INVESTIGADORES
ALVAREZ Diego Ezequiel
congresos y reuniones científicas
Título:
HIGH-THROUGHPUT SCREENING OF HOST FACTORS INVOLVED IN ACTIN-DEPENDENT SPREADING OF VACCINIA VIRUS
Autor/es:
DIEGO E. ALVAREZ; HERVE AGAISSE
Lugar:
San José
Reunión:
Encuentro; PEW Programs in the Biomedical Sciences 2010 Annual Meeting; 2010
Resumen:
Poxviruses
are large, enveloped, double-stranded DNA viruses. Vaccinia virus was
used to eradicate smallpox and constitutes the prototypic poxvirus. The
potential use of smallpox as a biological weapon, together with the
high rate of adverse effects associated with vaccination have raised
the need to find new therapeutic targets to treat poxvirus infections.
During replication in the host cell cytoplasm, three different viral
forms are generated: intracellular mature virions (IMV), intracellular
enveloped virions (IEV), and extracellular virions (EV). After their
morphogenesis, a subset of IMV migrates to the trans-Golgi network and
early endosome to acquire a two membrane wrapping and yield IEV. In
contrast to IMV, which mainly remain inside the infected cell, IEV are
transported on microtubules to the plasma membrane where fusion occurs
to release EV. This viral form has the ability to promote actin
polymerization beneath the plasma membrane facilitating cell-to-cell
spread of the virus. While several viral proteins have been identified,
the host cell machinery involved in viral actin-based motility is
poorly understood. We combined the RNA interference technology, with
high-throughput fluorescence microscopy and computer assisted image
analysis to image cells infected with recombinant vaccinia virus that
expresses a GFP-fusion membrane protein (vGFP). This system allowed us
to monitor vaccinia virus spread in epithelial cells. In order to
validate our screening conditions, we investigated the effect of
silencing the Arp2/3 complex components on viral spread. We infected
siRNA treated or untreated cells with vGFP and evaluated the ability of
the virus to spread from the primarily infected cell to the adjacent
cells. While spreading was evident in untreated cells, the virus
remained confined to single cells in Arp2/3-depleted cells. These
observations lay the basis to use the high-throughput siRNA screening
approach to identify novel cell factors required for the process of
actin-dependent cell-to-cell transmission of vaccinia virus. Our
systematic identification of host factors involved in viral
cell-to-cell spread may reveal novel targets for therapeutic
intervention.