This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The rate-limiting step for live-virus vaccine development is the identification of a suitable attenuated virus. Isolation of attenuated viruses is a random, slow process that often involves passage of the virus in foreign or non-permissive cell lines. The availability of a universal strategy for virus attenuation would remove this bottleneck in vaccine development, paving the way for rapid, effective response to viruses with regular intervals of antigenic shift (e.g. influenza virus), newly emerging and re-emerging viruses (e.g. SARS coronavirus, West Nile virus or Dengue virus), or agents of terror and biological weapons (e.g. ebola virus or smallpox virus). Genome replication of all viruses requires a polymerase. Although it is assumed that polymerase rate and fidelity are optimized for production of progeny genomes as fast as possible containing as few or as many errors as necessary for efficient transmission to the next cell or host, direct evidence in support of this belief is, at best, scarce. The Cameron laboratory at PennState has recently developed the tools for a model RNA virus, poliovirus (PV), that have permitted them to begin to evaluate the impact of replication rate and fidelity of the viral polymerase on replication capacity in cells and pathogenesis in animals. Surprisingly very subtle (2-3-fold) changes in polymerase speed and accuracy have very dramatic, attenuating effects on the virus. These observations have led them to hypothesize that well conserved residues in the polymerase active site or in active-site-interacting domains that modulate speed and/or accuracy may represent sites for universal, rational attenuation of viruses. Together, Camerons data are consistent with protein dynamics influencing the fidelity of nucleotide incorporation. We are excited about this possibility because it is becoming increasingly clear that coupled motions between remote sites and active sites can determine rate-limiting steps along a reaction coordinate. To date, there has been no investigation of the relationship between polymerase dynamics and polymerase function. We will use molecular dynamics simulations as a complementary approach to investigate the role for polymerase dynamics in fidelity. Our hope is that this approach will reveal correlations between dynamics and fidelity with known mutants. If so, then we can create a library of 3Dpol derivatives in silico to discover additional sites of 3Dpol that influence fidelity. This approach is particularly attractive because it can be applied quite easily to all polymerase systems for which structural information exists, permitting us to potentially extend what we learn with the RdRp to other classes of nucleic acid polymerases. We strongly believe that understanding polymerase dynamics will provide the next major breakthrough in understanding polymerase fidelity. We will employ and improve current theoretical methodology to assess the dynamical differences in a consistent manner using molecular dynamics (MD). The basis of our simulations will be the x-ray crystal structure (xrcs) obtained in Camerons lab and others. First, we will employ homology modeling to complete the xrcs structures, and then the AMBER 9/PMEMD9 molecular dynamics program will be used.
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