Current and Future Objectives
We have a number of
projects funded by NIH that investigates the different aspects of
alphavirus replication and pathogenesis. In addition, we have expanded
our research findings towards the development
of a new generation of human and veterinary vaccines against
alphavirus and flavivirus infections. These areas of our research
program are also supported by NIH under the biodefense-related
initiatives. Currently, our projects cover the following
broad areas of research.
1. The structure and function of the alphavirus replicative complexes using Sindbis virus (SINV) as a model.
We
have already isolated Sindbis virus-specific replicative complexes and
identified
several cellular components required for replication of viral genome
in both insect and vertebrate cells. We are analyzing the nature of the
replicative complexes formed in two fundamentally different types of
cells, namely, mosquito cells and vertebrate
cells. Ongoing research in this area involves comparative and
mechanistic studies aimed at defining the role(s) of each cellular
protein in complex formation and RNA synthesis. Our goal is to identify
the cellular protein factors that have a common
function in all the alphaviruses and also reveal the unique factors
that are specific for the different members of the alphavirus family. We
strongly believe that this information will not only greatly advance
our understanding of the mechanism of
alphavirus replication, but also ultimately lead to the development
of novel strategies for antiviral therapy.
2. Functional studies of the replicative complexes using siRNA technology
We
are using a 16,000-component
siRNA library that is specific to human genes to complement the
functional study of the replicative complexes. The siRNAs are tested for
their ability to suppress replication of VEEV replicons expressing the
luciferase gene. We plan to ultimately
identify i) cellular genes, whose products function in the formation
of the alphavirus replicative complexes (the corresponding siRNAs
inhibit replication of virus-specific RNAs and Luc expression by 5 to
100-fold), and ii) the genes whose expression
affect the early stages of VEE RNA replication (the corresponding
siRNAs increase RNA replication and Luc expression by more than
10-fold). The identified subset will need further characterization. This
project represents one of the components of
the long-term strategy of our research aimed at defining cellular
targets for antiviral therapeutic drug and development the new means of
antiviral treatment.
3. Investigate the major components of alphavirus-host cell interactions.
One
of the main, characteristic events in the alphavirus-infected cells is a
strong inhibition of cellular transcription. It plays a critical role
in downregulation of both the type I interferon response as well as the
activation of the interferon-inducible
genes. Our data indicate that both the New World and the Old World
alphaviruses have developed the same strategy to counter the host
response to virus replication; replication of the virus interferes
efficiently with the cellular transcription machinery.
However, the Old World and New World alphaviruses employ
different mechanisms, which involve different virus-specific proteins,
for the transcriptional shutoff induction. In at least two of the Old
World alphaviruses, SINV and SFV, which belong to different
serological complexes, the nsP2 protein is responsible for
transcription inhibition whereas in the New World alphaviruses, VEEV and
EEEV, it is mainly determined by the capsid protein, but not the nsP2.
We mapped the functional domain of the VEEV
and EEEV capsid proteins to the N-terminal, ~35-aa region that was
critical for the downregulation of cellular transcription and the
development of a cytopathic effect (CPE). This region includes two
domains with distinct functions: the a-helix domain,
helix I, which contains a nuclear import signal that is involved in
maintaining the critical balance between the levels of the protein in
the cytoplasm and nucleus, and the domain downstream of the helix I,
that contains functional nuclear localization
signal(s). The integrity of both domains determines not only the
intracellular distribution of the VEEV capsid, but is also essential for
its function in the inhibition of transcription. Our results suggested
that the VEEV capsid protein interacts
with the nuclear pore complex that was confirmed in a recent study,
where we demonstrated that VEEV capsid through its N-terminal region
efficiently inhibited nuclear import, mediated by different importins.
However, it does not affect passive diffusion
of small proteins to the nucleus. As expected, the capsid protein of
the Old World alphavirus, SINV, and the mutated VEEV capsid were found
to have no detectable effect on nuclear import. Interestingly, the VEEV
capsid did not noticeably interfere
with nuclear import in the mosquito cells, and this might play a
critical role in the ability of the virus to develop a persistent
infection in mosquito vectors. These unique findings have uncovered a
novel aspect of VEEV-host cell interactions and
viral pathogenesis at the molecular level that could be applicable
to other New World alphaviruses, such as eastern and western equine
encephalitis viruses.
Currently, we further investigate the
mechanism of capsid interaction with cell nuclei and test the VEEV
variants with modified capsids as vaccine candidates.
4. Role of the alphavirus nonstructural protein, nsP2, in downregulation of the innate immune response.
Our
recent data indicates that the role of nsP2 in the Old World
alphavirus-host cell interaction is significantly underestimated.
Besides the proteolytic function, which regulates the composition and
template preference of alphavirus replication
complexes, the New World alphavirus-derived nsP2 is also an IFN-a/b
antagonist. It accumulates not only in the cytoplasm, but also in the
nucleus and downregulates the transcription of cellular genes. Defined
mutations in nsP2, which affect its intracellular
distribution, affect the development of CPE, and also lead to the
establishment of persistent infection in some cell types or clearance of
virus from others. As it was indicated above, the nsP2 protein appears
to have very different functions during
replication of the New World and the Old World alphaviruses, and
this novel feature is now under careful investigation. However, in all
the alphaviruses, the adaptive mutations leading to a noncytopathic
phenotype occur in the same location present
in the carboxy terminal region of the protein, and the cytotoxicity
of this protein does not depend on its protease function. It is
reasonable to expect that the carboxy terminal domain (other than the
previously described helicase and protease domains)
plays a critical role(s) in virus-host interactions. Our data
indicate that the mutations have a deleterious effect on the ability of
alphaviruses to inhibit transcription of cellular mRNAs and rRNAs,
suggesting the interaction of nsP2 with cellular
transcription factors. Thus, our goal is to utilize the wt and
mutant nsP2 proteins to analyze the interaction with host proteins that
will define the exact mechanism of nsP2 function.
5. Investigate the role of mutations in the vaccine strain of VEEV in viral pathogenesis.
We
are interested in studying the effect of the mutations, specific to the
vaccine strain, on the translation of VEEV nsPs, VEEV RNA replication,
resistance of VEEV RNA replication to IFN-a/b and IFN induction. Based
on our recent data, the attenuated
phenotype of some of the VEEV strains is determined by a higher
level of their genomic RNA replication, which leads to an earlier
development of a more efficient antiviral response at the cellular
level. We are using the cDNA microarray technology
along with an arsenal of supplementary methods to define the
differences in cellular response to replication of i) VEEV and SINV; ii)
wt and the vaccine strain of VEEV iii) VEEV replicons that lack the
genes for structural proteins and iv) viruses
with different 5¹UTRs.
6. Rational design of novel recombinant vaccines and cell lines for specific detection of viral agents.
We
are developing different alphavirus replicons and packaging systems for
the large-scale production of recombinant
vaccines against yellow fever, Rift Valley fever virus, H5N1
influenza virus, chimeric alphaviruses (SIN/VEEV, SIN/EEEV, SIN/WEEV)
and alphaviruses with mosaic envelopes. To date, the designed vaccine
candidates against VEEV, EEEV and WEEV infections
are in pre-clinical trials. Currently, we are testing a new vaccine
against Chikungunya virus. In addition to the use of these developed
chimeras as excellent vaccines, these chimeric viruses also serve as
valuable models for studying the mechanism
of alphavirus pathogenesis because of their highly attenuated
phenotype.
We have also utilized the recently generated
noncytopathic alphavirus replicons for the development a new type of
vaccine against flavivirus infections. These defective pseudoinfectious
flaviviruses (PIVs) lack a functional copy of the capsid (C) gene
in their genomes and are incapable of causing secondary infections
in neighboring cells upon infection both in vivo and in vitro.
However,
they produce extracellular E protein in the form of secreted subviral
particles (SVPs) that are known to be effective immunogens. PIVs can be
efficiently propagated in trans-complementing cell lines producing high
levels of C or all three viral
structural proteins. Immunization with PIVs derived from YFV and WNV
produced high levels of neutralizing antibodies and elicited an
efficient protective immune response. These vaccines have demonstrated
high standards of safety as well. Such defective
flaviviruses can be produced in large scale under low biocontainment
conditions and should be useful for diagnostic or vaccine applications.
Such vaccines combine the efficiency of live vaccines and the safety of
subunit vaccines. Currently, we are
collaborating with industry to develop similar vaccine candidates
for dengue strains 1-4 and tick borne encephalitis viruses.
Our
research has a balanced combination of detailed molecular investigation
of different aspects of alphavirus replication and high-throughput
analyses of virus-host cell interactions aimed at generating a wealth of
valuable information that guides our
future research directions. Our research projects involve basic
research as well as applied studies that lead to the design of
recombinant vaccines and novel therapeutic strategies.