Identification and targeting of a pan-genotypic influenza A virus RNA structure that mediates packaging and disease
Rachel J. HageyMenashe ElazarSiqi TianEdward A. PhamWipapat KladwangLily Ben‐AviKhanh Cong NguyenAnming XiongMeirav RabinovichSteven SchaffertTalia AvisarBenjamin FramPing LiuPurvesh KhatriJeffery K. TaubenbergerRhiju DasJeffrey S. Glenn
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Abstract Currently approved anti-influenza drugs target viral proteins, are subtype limited, and are challenged by rising antiviral resistance. To overcome these limitations, we sought to identify a conserved essential RNA secondary structure within the genomic RNA predicted to have greater constraints on mutation in response to therapeutics targeting this structure. Here, we identified and genetically validated an RNA stemloop structure we termed PSL2, which serves as a packaging signal for genome segment PB2 and is highly conserved across influenza A virus (IAV) isolates. RNA structural modeling rationalized known packaging-defective mutations and allowed for predictive mutagenesis tests. Disrupting and compensating mutations of PSL2’s structure give striking attenuation and restoration, respectively, of in vitro virus packaging and mortality in mice. Antisense Locked Nucleic Acid oligonucleotides (LNAs) designed against PSL2 dramatically inhibit IAV in vitro against viruses of different strains and subtypes, possess a high barrier to the development of antiviral resistance, and are equally effective against oseltamivir carboxylate-resistant virus. A single dose of LNA administered 3 days after, or 14 days before, a lethal IAV inoculum provides 100% survival. Moreover, such treatment led to the development of strong immunity to rechallenge with a ten-fold lethal inoculum. Together, these results have exciting implications for the development of a versatile novel class of antiviral therapeutics capable of prophylaxis, post-exposure treatment, and “just-in-time” universal vaccination against all IAV strains, including drug-resistant pandemics. One Sentence Summary Targeting a newly identified conserved RNA structure in the packaging signal region of influenza segment PB2 abrogates virus production in vitro and dramatically attenuates disease in vivo .Keywords:
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A PB1-K577E Mutation in H9N2 Influenza Virus Increases Polymerase Activity and Pathogenicity in Mice
H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation.
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Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.
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Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.
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Influenza is caused by a virus with a segmented RNA genome. The virus can infect humans, pigs and other mammals and birds. The viral envelope contains two key proteins, haemagglutinin (HA) and neuraminidase (NA). There are sixteen known variants of the HA and nine of the NA. During an infection, HA binds to receptors on airway epithelial cells. Human influenza viruses and bird influenza viruses have different receptors. Some animals, such as pigs have both types of receptors. NA removes sialic acid residues from viral glycoproteins and plays a role in the infection process. The RNA polymerase of influenza is error-prone and this results in mutations in HA and NA (antigenic drift). Some of these mutants are more infectious and cause seasonal influenza. If an avian influenza virus and a human influenza virus infect an animal with both virus receptors, such as a pig, reassortment of the eight RNA segments from each virus can occur. If any of the 256 possible new combinations (antigenic shift) has increased virulence then it can cause a pandemic. Seasonal vaccination of those most at risk is the best preventative strategy but occasionally the virus changes in unexpected ways and the vaccines in use have reduced effectiveness.
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With the exception of phage Qbeta, the RNAs of all the other small icosahedral RNA viruses so far examined contain a poly (A) tract. This tract has been implicated in the infectivity of poliovirus RNA. We have now shown that Nodamura virus, a divided genome virus from which infective RNA can be extracted, does not contain any poly (A) tracts. This evidence with Nodamura virus shows that poly (A) is not a necessary requirement for the infectivity of virus RNA molecules.
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Various methods of handling samples of avian influenza prior to detecting influenza viruses can significantly influence both, the detection of the virus and the quantification of viral nucleic acids. The quantity of influenza viral RNA remaining in different collecting buffers and kept at temperatures of -20°C, +4°C or +22°C for various lengths of time, was determined. The quantity of viral RNA remained the same for 120 days at -20°C, but decreased when the samples were stored at either +4°C or +22°C. The quantity of RNA was influenced by the composition of the collecting buffer. The influenza virus sample that is to be used for RNA quantification can be stored at +4°C and freeze and thaw cycles should be avoided during transport. Our results clearly indicate that the quality and quantity of influenza virus nucleic acid depends on the chemical composition of used buffer and also that the samples can be protected from degradation even if they are not stored at ultra-low temperatures. However, repeated thaw and freeze cycles will damage viral RNA even if kept in stabilizing buffers.influenza virus; degradation; RNA; buffer.
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