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PROJECT
SUMMARY
BSL-4
pathogens have been notoriously difficult to study due to
strict biocontainment issues. The Nipah (NiV) and Hendra viruses,
within a new genus (Henipahvirus) of paramyxoviruses, are
currently the only members of the family classified as BSL-4
pathogens. Plasmid-based transfection systems for recovering
recombinant paramyxoviruses have been recently established
and I am attempting to engineer functional T7-driven and RNA
POL I-driven reverse genetics systems that would allow for
the generation of viral-like particles (VLPs) carrying different
Nipah minigenome constructs.
Using this system, we can analyze
unique aspects of Nipah virus biology in BSL-2/2+ conditions.
These aspects include, but are not limited to, the interactions/functions
of the nucleoprotein (N), the phosphoprotein (P), and the
RNA-dependent RNA polymerase (L) within the viral ribonucleoprotein
(vRNP) complex that's responsible for viral gene transcription
and genome replication. In addition, we can also use this
system to study the unique genetic determinants of this negative
strand, non-segmenented, RNA genome such as the unique untranslated
regions (UTRs) juxtaposed each gene. We can analyze these
functional aspects by introducing markers like RFP and firefly
luciferase into the minigenome. In addition, with other novel
reporters such as beta-lactamase, we can even begin to study
the real-time kinetics at the level of viral entry under different
minigenomic backgrounds.
Another project seeks to develop
and define the ability of small molecule antagonists to block
NiV-G's (the NiV receptor-binding protein) interaction with
its cognate cellular receptor, EphrinB2. Using ELISAs and
psuedotyped-NiV expressing renilla luciferase, we can
conduct robotics-based screens at the UCLA Molecular Screening
Shared Resource (MSSR) core facility. This facility allows
us to perform high-throughput screens with 30, 000 compound
libraries and smaller, targeted libraries to find small molecules
that may block Nipah virus infection at varying points along
the viral life cycle.
Recently, I've also began work on
the Nipah Matrix protein. Using state-of-the-art in vivo
labelling techniques, we can track matrix as it participates
in the viral life cycle. Paramyxovirus matrix proteins
have the reputation of being involvled in multiple aspects
of viral entry, budding, and pathogenesis. It will be very
interesting to elucidate the Nipah-M's involvement in these
areas.
Finally, my initial work in searching
for a specific small molecule inhibitor of NiV infection resulted
in the discovery of a novel, potent (IC50 = submicromolar)
broad-spectrum antiviral targeting all enveloped viruses tested
to date (Ebola, SV5, Marburg, Flu, HIV-1, Nipah, VSV, Vaccinia,
Cowpox, RSSEV, Junin, La Crosse, RVFV...etc); interestingly,
the compound does not inhibit naked (non-enveloped) viruses
such as CoxsackieB and Adenovirus. The compound, LJ001 (US
Patent Serial No. 61/073,448), does not exhibit an overt toxicity
in animal models while preventing lethal infection of Ebola
and Rift Valley Fever viruses during murine challenge experiments.
I am seeking to further characterize the compounds mechanism
of action (specific targeting of the viral membrane) while
conducting pharmacokinetic studies and preclinical trials.
I also design and maintain this
website, in my decreasingly spare time. :)
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