The Hierarchical Sequence Requirements of the H1 Subtype-Specific Noncoding Regions of Influenza A Virus

ABSTRACT The genome of influenza A virus consists of eight single-stranded viral RNA (vRNA) segments. The nonconserved noncoding regions (NCRs) at the 3′ and 5′ termini of each segment show extremely low divergence and mutation rate. They appear as segment specific among the eight segments and also subtype specific among different subtype-determinant hemagglutinin (HA) and neuraminidase (NA) segments. In order to acquire in-depth knowledge on the sequence requirements of the segment-specific or subtype-specific NCRs (ssNCRs), we, in the context of WSN (H1N1) reverse genetics, designed a virus random nucleotide selection assay (vRNSA) in which we generated pHW2000-HA plasmid libraries with random nucleotides in each grouped nucleotide positions in the 3′ and 5′ H1-ssNCRs, followed by virus rescue, serial passage, and deep sequencing. The resulting sequence logos present a visualized dynamic overview of the hierarchical sequence requirements of the 3′ and 5′ H1-ssNCRs. It showed that, in the process of continuous passage, the 3′ H1-ssNCR, in general, stabilized more quickly than the 5′ H1-ssNCR. The nucleotides close to the highly conserved 3′ and 5′ promoter regions showed higher sequence stringency than nucleotides away from the promoter regions. All stabilized sequences displayed a common feature of high A/U ratios. Especially with our mutational function analyses, we demonstrate that the 3′ promoter-proximal nucleotides could cooperatively exert a direct effect on the transcription and replication of the HA segment. Together, these results provide in-depth knowledge for understanding the NCRs of influenza A virus. IMPORTANCE The segment-specific and subtype-specific nonconserved noncoding regions (ssNCRs) at both 3′ and 5′ ends of viral RNA segments of influenza A virus are largely conserved among the same segments of different viruses. However, the function-related sequence requirements of these ssNCRs remain unclear. In this study, through a novel self-designed vRNSA approach, we present a visualized dynamic overview diagram directly reflecting the hierarchical sequence requirements within and between the 3′ and 5′ H1-ssNCRs. The in-depth functional mutagenesis analyses further revealed that specific nucleotides in the 3′ promoter-proximal region could cooperatively exert a direct effect on viral RNA transcription and replication. This work further advanced our knowledge in understanding the nonconserved noncoding regions of influenza A viruses.

I nfluenza A viruses are highly infectious respiratory pathogens posing a great threat to public health (1). Influenza A virus is an eight-segmented, negative-sense RNA virus of the family Orthomyxoviridae. Within the virions, each viral RNA segment is present in the form of viral ribonucleoprotein complexes (vRNPs) in which the RNA associates with nucleoprotein (NP) oligomers and a heterotrimeric RNA-dependent RNA polymerase formed by polymerase basic 1 (PB1), PB2, and polymerase acid (PA) (2). The vRNPs are the minimal functional unit required for viral RNA transcription (vRNA!mRNA) and replication (vRNA$cRNA) to occur in the nucleus of infected cells (3). The strategies used by the viral RNA polymerase to initiate and terminate the transcription and replication are very different (4). The initiation of vRNA!mRNA synthesis is primer dependent involving a host cap-snatching process while the initiations of vRNA$cRNA syntheses are primer independent but with internal initiation on the cRNA promoter and terminal initiation on the vRNA promoter (5)(6)(7). The termination of mRNA synthesis occurs by repeatedly copying a stretch of U close to the 59 end of vRNA leading to viral mRNA polyadenylation (8), while the replication is the full-length copy between vRNA and cRNA. Both vRNAs and cRNAs are assembled into vRNPs and cRNPs along with their syntheses (9). When sufficient vRNPs are produced in the nucleus, they get exported to the cytoplasm and travel across cytoplasm to the plasma membrane where they are selectively packaged into progeny virions (10,11).
The eight vRNA segments of the influenza genome, designated polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acid (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and nonstructural protein (NS), contain one or two major open reading frames (ORFs) in negative sense, which are flanked by noncoding regions (NCRs) at both ends. The NCRs of each segment consist of a highly conserved promoter region (12 and 13 nucleotides from the 39 and 59 ends, respectively) followed by the nonconserved noncoding region that are segment specific among the eight segments and subtype-specific NCRs among the two subtype-determinant HA and NA segments (12,13). These segment-specific or subtype-specific NCRs (ssNCRs) vary significantly in sequence and length (from 5 to 45 nucleotides), at the 39 and 59 ends of different segments (13)(14)(15)(16). It has been reported that, compared with the coding region, these ssNCRs are largely conserved in the same vRNA segments of different influenza virus strains with extremely low divergence and evolutionary rate (17).
It has been demonstrated that segment-specific NCRs play multiple roles in the life cycle of influenza A viruses. The 39-and 59-segment-specific NCRs together with adjacent terminal coding regions have been reported for each segment acting as selective packaging signals (18)(19)(20)(21)(22)(23)(24)(25)(26)(27). The replacement of H1-subtype-specific NCRs with other subtypespecific NCRs at both ends or at single ends in the context of H1N1 virus have suggested that the 39 HA ssNCR plays a more critical role than the 59 HA ssNCR in regulating virus multiplication, and no stringent compatibility between the two ends is required (13). In addition, earlier studies have shown that the simultaneous deletions of ssNCRs at both ends in the NA segment of the influenza virus A/WSN/33 (H1N1) significantly reduced the replication of this segment (28). Moreover, the HA-specific nucleotides in the 39 and 59 ssNCRs forming the extended duplex region of the promoters were found to play dual roles in regulating RNA syntheses and controlling virus selective genome packaging (16). Recently, we have reported that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multisegment environment (29). However, the sequence requirements of these ssNCRs remain unclear.
In this study, we aimed to characterize the sequence requirements of the ssNCRs in the context of the H1 segment of influenza A virus. We designed a novel virus random nucleotide selection assay (vRNSA) to explore the nucleotide selectivity of the H1-ssNCRs during virus passage. Through this assay and our in-depth functional studies, we have obtained in-depth knowledge in understanding the basic characteristics of H1-ssNCRs. An A19U mutant and the appearance of its recovery U23G mutation further highlight the essentiality of the 39 promoter-proximal nucleotides in regulating viral RNA transcription and replication.

RESULTS
The hierarchical sequence requirements within and between the 39 and 59 H1-ssNCRs upon virus serial passage. To explore the sequence requirements of H1-ssNCRs for virus propagation, in the context of WSN (H1N1) reverse genetics, we designed a virus random nucleotide selection assay (vRNSA) in which we provided random nucleotides at each nucleotide position in the 39 and 59 H1-ssNCRs, followed by virus rescue passage 0 (P0); six generation passages in Madin-Darby canine kidney (MDCK) cells; and deep sequencing at P0, P2, P4, and P6 (Fig. 1A). Considering the limitations of transformation efficiency in plasmid preparation and transfection efficiency during virus rescue, we divided the 39 and 59 H1-ssNCR into three groups. Each 39 H1-ssNCR group contained six random nucleotides ( (Fig. 1B). For those nucleotides with essential functions were kept unchanged, such as a U nucleotide at 23 position before the start codon, the HAspecific CC nucleotides adjacent to the 39 promoter region, and the stretch of U, the HA-specific GUG nucleotides adjacent to the 59 promoter region (12,16,30). Based on this principle, we generated six pHW2000-HA rescue plasmid libraries (3'G1, 3'G2, 3'G3, 5'G1, 5'G2, and 5'G3) with random nucleotides in each group by using primers containing degenerate bases in each nucleotide position.
We then conducted the vRNSA with these plasmid libraries in two independent experiments. Considering that the untranslated regions of influenza viruses have an extremely low divergence and evolutionary speed (17), together with the fact that they are very stable during passages in cell culture, we did not perform next-generation sequencing (NGS) for the wild-type (WT) WSN virus. The NGS results of the rescued viruses with randomly mutated NCRs showed that, in the first round of the virus rescue (P0), the nucleotides in 5'G3 and 5'G2 at P0 are completely in a disordered state with the relatively equal ratio of the four types of nucleotides (Fig. 2B), confirming that the nucleotide randomization has worked well and the mutagenesis was not biased. The relatively unequal ratios of the four nucleotides shown in 3'G1, 3'G2, 3'G3, and 5'G1 indicate that the selection has obviously occurred during the initial virus rescue step ( Fig. 2A and B). Fig. 2A showed that, within the 39 H1-ssNCR, all nucleotides in the three 3'G1, 3'G2, and 3'G3 groups were stabilized into a constant sequence at P6. However, the stabilization speed of the nucleotide sequence in 3'G1 was much faster than that in 3'G2 and 3'G3. Moreover, the stabilized sequence in 3'G1 was exactly the same as that in the wild-type virus in both replicates, whereas the stabilized sequences in 3'G2 and 3'G3 not only differed from the wild-type sequence but also differed in the two replicates. Fig. 2B showed that, within the 59 H1-ssNCR, only the nucleotides in 5'G1 were stabilized to a constant sequence at P6 in which the two replicates showed one wild-type sequence and one non-wild-type sequence. For those nucleotides distant from the 59 promoter in 5'G2 and 5'G3, they were still in chaotic sequence. These results suggest strongly that the nucleotides close to the 39 and 59 promoters require higher sequence stringency than those nucleotides distant from the 39 and 59 promoter.
On the other hand, the extents of sequence flexibility were significantly different between the 39 and 59 H1-ssNCRs (compare Fig. 2A and B) in which the 59 H1-ssNCR showed much higher sequence variation than the 39 H1-ssNCR. This result unveils the hierarchical sequence requirements within and between the 39 and 59 H1-ssNCRs.
The stabilized sequences are sufficient for virus replication in cell culture and show a feature of high A/U ratios. Since the stabilized sequences in 3'G2, 3'G3, and 5'G1 showed different sequences from the wild type, we then went on to examine the effects of these non-wild-type sequences on virus replication efficiencies and their stability along serial passage in cell culture. With the reverse genetics, we generated five mutant viruses containing the stabilized non-wild-type sequences as shown in Fig. 3A. The simultaneously rescued wild-type and mutant WSN viruses were then passaged for two generations in MDCK cells, and the replication kinetics of wild-type and mutant viruses was examined in MDCK or A549 cells. As expected, the growth curves of all mutant WSN viruses in MDCK and A549 cells were similar to those of the wild-type virus (Fig. 3B). We subsequently plaque purified the mutant viruses, passaged them for six generations, and sequenced both the 39 and 59 NCRs of the HA segment. The results showed that the mutant sequences remained stable and no other mutations were generated in the 39 and 59 NCRs of the HA segment (Fig. 3C). These results illustrate that the stabilized non-wild-type sequences selected by the virus are sufficient for efficient virus replication in the cell culture.
Next, we analyzed the nucleotide composition of these stabilized non-wild-type sequences. The results showed that the proportion of A and U bases to the total bases (A/ U ratio) was above 4/6 in the stabilized mutant sequences in the 39 and 59 H1-ssNCRs, e.g., the 3'G3-1 group was entirely composed of A and U bases (Table 1). In addition, the A/U ratios of the corresponding wild-type sequences were all higher than 4/6. Overall, the results above suggest that the H1-ssNCR nucleotides display a higher requirement of the proportion of A and U bases than the actual sequences.
A point mutation introduced at a fast-stabilized position A19U affects viral replication efficiency. Since the wild-type sequence was selected with an extremely high A/U ratio in 3'G1 at P6 in the two independent replicates, together with the observation that 15U, 17U, and 19A in 3'G1 were the fastest stabilized in P2 and remained stable from P2 to P6 passages, we were interested in examining the effects of these As or Us in regulating virus replication. To avoid changing the A/U ratio mentioned earlier, we mutated nucleotides 15U, 17U, and 19A into 15A, 17A, and 19U, respectively.
First, we examined the effects of these mutants on viral RNA synthesis and protein production in an RNP reconstitution system derived from the WSN virus (31). The wildtype or mutant pPOLI-HA RNA-expressing plasmid was individually cotransfected with pcDNA-PB2, pcDNA-PB1, pcDNA-PA, and pcDNA-NP plasmids in 293T cells. At 24 h posttransfection, the total RNA of the cells was extracted, and the levels of three viral RNA species (mRNA, cRNA, and vRNA) were detected by primer extension analysis. As shown in Fig. 4A and B, U15A and U17A mutations caused a slight reduction in mRNA and cRNA levels and had no effect on vRNA level, whereas the A19U mutation significantly attenuated the level of all three species of viral RNAs; in particular, cRNA showed 10% of the wild-type cRNA level. We also performed Western blotting to examine the protein production with a monoclonal anti-HA antibody. Consistent with the mRNA levels, U15A and U17A mutations caused a slight decrease in the levels of HA protein, while the A19U mutation caused significantly reduced HA protein levels (.50%) ( Fig. 4C and D). Next, we examined the effect of these mutations on viral replication efficiency. The wild-type and mutant viruses were rescued with the WSN reverse genetics, and the titers of the rescued viruses (P0) were examined by plaque assay. The U15A and U17A mutant viruses showed similar titers to the wild-type virus, while the viral titer of A19U decreased approximately 10-fold compared with that of the wild type (Fig. 4E). To further characterize these mutant viruses, the rescued wild-type or mutant WSN virus (P0) was then passaged for two generations in MDCK cells, and the growth kinetics of the wild-type and mutant viruses (P2) were determined in MDCK cells. The results showed that the growth kinetics of U15A and U17A viruses were similar to that of the wild-type virus; interestingly, the titer of A19U virus decreased by only 4-fold compared with the wild-type virus (Fig. 4F). To address the molecular basis of the improved growth, we sequenced the HA vRNA of the A19U virus (P2). In the A19U virus, point mutation U23G (A23C in positive sense) was found (Fig. 4G). Subsequently, we plaque purified the P2 A19U mutant viruses and sequenced the HA segment individually. We found that the U23G mutation exists simultaneously with the A19U mutation, and no individual U23G mutant virus was found. Taken together, these results suggest that the 39 19A nucleotides in H1-ssNCR are under the highest selection pressure during virus propagation. In addition, the A19U mutant virus is unstable and can induce a mutation at its adjacent position upon passaging, which could improve its growth.
A reverted U23G mutation neutralizes the effects of the A19U mutation during virus replication. In order to assess the effect of the point mutation U23G, we constructed the U23G and A19U1U23G mutations in the pPOLI-HA plasmid and examined the effects of these mutants on viral RNA synthesis and protein production in the RNP reconstitution system as described earlier. In general, the results were consistent with the results observed above; the point mutation A19U significantly attenuated the level of all three species of viral RNAs, while the A19U1U23G mutation significantly  increased the HA vRNA and mRNA levels compared with A19U mutation (Fig. 5A and  B). Of note, we found that although the A19U1U23G mutation restored both HA mRNA and vRNA levels to the wild-type level, the cRNA level was still lower than that of the wild type (Fig. 5A). We speculated that the reduced cRNA level is sufficient to produce vRNA and the mutations have a specific impact on vRNA!cRNA synthesis. In terms of protein production, the A19U1U23G mutation significantly increased the level of HA protein compared with the A19U mutation which correlates well with the mRNAs of the mutants (Fig. 5C and D). In contrast, the U23G mutation alone did not have any effect on HA RNAs or protein levels ( Fig. 5A to D). Since the A19U mutation was introduced in the 39 H1-ssNCR of vRNA, it would be used as the template to conduct both viral RNA transcription (vRNA!mRNA) and replication (vRNA$cRNA); next, we wanted to explore the effects of the A19U mutation on viral RNA transcription and replication, respectively. We first investigated the effect of the A19U mutation on transcription through an RNP reconstitution system which contains a replication-deficient polymerase (PA-C95A) (32) to avoid the generation of cRNA and vRNA. We found that the A19U mutation resulted in a significant decrease in mRNA synthesis, and the U23G mutation rescued the level of mRNA ( Fig. 5E and F). Next, we used an RNP reconstitution system, which contains a transcription-deficient polymerase (PA-D108A) (33) to avoid the generation of mRNA, to examine the effect of the A19U mutation on replication. The results showed that the A19U mutation Hierarchical Sequence Requirements of the H1-ssNCRs Microbiology Spectrum significantly reduced the level of vRNA, and the U23G mutation rescued the level of vRNA ( Fig. 5G and H). Besides, given the effects of the three mutant templates (A19U, U23G, and A19U1U23G) on cRNA levels, we could further induce that, during viral RNA replication, A19U may have a major defect in vRNA!cRNA synthesis.
To further assess the effect of the U23G mutation on WSN virus growth efficiency, we generated U23G and A19U1U23G recombinant viruses using the reverse genetics and examined the titers of the rescued viruses (P0) by plaque assay. Unlike the significant attenuation (1 log reduction) observed for the A19U mutant virus, the titer of the A19U1U23G mutant virus was similar to that of the wild-type virus (Fig. 5I). In order to prevent the emergence of the U23G mutation in the A19U mutant virus and to examine the effects of the actual mutation on virus growth, we detected the growth kinetics of the P0 generation wild-type and mutant viruses. The results showed that the titer of the A19U mutant virus decreased about 0.7-log compared with that of the wild-type virus, while there was no significant difference between wild-type and A19U1U23G mutant viruses (Fig. 5J). Together, these results show that the A19U mutation has affected both viral RNA transcription and replication, and the revertant U23G in the H1-ssNCR neutralizes the decrease of the HA RNA levels and virus titers caused by the point mutation A19U.

DISCUSSION
The segment-specific and subtype-specific NCRs of influenza A virus are highly conserved within each segment but vary significantly in length and sequences between different segments. However, the biological significance of these variations and the sequence requirements remain poorly understood. In this study, we investigated the sequence requirements of the 39 and 59 H1-ssNCRs in the context of H1N1 WSN virus. By performing the self-designed vRNSA, it provides, for the first time, a visualized dynamic overview directly reflecting the hierarchical sequence requirements within and between the 39 and 59 H1-ssNCRs. The results showed that, between the 39 and 59 H1-ssNCRs, the sequence requirements of the 39 H1-ssNCR in general was more stringent than that of the 59 H1-ssNCR; within the 39 and 59 H1-ssNCRs, sequence stringency is declining with the locations of the nucleotides away from the promoter. Moreover, the stabilized sequences presented a common feature of high A/U ratios. More importantly, we reported that the identities of nucleotides close to the 39 vRNA promoter cooperatively play a role in regulating viral RNA transcription and replication.
It is widely accepted that the key packaging signal of each segment is composed by ssNCRs and its adjacent terminal coding sequences at both ends. In the studies of packaging signal identifications for each segment of WSN virus through serial truncation of the terminal coding sequences, the authors mentioned that the 39-terminal coding region is more important than the 59-terminal coding region toward the efficient packaging of PB2, HA, NA, and NS segments (24)(25)(26)(27). Consistent with these findings, we previously replaced the wild-type H1-ssNCRs at the 39 or 59 ends with the corresponding ssNCRs of the other HA subtypes (H2-to H7-and H9-ssNCRs) individually and found that the 39 H1-ssNCR plays a more critical role than the 59 H1-ssNCR on HA vRNA virion incorporation (13). In this report, our sequence logos produced by the vRNSA further show that the speed of the sequence stabilization in the 39 H1-ssNCR is generally faster than that of the 59 H1-ssNCR. Together, these results strongly indicate that the 39 H1-ssNCR plays a more important role than the 59 H1-ssNCR for efficient virus replication.
In addition to the disparity on stabilization speed, the actual sequence stringency displayed in the vRNSA logos is also significantly different between the 39 and 59 H1-ssNCRs. The nucleotides in 3'G1 showed the exact wild-type sequence in the both replicates at P6 and the nucleotides in 3'G2 and 3'G3 showed constant but non-wild-type sequences. In contrast, the nucleotides in 5'G1 showed one wild-type and one non-wild-type constant sequence and the nucleotides in 5'G2 and 5'G3 were still in disordered status at P6. These results clearly demonstrate that the sequence requirements in the 39 H1-ssNCR are, in general, more stringent than that those in the 59 H1-ssNCR; especially, the promoter-proximal nucleotides at both ends are of the greatest importance for the virus to survive. Our indepth in vivo functional analyses demonstrate that the nucleotides adjacent to the promoter region in the 39 H1-ssNCR cooperatively regulate the HA vRNA synthesis, further highlighting the importance of 39 promoter-proximal nucleotides in regulating virus replication efficiency. Moreover, we previously studied the functions of the 39 H1-ssNCR with a series of truncation mutants, and we found that the truncation with these six nucleotides or less (39 15 to 20) left could work with its adjacent coding nucleotide toward regulating HA vRNA template preference in a multisegment environment (29). Of note, in our vRNSA experiment, we performed NGS for either the 39 NCR or 59 NCR. We could not exclude the possibilities that compensatory mutations in the entire viral genome could occur, which, however, is beyond the scope of this study. Moreover, we showed evidence from both the vRNSA experiment and the A19U mutant virus that the identity of a specific nucleotide (A19) in the 39 promoter-proximal region plays an important role during viral RNA synthesis. Together, we propose that the nucleotides adjacent to the 39 promoter region play an essential role in regulating viral RNA synthesis. Nevertheless, further investigation is needed to clarify the detailed mechanisms by deep sequencing and function-related highresolution RNA structure studies.
Interestingly, our vRNSA logos showed that all stabilized sequences in the 39 and 59 H1-ssNCRs at P6 have a common feature of high A/U ratios (.66%) ( Table 1). We then checked the sequences of all segment-specific and subtype-specific NCRs and found that all ssNCRs contain A/U nucleotides in a range of 60% to 100%. However, the underlying mechanism(s) remain unclear. Dadonaite et al. (34) investigated the global high-resolution structure of the influenza A virus genome and found that vRNA in the context of vRNP is capable of accommodating secondary RNA structures through extensive base pairing. We speculate that the high occupancy of A and U bases may increase the free energy of the ssNCR structures and thus facilitate the polymerase to unravel the structures to initiate viral RNA transcription and replication.
In summary, by using the self-designed vRNSA, we generate, for the first time, the visualized dynamic sequence selection logos for the 39 and 59 H1-ssNCRs during virus rescue and serial passage. It clearly differentiates the importance of the 39 H1-ssNCR versus the 59 H1-ssNCR and also demonstrates hierarchical sequence requirements of the nucleotides within the 39 and 59 H1-ssNCRs. Our functional assays further pinpoint the essentiality of the promoter-proximal nucleotides in regulating influenza virus replication. These data will not only broaden our knowledge in understanding the nonconserved noncoding region of influenza A virus but also may provide basic information to design the antisense oligonucleotide (ASO) in targeting key viral RNA sequences.
Reverse genetics. Virus rescue was conducted by the previously described protocol for the eight-plasmid rescue system (37). Briefly, approximately 10 6 293T and MDCK cells (at a ratio of 2:1) in a 6-well plate were transfected with 0.5 mg of each plasmid of the influenza virus A/WSN/33 (H1N1) reverse genetic system (pHW2000-PB2, -PB1, -PA, -HA, -NP, -NA, -M, and -NS) using Lipofectamine 2000 and Opti-MEM according to the manufacturer's instructions. At 24 h posttransfection, the medium was replaced with DMEM containing 0.5% FBS and 0.5 mg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). The virus supernatant was harvested 48 h after the medium was changed, and the viruses were plaque purified. The purified viruses were passaged for two generations in MDCK cells and subjected to virus genome sequencing. The virus titers were then determined by a standard plaque assay.
Virus random nucleotide selection assay.  (3'G1 forward primer, 59-GCGCGTCTCCGGGAGCAAAAGCAGGGGNNNNNNAAAACAACCAAAATGAAGGCAAAAC  TACTGGTCCTGTTATATGCATTTG-39; 3'G2 forward primer, 59-GCGCGTCTCCGGGAGCAAAAGCAGGGGAAAATAN  NNNNNACCAAAATGAAGGCAAAACTACTGGTCCTGTTATATGCATTTG-39; 3'G3 forward primer, 59- To identify the HA ssNCR sequences required for efficient virus replication selected by this method, viral supernatants of passage 2, 4, and 6 were clarified by centrifugation at 3,000 rpm for 20 min. The supernatant was further centrifuged at 10,000 rpm for 30 min at 4°C. Then, the clarified supernatant was collected and loaded onto a 30% sucrose cushion and further centrifuged at 25,000 rpm for 2.5 h at 4°C. The RNA of the virus pellet was extracted using TRIzol reagent (Invitrogen) and reverse transcribed according to the following method of virus genome sequencing. The reverse transcription (RT) products were then amplified together by PCR with a high-fidelity enzyme using HA 39 H1-ssNCR-specific primers with different barcode sequences. To avoid excessive cycles of PCR that may affect the actual proportion of bases at different positions of the 39 and 59 H1-ssNCRs, we performed quantitative PCR (qPCR) at first with RT products that were diluted 5 times as the template to choose a suitable number of cycles for PCR and make sure that the PCR products were in exponential phase. After performing DNA gel electrophoresis and confirming the locations of lines, we recycled the PCR products and subjected them to deep sequencing. The ratios of different nucleotide identities at respective sites of HA ssNCRs were calculated and analyzed. The ssNCR sequence logos for each library showing the nucleotide frequency were constructed by the online Web server WebLogo (http://weblogo.berkeley.edu/logo.cgi) (38).
Growth curve analysis. MDCK or A549 cells were infected with wild-type or mutant viruses at a multiplicity of infection (MOI) of 0.001. At time points 24, 36, 48, 60, and 72 h postinfection (p.i.), the supernatants were collected. The virus titers were determined by plaque assay in MDCK cells. The growth curves shown are the average results of three independent experiments.
Western blot and primer extension analysis. Approximately 10 6 HEK-293T cells were transfected with 0.5 mg each of four protein expression plasmids (pcDNA-PA, -PB1, -PB2, and -NP) and pPOLI-HA (wild type or mutant) using Lipofectamine 2000 and Opti-MEM according to the manufacturer's instructions. Cells were harvested 24 h posttransfection. For Western blot analysis, the cells were lysed with Cytobuster lysis buffer (Novagen), followed by Western blotting with a monoclonal anti-HA antibody, with anti-b-actin antibody as an internal control. Protein expression levels were visualized with an Odyssey infrared imaging system (Li-Cor Biosciences, USA). The relative protein expression level was analyzed using the integrated software of the Odyssey system. For primer extension analysis, total RNA was extracted using TRIzol reagent (Invitrogen). The levels of mRNA, cRNA, and vRNA of the HA template were analyzed by primer extension with two 32 P-labeled HA-specific primers, as follows: 59-TACTCAACTG TCGCCAGTTC-39 was used to detect HA vRNA and 59-GTCACTGCCACATTCTTCTC-39 was used to detect HA mRNA and cRNA. The primer used for detecting internal control 5S rRNA was 59-TCCCAGGCGGTC TCCCATCC-39. Primer extension products were analyzed by 6% denaturing PAGE with 7 M urea in Trisborate-EDTA (TBE) buffer and visualized by phosphor-imaging on an FLA-5000 scanner (Fuji). ImageJ was used to analyze the 32 P-derived signal (39).
Hierarchical Sequence Requirements of the H1-ssNCRs Microbiology Spectrum Statistical analysis. GraphPad Prism, version 7, software was used for statistical analysis. Two-way analysis of variance (ANOVA) with Dunnett correction was used for two-variable comparisons, while one-way ANOVA with Dunnett correction was used for one-variable comparisons. P values of ,0.05 were considered to be significant.