A Conserved Secondary Structural Element in the Coding Region of the Influenza A Virus Nucleoprotein (NP) mRNA Is Important for the Regulation of Viral Proliferation

Abstract

Influenza A virus is a threat to humans due to seasonal epidemics and infrequent, but dangerous, pandemics that lead to widespread infection and death. Eight segments of RNA constitute the genome of this virus and they encode greater than eight proteins via alternative splicing of coding (+)RNAs generated from the genomic (-)RNA template strand. RNA is essential in its life cycle. A bioinformatics analysis of segment 5, which encodes nucleoprotein, revealed a conserved structural motif in the (+)RNA. The secondary structure proposed by energy minimization and comparative analysis agrees with structure predicted based on experimental data using a 121 nucleotide in vitro RNA construct comprising an influenza A virus consensus sequence and also an entire segment 5 (+)RNA (strain A/VietNam/1203/2004 (H5N1)). The conserved motif consists of three hairpins with one being especially thermodynamically stable. The biological importance of this conserved secondary structure is supported in experiments using antisense oligonucleotides in cell line, which found that disruption of this motif led to inhibition of viral fitness. These results suggest that this conserved motif in the segment 5 (+)RNA might be a candidate for oligonucleotide-based antiviral therapy.

Fig 1. Secondary structure of M121 predicted by RNAalifold and bioinformatics calculations for possibility of each base pair.

A. Base pairs in secondary structure are colored according to % of canonical base pairs calculated for type A influenza (last column of table): red >98%; 95 ≤ orange < 98%; 90 ≤ green < 95%; 85 ≤ blue < 90%. B. In table are marked preserving mutations: indicated by green shadow are evidence of compensatory mutation.

Fig 2. Secondary structure of influenza RNA motif.

A. M121, isolated RNA with consensus sequence; secondary structure was predicted by RNAstructure 5.5 using chemical mapping results from SHAPE. B. Chemical mapping results of M121 in entire segment 5 (+)RNA (A/VietNam/1203/2004 (H5N1)); in blue is marked difference in sequence comparing to M121 on panel A.

Fig 3. Predicted structure probability of M121 by RNAstructure 5.5 program.

Fig 4. Enzymes mapping of M121.

All reactions were conducted in 23°C for 30 min. Lane 1 –control reaction: M121 incubated in 100 mM KCl and 5 mM MgCl₂, 10 mM Tris-HCl, pH 7, for 30 min in 23°C. Lanes 2–4—RNase V1 cuts in increasing concentration of enzyme: 0.5x10^-3 U/μl, 1x10^-3 U/μl and 3x10^-3 U/μl, respectively. Lanes 5–7—RNase T1 cuts in increasing concentration of enzyme: 0.15 U/μl, 0.25 U/μl and 0.75 U/μl, respectively. Lanes 8–10—RNase S1 cuts in increasing concentration of enzyme: 0.05 U/μl, 0.3 U/μl, 1 U/μl, respectively, Lane 11—formamide ladder, Lane 12—RNase T1 ladder.

Fig 5. Lead ion cleavage of M121.

RNA was incubated with 1 mM Pb(OAc)₂, 100 mM KCl and 5 mM MgCl₂, 10 mM Tris-HCl pH 7 in time course: lanes 1–6 - 0, 1, 5, 15, 30 and 60 min, respectively. Lane 7—control reaction: M121 incubated in 100 mM KCl and 5 mM MgCl₂, 10 mM Tris-HCl pH 7 for 60 min. Lane 8—RNase T1 ladder. Lane 9—formamide ladder.

Fig 6

A. (+) RNA5 motif with marked complementary region to anisense oligonucleotides. In green were marked differences in sequence between A/California/04_NYICE_E3/2009 strain and consensus sequence of M121. B. Effect of antisense oligonucleotides targeting M121 motif of scIAV A/California/04_NYICE_E3/2009 in cell line MDCK-HA. C—control; L—control with lipofectamine, R—positive control with ribavirin; N—negative control with oligonucleotide N.