Last year, Mehle and Doudna showed that human cells apparently prevent the three subunits of bird virus polymerases from assembling into a functioning enzyme. A single amino acid switch at position 627 on the second subunit of the polymerase overcomes that inhibition and allows the virus to replicate. Apparently, Mehle said, when the amino acid glutamic acid - typical of most bird virus polymerases - is changed to a lysine, typical of human polymerases, the surface charge of the subunit changes from acidic (negatively charged) to basic (positively charged) and allows assembly of the subunits. Previous studies in mammals have shown that a lysine in that position enhances polymerase activity, increases viral replication and transmission, and in some cases, is associated with increased pathogenicity and death.

In their new study, Mehle and Doudna found that H1N1 has two rare mutations in the second subunit: a serine at position 590 and an arginine at position 591. This combination, which is most common in pigs, apparently has the same effect on surface charge as the mutation at position 627, allowing the polymerase complex to form and function in human cells.

Mehle noted that, in addition to such point mutations, flu viruses also mix and match the three subunits. Both the 1957 and 1968 viruses had polymerases composed of a first subunit from a bird and the other two subunits from humans. H1N1 has a human-like first subunit, while the second and third are bird-like - hence the need for a mutation in the second subunit to make it more human-like.

To see which other combinations might make H1N1 more virulent, they mixed human, avian and pig subunits in culture, replicating the pig "mixing vessel," Mehle said. Several combinations with a human third subunit increased the activity of the polymerase enzyme when other mutations were not present in the second subunit. Viruses with this alteration are now being tested in human cell culture to see if they are more virulent.

"In addition to having individual amino acid changes affecting the ability of the virus to transmit across species and be more pathogenic, we need to think about these entire gene segments being exchanged back and forth," said Doudna, who also is a faculty affiliate of the California Institute for Quantitative Biosciences (QB3). "Those will affect the outcome of disease."

"We are very hopeful that the kind of basic science that we are doing here will have an impact on human health, either at the level of diagnostics or thinking forward to development of antiviral therapeutics," she added.

Mehle and Doudna continue to explore the polymerase to discover what in human cells prevents the assembly of the bird polymerase, and to determine the three-dimensional structure of the enzyme and its three subunits.

Source: University of California - Berkeley