Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.     

Nuclear export and import of human hepatitis B virus capsid protein and particles

It remains unclear what determines the subcellular localization of hepatitis B virus (HBV) core protein (HBc) and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD) of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS), while ARD-II and ARD-IV behave like two independent nuclear export signals (NES). This conclusion is based on five independent lines of experimental evidence: i) Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii) These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT). iii) By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv) We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1), which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v) HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel TAP-dependent NES.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Nuclear localization of Sindbis virus nonstructural protein nsP2

In early infection, approximately 10% of nonstructural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear asP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile. a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were diseussed in relation to the inhibition of host macromolecular synthesis.     

Complete inactivation of Venezuelan equine encephalitis virus by 1,5-iodonaphthylazide

Hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA) partitions into biological membranes and accumulates selectively into the hydrophobic domain of the lipid bilayer. Upon irradiation with far UV light, INA binds selectively to transmembrane proteins in the viral envelope and renders them inactive. Such inactivation does not alter the ectodomains of the membrane proteins thus preserving the structural and conformational integrity of immunogens on the surface of the virus. In this study, we have used INA to inactivate Venezuelan equine encephalitis virus (VEEV). Treatment of VEEV with INA followed by irradiation with UV light resulted in complete inactivation of the virus. Immuno-fluorescence for VEEV and virus titration showed no virus replication in-vitro. Complete loss of infectivity was also achieved in mice infected with INA treated plus irradiated preparations of VEEV. No change in the structural integrity of VEEV particles were observed after treatment with INA plus irradiation as assessed by electron microscopy. This data suggest that such inactivation strategies can be used for developing vaccine candidates for VEEV and other enveloped viruses.     

Nuclear import and export of plant virus proteins and genomes

Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the host's nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.     

Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis

The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which migrate to the draining lymph node following activation. A point mutation in the E2 glycoprotein gene of VEE that renders the virus avirulent and compromises its ability to spread beyond the draining lymph blocked the appearance of virally infected dendritic cells in the lymph node in vivo. A second-site suppressor mutation that restores viral spread to lymphoid tissues and partially restore virulence likewise restored the ability of VEE to infect dendritic cells in vivo.     

Combinatorial selection of a RNA thioaptamer that binds to Venezuelan equine encephalitis virus capsid protein

A phosphorothioate RNA aptamer (thioaptamer) targeting the capsid protein of Venezuelan equine encephalitis virus (VEEV) was isolated by in vitro combinatorial selection. The selected thioaptamer had a strong binding affinity (approximately 7nM) and high specificity for the target protein. For the binding to the protein, the overall tertiary structure of the thioaptamer is required. We introduce two theoretical methods to examine the effect of phosphorothioate modification on the enhancement of binding affinity and one experimental method to examine the nature of the multiple bands of thioaptamer in a native gel.     

Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1–E1 and E1–E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.     

Specific in vitro lymphocyte transformation with Venezuelan equine encephalitis virus.

Specific lymphocyte transformation to viral antigen was detected in individuals vaccinated with live, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain TC-83. Suspensions of purified, inactivated virus were used an antigen in 250-μ1 lymphocyte cultures, and under optimal conditions the assay demonstrated 10-fold greater incorporation of ¹⁴C-thymidine by lymphocytes from immune than from nonimmune people. The rise in lymphocyte transformation response occurred 1 week after vaccination. The magnitude and range of the lymphocyte transformation response to the VEE viral antigen were similar to the responses seen using antigens derived from five other microbial sources: Francisella tularensis, Coccidioides immitis, Mycobacterium tuberculosis, Streptococcus, and parainfluenza virus. Autologous plasma containing antibody exerts an inhibitory effect on cultures from immune individuals. The onset, magnitude of response, and specificity of this in vitro assay are correlated with the clinical and pathological events of VEE virus infection.     

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.     

Vector infection determinants of Venezuelan equine encephalitis virus reside within the E2 envelope glycoprotein

Epizootic subtype IAB and IC Venezuelan equine encephalitis viruses (VEEV) readily infect the epizootic mosquito vector Aedes taeniorhynchus. The inability of enzootic subtype IE viruses to infect this mosquito species provides a model system for the identification of natural viral determinants of vector infectivity. To map mosquito infection determinants, reciprocal chimeric viruses generated from epizootic subtype IAB and enzootic IE VEEV were tested for mosquito infectivity. Chimeras containing the IAB epizootic structural gene region and, more specifically, the IAB PE2 envelope glycoprotein E2 precursor gene demonstrated an efficient infection phenotype. Introduction of the PE2 gene from an enzootic subtype ID virus into an epizootic IAB or IC genetic backbone resulted in lower infection rates than those of the epizootic parent. The finding that the E2 envelope glycoprotein, the site of epitopes that define the enzootic and epizootic subtypes, also encodes mosquito infection determinants suggests that selection for efficient infection of epizootic mosquito vectors may mediate VEE emergence.     

Production of Venezuelan equine encephalitis virus in cells grown on artificial capillaries

Primary cell cultures, a continuous cell line, and a diploid cell line were grown on an artificial capillary system. The cells were subsequently infected with Venezuelan equine encephalitis virus, and viral replication was studied. Extracellular fluids harvested from this system contained high titers of virus and were relatively free of cell debris.     

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.