Influenza A viruses spawn epidemics – scientists discovered vulnerable targets in RNA polymerase

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Influenza A viruses spawn epidemics, global pandemics and sweeping outbreaks that kill millions of birds, yet only now have flu scientists obtained comprehensive, high-resolution structural data on a protein that is key to the very survival of an influenza virus.

The highlighted protein is RNA polymerase, which transcribes and replicates the viral genome in the nucleus of host cells.

Dr. Ervin Fodor and colleagues at Oxford University have shed new light on the protein in groundbreaking research that also has uncovered new sites for antiviral drugs that can take aim at vulnerable targets in RNA polymerase, a protein Fodor calls FluPolA.

“All these sites are well-conserved regions,” Fodor said in an email, referring to possible new flu-drug targets that could function across a range of flu viruses.

“FluPolA is one of the most conserved proteins in flu in general.

Hence, it is a good target for antiviral drug development.”

FluPolA is a major driver in the flu infection process.

Without it, a flu virus can’t copy its genetic information and commandeer that of the host cells.

But more data had been collected over the years on the structure of FluPolA derived from an influenza A virus circulating among bats, Fodor had found, than FluPolA in H3N2, which frequently affects vast swaths of the human population.

There also wasn’t much FluPolA structural information for H5N1, a noteworthy flu menace in birds. 

Fodor and his Oxford colleagues have now cast a bright spotlight on FluPolA and its multiple functions by revealing the arrangement of atoms within the critical protein for both H3N2 and H5N1.

The team achieved new insights into the structure of FluPolA for both viruses by using X-ray crystallography and cryo-electron microscopy.

Writing in a recent issue of the journal Nature, Fodor defines the viruses involved in the FluPolA research as the human flu virus, A/1968/H3N2, the strain that circled the globe in the Hong Kong flu pandemic 51 years ago.

The other FluPolA for which the team obtained high-resolution structural data is A/duck/Fujian/H5N1, a 2002 strain—a particularly nasty killer of domesticated birds.

New hurdles had to be surmounted to provide what is now an extensive amount of data on FluPolA for both H3N2 and H5N1, the Nature paper revealed.

“There were many technical difficulties to overcome,” Fodor said.

“The influenza A virus polymerase – FluPolA – is difficult to express and purify in large amounts.

One issue is that three subunits need to be co-expressed and purified.

A second issue is that FluPolA is particularly unstable, it has highly flexible domains which make crystallization-based approaches very difficult.

“We opted for an H3N2 polymerase as a representative of a human influenza virus; H3N2 strains currently circulate in the human population, although we went for an old strain from 1968, which our groups studied previously,” he said. 

“We picked an H5N1 avian FluPolA because we wanted to compare avian and human influenza A virus polymerases – and we also studied this particular avian strain before.

Human and avian FluPolA differ in sequence and we wanted to know exactly how these differences affect the structure.

Avian FluPolA works poorly in human cells and the avian virus needs to adapt to the mammalian cell environment which involves adaptive mutations in FluPolA,” Fodor said.

Influenza A viruses – as well as their B counterparts – have a genome made up of eight RNA segments.

These strands carry all the information a flu virus needs to replicate in a host. The RNA segments are copied by RNA polymerase.

The flu is a respiratory illness in humans that can be caused by A or B viruses. B influenza causes infection only in humans. Both types can cause serious respiratory complications.

Influenza A viruses have driven major epidemics as well as global pandemics.

The deadly 1918 flu, the worst in recorded history, was caused by an A influenza virus.

Statisticians at the U.S. Centers for Disease Control and Prevention estimate that anywhere from 291,000 to 646,000 people die annually around the world because of seasonal flu-related complications.

H5N1, often referred to as an HPAI, or highly pathogenic avian influenza, primarily affects birds. Other HPAIs affect birds as well.

The spread of H5N1, which is lethal in domestic birds, can have tremendous economic consequences. Knowing more about how structure influences function can lead to new ways of disrupting the infection process in humans and birds, global health experts say. 

Among the vulnerable sites that are potential antiviral targets in influenza A viruses, are those that hinge on a key discovery in the Oxford research: FluPolA exists as a monomer or dimer, a small molecule or molecular complex.

“We identified several sites, including the FluPolA dimerization interface and a binding site for viral RNA,” Fodor said of possible antiviral sites.

“These sites could be targeted by small compounds.

The idea is that if such compounds prevented FluPolA dimerization or RNA binding, these would act as antivirals,” he said.

“We also identified a nanobody, a small antibody, that binds at a particular site on FluPolA, preventing FluPolA dimerization. When we express this nanobody in cells, the cells produce less virus, indicating it is inhibitory.”


nfluenza A virus (IAV) causes respiratory disease in swine and is a pathogen shared between humans and pigs. Genetic drift and spillover of human IAV with subsequent reassortment may result in human-like IAV strains novel to swine.

We describe here two novel IAV strains detected in swine in 2017 that contain human seasonal influenza virus gene segments potentially transmitted through reverse zoonosis.

Nasal swabs originating from one swine production system, but separate locations in Oklahoma, were submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) in 2017. An H3N2 virus with a novel human-like hemagglutinin (HA) sequence was detected using the VetMAX Gold swine influenza virus (SIV) subtyping real-time PCR (Thermo Fisher Scientific, Waltham, MA).

Whole-genome sequencing was performed on strains A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), isolated in Madin-Darby canine kidney cells. Nucleic acids were extracted using the MagMAX pathogen RNA/DNA kit (catalog number 4462359) and a KingFisher Flex system (both Thermo Fisher Scientific) to construct sequencing libraries using TruSeq (Illumina, Inc., San Diego, CA).

Sequencing was performed on a MiSeq system (Illumina, Inc.) following standard Illumina protocols at the ISU VDL (12).

Approximately 2,000,000 raw sequencing reads per sample were preprocessed using Trimmomatic version 0.36 and subjected to sequencing quality analysis with FastQC (34).

Quality-trimmed total reads were mapped against reference sequences downloaded from the NCBI Influenza Sequence Database (ftp://ftp.ncbi.nih.gov/genomes/INFLUENZA/) using BWA-MEM (5). Mapped reads were extracted using SAMtools (6) and used for de novo assembly.

For each segment, contigs were assembled using ABySS (7) and SPAdes (8). The resulting contigs were manually curated in SeqMan Pro to remove contamination and trim chimeric contigs, thus generating a consensus sequence per segment.

A comparison of the nucleotide sequences of both strains demonstrated that the HA, NA, and M sequences were 99.5%, 99.9%, and 99.5% similar, respectively.

The NS1 sequences were identical, and the PB1, PB2, and PA sequences had greater than 99.7% identity. The HA sequences of both isolates demonstrated 99% nucleotide (nt) identity to that of the human IAV strain A/Baltimore/0294/2017 (H3N2) (GenBank accession number KY949654).

Each isolate had 10 amino acid substitutions in the HA compared to the most similar human strain. The NA sequence of A/swine/Oklahoma/65980/2017 was most similar (99% nt identity) to that of human IAV strain A/Tennessee/06/2017 (H3N2) (GenBank accession number CY226641), and the NA sequence of A/swine/Oklahoma/65260/2017 was most similar (99% nt identity) to that of human IAV strain A/Baltimore/0223/2017 (H3N2) (GenBank accession number KY950122).

PB2, PB1, PA, NP, and NS genes were similar to swine-origin triple-reassortant IAV. Phylogenetic analysis indicates that the M gene was derived from the 2009 H1N1 pandemic matrix circulating in swine. The HA and NA were nested within a monophyletic clade of 2016 to 2017 human seasonal H3 IAV, suggesting novel human-to-swine transmission (9).

This study documents a human-to-swine spillover and the potential for human seasonal IAV to cross the species barrier and infect swine. The ISU VDL has detected 21 genetically similar human-like H3 strains since the fall of 2016 (http://influenza.cvm.iastate.edu/correlation.php). The viruses have acquired a swine internal gene constellation through reassortment (1011), with at least 10 amino acid mutations in the HA suggesting adaptation and transmission in swine. The USDA swine surveillance system also reported similar human seasonal IAV designated “human-like 2016” in the Influenza A Virus in Swine Surveillance report (http://www.aphis.usda.gov/animal_health/animal_dis_spec/swine/downloads/fy2018quarter1swinereport.pdf).


More information: Haitian Fan et al. Structures of influenza A virus RNA polymerase offer insight into viral genome replication, Nature (2019). DOI: 10.1038/s41586-019-1530-7

Journal information: Nature

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