Towards solving the three dimensional structure of the influenza virus RNA polymerase

?Bird flu? has hit the headlines over the last year as concerns have grown amongst influenza experts that there is a serious problem in controlling the disease in poultry in S.E Asia, China and Turkey. Moreover, more and more poultry seem to be affected despite widespread attempts to control the spread of the disease. In parallel, more cases of human infection and, tragically, human deaths have been reported in the last year from H5N1 bird flu. Nearly 250 people have been infected and at least 140 have died. However, these numbers could pale into insignificance if bird flu were to adapt by genetic change into a truly human virus. Given the known high rate of genetic change possible in influenza virus the fear, now, is that the H5N1 virus will mutate or combine with a human-adapted influenza, such as a conventional seasonal influenza, enabling effective human to human transmission. If this occurred - given the 50% mortality of H5N1 flu ? no-one can be complacent. Certainly many millions of people could die, even in the UK alone. Indeed some argue that it is inevitable that a global H5N1 pandemic will occur sometime although the time scale is almost impossible to predict. Governments in many countries, including the UK have responded with measures to stockpile an antiviral - ?Tamiflu? - in case a global pandemic emerges. However, new drugs are needed because of the emergence of virus strains resistant to ?Tamiflu?. Our research, carried out at Oxford University by George Brownlee FRS and Ervin Fodor at the School of Pathology and David Stuart FRS and Jonathan Grimes at the Wellcome Trust Centre for Human Genetics, aims ultimately to make it possible to find new antivirals targeted to an enzyme - the RNA polymerase - central to the influenza virus life cycle. By blocking the activity of an enzyme crucial for viral multiplication, we hope that such drugs will be particularly effective against this disease.

The potential for H5N1 avian influenza (bird flu) to cause human disease is now undeniable, since over 140 people have now died worldwide. At the moment the H5N1 virus is not well adapted to human-human transmission, although many influenza experts believe it is only a matter of time before this adaptation occurs and a new global pandemic occurs. The number of available drugs to treat bird flu is very limited. Combination drug therapy - incorporating new antivirals, is now urgently required to combat documented drug resistance to existing antivirals and to protect the population against a future pandemic. The immediate purpose of this proposal, involving collaboration between two Oxford academic laboratories is to bring together two groups - one expert in the molecular biology of influenza, the other in the structural biology of polymerases and viruses with the ultimate aim of solving the 3D structure of the influenza RNA-dependent RNA polymerase. Structural studies are challenging because the polymerase is a heterotrimeric complex of PB1, PB2 and PA. Here we will initiate a study of the co-expression and purification of the polymerase complex derived from a classic (H1N1) human influenza strain (A/WSN/33), using Baculovirus technology in insect cells, for analysis by high resolution X-ray crystallography. Encouraging preliminary progress in purifying PA monomers and [PB1, PA] dimers has already been achieved, serving as a model for the purification of the trimeric polymerase complex. Knowledge of polymerase structure would allow future rational structure-based design of antivirals, targeted to the polymerase active site and/or endonuclease and/or cap-binding sites of the enzyme. Also, a future comparison of the structure of a classic human influenza polymerase, as studied here, with a modern H5N1 avian polymerase is needed to establish the structural basis of the adaptation of the avian polymerase to mammalian cells.