Project Summary / Abstract.
RNA viruses like SARS-CoV-2 (family Coronaviridae) and dengue virus (DENV; family Flaviviridae) cause
significant disease globally. The rise of SARS-CoV-2 variants of concern has highlighted the need to understand
the viral genetic determinants underlying disease severity, transmission potential, and ability to evade immunity,
which requires molecular tools to make viral mutants. However, current approaches to constructing and
manipulating viral infectious clones rely on a living host (bacteria or yeast), which can lead to unwanted mutations
and deletions in the viral genome, especially for larger viruses like coronaviruses and flaviviruses. Due to these
issues, few labs can successfully perform this work. Since the host causes these unwanted mutations, a strategy
that removes the need for a living host represents the best way forward.
Our long-term goal is to create simple-to-use tools that facilitate fundamental virology research aimed at
reducing disease burden, in line with NIH’s mission. This project aims to develop and optimize a paradigm-
shifting but straightforward host-free approach for constructing and manipulating viral infectious clones,
facilitating studies seeking to identify viral genetic determinants of disease, transmission, and other phenotypes.
This approach can also be used to construct recombinant vaccine viruses and diagnostic tools like reporter
viruses. We will use an exciting technique—replication cycle reaction (RCR)—which reconstitutes the E. coli
DNA replication machinery in a tube. RCR can efficiently amplify a single DNA molecule of up to 1 Mb with high
fidelity in a simple-to-use format. Here, we propose developing this RCR-based system to construct new
infectious clones (Aim 1) and manipulate existing ones (Aim 2). Aim 1 will use chemically synthesized DNA to
create infectious clones for the rapidly spreading SARS-CoV-2 Omicron variant and DENV strain Puo-218, a
component of the approved DENV vaccine, Dengvaxia. The impact of this aim will be an innovative system to
generate new infectious clones. Aim 2 will use the RCR-based system to make mutations and deletions in the
SARS-CoV-2 Omicron variant in an existing infectious clone; we will then study the impact these mutations have
on viral replication in primary human cells. The impact of this aim will be a straightforward method to generate
viral mutants, facilitating mechanistic studies to understand the viral genetic determinants of pathogenesis,
transmission, and immune evasion. These studies will also identify mutations outside Spike that impact viral
replication. We expect these studies will have a sustained impact on molecular virology by empowering more
labs to construct and manipulate infectious clones. We anticipate the techniques generated here to be broadly
applicable to other positive-sense RNA viruses and negative-sense RNA and DNA viruses as well. Finally, we
will share all the methods and tools generated here openly and without restriction.