar titers in eggs and similar plaque phenotypes in MDCK cells (Fig.
1E and F). The −PB1(ps)+GFP virus was stable, and GFP expression in infected cells (Fig.
1G) was maintained over 5 passages in eggs by the limiting dilution technique. The percentage of GFP-expressing plaques formed by the −PB1(ps)+GFP virus also did not change over 5 passages in eggs (see the supplemental material).
Following the same strategy, the packaging signals of the PB2 and PA segments were also each replaced with those of NA. Chimeric constructs NA-PB2mut-NA and NA-PAmut-NA were generated (Fig.
1A, left). The PB2mut ORF had 13-nt synonymous changes in the 3′ end and 36 nt in the 5′ end to inactivate the PB2 ORF region packaging signals, and the PAmut ORF region carried 19-nt synonymous changes in the 3′ end and the same number of changes in the 5′ end to inactivate the PA ORF region packaging signals (Fig.
1A, left). The two chimeric GFP constructs PB2-GFP-PB2 and PA-GFP-PA, which respectively carried PB2 and PA segment-specific packaging sequences, were made using the same method utilized to produce the PB1-GFP-PB1 construct (Fig.
1A, right). The 3 ATGs in the 3′-end 158-nt PB2 packaging sequences of the PB2-GFP-PB2 and 3 ATGs in the 3′-end 129-nt PA packaging sequences of the PA-GFP-PA construct were all mutated to TTGs in order for the GFP gene to utilize its own initiation codon (Fig.
1A, right). For the PB2 segment, we were unable to rescue a virus that has seven wild-type A/PR/8/34 RNA segments (PB1, PA, HA, NP, NA, M, and NS) and one chimeric segment, NA-PB2mut-NA. However, when a ninth PB2-GFP-PB2 construct was added, the −PB2(ps)+GFP virus was successfully rescued (Fig.
1C). The −PB2(ps)+GFP virus grew in eggs to a titer similar to that of the −PB1(ps)+GFP virus (Fig.
1E), but it produced slightly smaller plaques in MDCK cells (Fig.
1F). The expression of GFP in infected cells (Fig.
1G) and the percentage of GFP-expressing plaques (see the supplemental material) were also stably maintained over at least five passages in embryonated chicken eggs by the limiting dilution technique. For the PA segment, we successfully rescued a −PA(ps) virus that has seven wild-type A/PR/8/34 segments (PB2, PB1, HA, NP, NA, M, and NS) and one chimeric segment, NA-PAmut-NA (Fig.
1D). The −PA(ps)+GFP virus carrying the ninth PA-GFP-PA segment was also successfully rescued (Fig.
1D). The −PA(ps) and −PA(ps)+GFP viruses were more attenuated than the −PB1(ps), −PB1(ps)+GFP, and −PB2(ps)+GFP viruses, growing to lower titers in eggs (data not shown) and generating smaller plaques in MDCK cells (Fig.
1F). Due to small plaque size, the infectious titers of the −PA(ps) and −PA(ps)+GFP viruses could not be accurately measured and we therefore did not further characterize their growth rates in eggs. The GFP expression by the −PA(ps)+GFP virus in infected cells (Fig.
1G) was, however, stably maintained over at least five passages in embryonated chicken eggs. Finally, although the infectious titers of the −PB1(ps), −PB1(ps)+GFP, and the −PB2(ps)+GFP viruses from eggs were much lower than that of recombinant A/PR/8/34 (rA/PR/8/34) virus (Fig.
1E), their hemagglutination assay titers were comparable to that of the rA/PR/8/34 virus 2 and 3 days postinoculation (Fig.
1H), suggesting that these viruses produced more defective virions than does the wild-type virus.
It should be noted that the number of synonymous mutations introduced to disrupt the packaging signals in the ORF region and the length of the flanking packaging sequences used in the chimeric constructs (Fig.
1A) were decided upon previous characterization of the A/WSN/33 viral RNA packaging signals (
5,
10,
11,
13,
15).
In conclusion, we generated a novel approach to construct several nine-segment influenza viruses simply by manipulating the RNA packaging sequences. The resulting viruses were genetically stable and carried an extra GFP segment. Linearity between dilutions and plaque numbers was also observed for these nine-segment viruses, suggesting indeed more than eight RNAs can be incorporated into one particle (data not shown).
Generation of recombinant influenza viruses carrying both A/PR/8/34(H1N1) and A/HK/1/68(H3N2) HAs.
We next sought to determine whether our method for generating the nine-segment GFP-expressing virus could be used to generate influenza viruses coding for two subtypes of HA: the A/PR/8/34(H1N1) HA and the HA from A/HK/1/68(H3N2). To do this, the GFP ORF regions of the PB1-GFP-PB1 and PB2-GFP-PB2 constructs (Fig.
1A, right) were each replaced by the A/HK/1/68 HA ORF, generating the PB1-HA(HK)-PB1 and PB2-HA(HK)-PB2 constructs (Fig.
2A). Using reverse genetics, we were able to successfully rescue two nine-segment viruses named −PB1(ps)+HK HA (Fig.
2B) and −PB2(ps)+HK HA (Fig.
2C). The −PB1(ps)+HK HA virus and the −PB2(ps)+HK HA virus had similar growth characteristics to the −PB1(ps)+GFP and −PB2(ps)+GFP viruses (Fig.
1E and
2D), respectively. In order to show that both the A/PR/8/34 and the A/HK/1/68 HAs were incorporated into particles of the −PB1(ps)+HK HA and −PB2(ps)+HK HA viruses, four viruses (rA/PR/8/34; X31, which has six A/PR/8/34 internal genes and the A/HK/1/68 HA and NA genes; −PB2(ps)+HK HA; and −PB1(ps)+HK HA) were grown in eggs and concentrated by passing through a sucrose cushion. Western blotting was then performed to detect the A/PR/8/34 and A/HK/1/68 HAs in purified virions (Fig.
2E). The results showed that when the same amounts of virus proteins were loaded, the −PB1(ps)+HK HA and −PB2(ps)+HK HA viruses had similar levels of A/PR/8/34 HA protein compared with the wild-type rA/PR/8/34 virus; this includes uncleaved HA0 and cleaved HA1 detected by the mouse MAb PY102 (Fig.
2E). Also, when comparable amounts of virus proteins were loaded, rA/PR/8/34 and X31 had the same amount of NP protein detected by MAb HT103 (Fig.
2E). However, for the −PB1(ps)+HK HA and −PB2(ps)+HK HA chimeric viruses, the NP levels were about five times lower than those of rA/PR/8/34 and X31 viruses (Fig.
2E), indicating a less-efficient RNP incorporation by the nine-segment viruses. Both HA0 and HA1 from A/HK/1/68 were detected in the −PB1(ps)+HK HA and the −PB2(ps)+HK HA virus particles using MAb 66A6; notably, when normalized for total protein, H3 HA incorporation by the chimeric viruses was much lower than incorporation by the X31 virus, with the lowest levels seen in the −PB1(ps)+HK HA virus (Fig.
2E). The Western blot using MAb 12D1 to detect A/HK/1/68 HA0 and cleaved HA2 showed similar results (Fig.
2E). We then used Western blotting to detect the expression of both A/PR/8/34 and A/HK/1/68 HAs by the −PB1(ps)+HK HA and −PB2(ps)+HK HA viruses in infected cells (Fig.
2F). As expected, both A/PR/8/34 and A/HK/1/68 HAs were detected in MDCK cells infected by these viruses (Fig.
2F, lower panel). In contrast, as with Fig.
2E, cells infected with rA/PR/8/34 virus expressed only A/PR/8/34 HA and the X31 virus-infected cells expressed only H3 HA (Fig.
2F, upper panel).
Finally, a sandwich ELISA was performed to confirm that both subtype H1 and H3 HA proteins were incorporated into the nine-segment virus particles (Fig.
2G). Ninety-six-well plates were coated with MAb 66A6 (
27) to capture intact virus particles in an H3-dependent manner. Virus particles were then probed for H1 content with MAb C179, an antibody with activity against subtype H1 and H2 HA but which does not react with H3 HA (
20). Signals were detected for the two nine-segment viruses, indicating that indeed two types of HA proteins were incorporated into the virus particles. In contrast, both rA/PR/8/34 and X31 viruses gave negative results (Fig.
2G).
In conclusion, we successfully rescued two recombinant viruses, each of which carried two subtypes of HA: one A/PR/8/34(H1N1) HA and one A/HK/1/68(H3N2) HA. Both HAs were incorporated into virus particles and were expressed in virus-infected MDCK cells.
To determine the RNA packaging efficiencies of the recombinant −PB1(ps)+HK HA and −PB2(ps)+HK HA viruses, RNA was isolated from the purified viruses and resolved on a 2.8% acrylamide gel followed by silver staining (Fig.
2H). The X31 virus has six A/PR/8/34 internal genes along with the A/HK/1/68 HA and NA segments which migrated to distinct positions from those of the A/PR/8/34 HA and NA (Fig.
2H). By comparing densities of bands, we observed that the −PB1(ps)+HK HA virus inefficiently incorporated the NA-PB1mut-NA segment. The PB1-HA(HK)-PB1 segment was also packaged somewhat inefficiently when compared with the A/PR/8/34 HA segment (Fig.
2H). For the −PB2(ps)+HK HA virus, the NA-PB2mut-NA segment was inefficiently packaged. In contrast, the PB2-HA(HK)-PB2 segment was packaged efficiently, at a level similar to that of A/PR/8/34 HA (Fig.
2H).
Immunization of mice with a recombinant nine-segment virus with two HAs conferred protection from lethal challenges of rA/PR/8/34 and X31 viruses.
To test whether the nine-segment influenza viruses carrying two subtypes of HA could be used as live vaccines, mouse challenge experiments were conducted. The −PB1(ps)+HK HA virus was arbitrarily chosen for the study. As a negative control immunogen, we used the −PB1(ps)+Luc virus, which carries a ninth PB1-Luc-PB1 instead of a PB1-HA(HK)-PB1 segment (Fig.
2B; see the supplemental material and Materials and Methods). Both −PB1(ps)+Luc and −PB1(ps)+HK HA viruses grew to similar titers as the −PB1(ps) virus in eggs (Fig.
3A). To test whether the nine-segment viruses were pathogenic in mice, groups of 8-week-old female C57BL/6 mice were given PBS, −PB1(ps)+HK HA virus, or −PB1(ps)+Luc virus, at either 10
3 or 10
4 PFU by intranasal administration (Fig.
3B). The mice infected with 10
4 PFU of either −PB1(ps)+Luc or −PB1(ps)+HK HA virus died or lost more than 25% of their initial body weight by day 8 postinfection (Fig.
3B). The group of mice given 10
3 PFU of −PB1(ps)+Luc exhibited little or no weight loss and exhibited no signs of disease, similar to the PBS group (Fig.
3B). The group of mice given 10
3 PFU of −PB1(ps)+HK HA virus lost approximately 5% of their body weight by day 7 postinfection followed by full recovery within 3 days; no other signs of disease were observed (Fig.
3B). Since administration of 10
3 PFU of either chimeric virus caused very little or no changes associated with illness, we considered exposure to this dose to be analogous with vaccination.
Three weeks postinfection, lethal virus challenge experiments were performed on the groups of mice infected with 10
3 PFU of −PB1(ps)+Luc virus, 10
3 PFU of −PB1(ps)+HK HA virus, or mice that were mock vaccinated with PBS. Mice were given 3,000 PFU (100 MLD
50) of rA/PR/8/34 virus by intranasal administration (Fig.
3C). In contrast to the PBS group, the groups vaccinated with either the −PB1(ps)+Luc or the −PB1(ps)+HK HA viruses were completely protected from lethal challenge: no loss of body weight or signs of disease were observed (Fig.
3C). Following the same methods, 10
7 PFU (33 MLD
50) of X31 virus was administered intranasally to a second set of mice that were mock vaccinated (PBS group), vaccinated with 10
3 PFU −PB1(ps)+Luc, or vaccinated with 10
3 PFU −PB1(ps)+HK HA virus (Fig.
3D). The groups of mice that were mock or −PB1(ps)+Luc vaccinated quickly lost 25% of their body weight in 3 days and were sacrificed. Although previous findings showed that cellular responses to the internal NP and M proteins conferred some protection against heterologous challenges (
30), no protection was observed in the −PB1(ps)+Luc-vaccinated group, possibly due to the high dosage of challenge virus used. In contrast, vaccination with 10
3 PFU of −PB1(ps)+HK HA virus protected the mice from the lethal challenge with X31 virus. Average body weight was reduced by 10% on the day following challenge, and all mice quickly recovered (Fig.
3D).
Analysis of serum samples from this experiment indicated that by day 21 postvaccination, all animals vaccinated with 10
3 PFU of −PB1(ps)+HK HA virus produced hemagglutination-inhibiting antibodies against rA/PR/8/34 virus, with titers ranging from 320 to 640. Four out of five animals produced a low but detectable level of hemagglutination-inhibiting antibodies against X31 virus, with titers ranging from 20 to 40 (Table
1). As expected, animals vaccinated with 10
3 PFU of −PB1(ps)+Luc virus had only hemagglutination-inhibiting antibodies against rA/PR/8/34 virus, with titers ranging from 160 to 320 (Table
1). No hemagglutination-inhibiting antibodies against either rA/PR/8/34 or X31 virus were detected in serum from animals mock vaccinated with PBS.
In conclusion, we have shown that vaccination with 103 PFU of −PB1(ps)+HK HA virus was protective in mice against lethal challenge with influenza viruses from two separate subtypes: one H1N1 subtype (rA/PR/8/34) and one H3N2 subtype (X31).