Education: PhD, 1988, Institute of Molecular Biology, SPA "Vector", Koltsovo, Russia
BS and MS, 1982, Novosiirsk State University and Institute of Cytology and Genetics, Novosibirsk, Russia
Overview: Molecular biology and molecular virology.
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
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
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
Currently, we further investigate the mechanism of capsid interaction
with cell nuclei and test the VEEV variants with modified capsids as
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
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.
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