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.