Watching conformational rearrangements in picornavirus replication proteins

Project Summary
Some of the most important new and (re)emerging pathogens are positive-strand RNA viruses,
including coronavirus and picornaviruses Enterovirus D68, Enterovirus A71 and even poliovirus.
These viruses can directly use their RNA genome to guide the synthesis of a large polyprotein, which
must then be proteolyzed into its component parts, including the capsid proteins and enzymes
important for genome replication and encapsidation. Virus RNA genomes are rather small, and so
these viruses have evolved strategies to essentially expand their functional proteomes. For example,
the picornavirus 3C protein is a multi-functional protein that has protease activity, binds RNA control
sequences important for coordinating replication and translation processes, and binds
phosphoinositide lipids found in virus “replication organelles”, which act to protect the virus from host
cell defenses. All of these activities are encoded within its small 20 kDa structure. Another strategy to
expand functional protein content is for proteolytic precursors to have different functions than their
fully processed counterparts. For example, 3C is also found as part of the 3CD protein, but the 3CD
protein has different protease specificity, and different RNA and lipid binding affinities. The 3CD
protein also has a 3D domain; the 3D protein is the RNA-dependent RNA polymerase but 3CD does
not possess polymerase activity. By itself, 3CD also upregulates phosphoinositide lipid production
and induces membrane proliferation, events important for replication organelle biogenesis. How the
different and emergent functions of 3CD arise is poorly understood; X-ray crystal structures indicate
that 3CD is merely a composite of the 3C and 3D proteins joined together by a small flexible linker.
We propose that structural dynamics, that is, the ability to sample multiple structural conformations, is
the missing ingredient in understanding virus protein function. We propose that 3C fluctuates among
many conformations, providing 3C the ability to access and coordinate its many functions, and we
propose that 3CD fluctuates into different conformations, providing it with alternative functions. These
dynamic excursions can be further modified by interactions with RNA, lipids and protein binding
partners to coordinate virus protein function. We will evaluate these protein structural dynamics
through solution-state nuclear magnetic resonance spectroscopy, which provide atomic-level detail of
protein motions from the picosecond to second timescales, and complement these studies with
mutagenesis studies, functional assays and cell-based approaches to better understand the roles of
protein structural dynamics in the virus life cycle. The completed work will provide new opportunities
for rational anti-viral strategies, for example, by finding molecules that bind to alternative protein
conformations and/or disrupt functionally-important motions, as already validated for 3D.

Grant Number: 5R01AI104878-08
NIH Institute/Center: NIH
Principal Investigator: David Boehr