Mutations in the E2 Glycoprotein of Venezuelan Equine Encephalitis Virus Confer Heparan Sulfate Interaction, Low Morbidity, and Rapid Clearance from Blood of Mice

AbstractThe arbovirus, Venezuelan equine encephalitis virus (VEE), causes disease in humans and equines during periodic outbreaks. A murine model, which closely mimics the encephalitic form of the disease, was used to study mechanisms of attenuation. Molecularly cloned VEE viruses were used: a virulent, epizootic, parental virus and eight site-specific glycoprotein mutants derived from the parental virus. Four of these mutants were selected in vitro for rapid binding and penetration, resulting in positive charge changes in the E2 glycoprotein from glutamic acid or threonine to lysine (N. L. Davis, N. Powell, G. F. Greenwald, L. V. Willis, B. J. Johnson, J. F. Smith, and R. E. Johnston, Virology 183, 20–31, 1991). Tissue culture adaptation also selected for the ability to bind heparan sulfate as evidenced by inhibition of plaque formation by heparin, decreased infectivity for CHO cells deficient for heparan sulfate, and tight binding to heparin–agarose beads. In contrast, the parental virus and three other mutants did not use heparan sulfate as a receptor. All eight mutants were partially or completely attenuated with respect to mortality in adult mice after a subcutaneous inoculation, and the five mutants that interacted with heparan sulfate in vitro had low morbidity (0–50%). These same five mutants were cleared rapidly from the blood after an intravenous inoculation. In contrast, the parental virus and the other three mutants were cleared very slowly. In summary, the five VEE viruses that contain tissue-culture-selected mutations interacted with cell surface heparan sulfate, and this interaction correlated with low morbidity and rapid clearance from the blood. We propose that one mechanism of attenuation is rapid viral clearance in vivo due to binding of the virus to ubiquitous heparan sulfate

West Nile virus methyltransferase domain interacts with protein kinase G

Background: The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods: We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results: We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions: PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication

Persistence of virus-specific immune responses in the central nervous system of mice after West Nile virus infection

<p>Abstract</p> <p>Background</p> <p>West Nile virus (WNV) persists in humans and several animal models. We previously demonstrated that WNV persists in the central nervous system (CNS) of mice for up to 6 months post-inoculation. We hypothesized that the CNS immune response is ineffective in clearing the virus.</p> <p>Results</p> <p>Immunocompetent, adult mice were inoculated subcutaneously with WNV, and the CNS immune response was examined at 1, 2, 4, 8, 12 and 16 weeks post-inoculation (wpi). Characterization of lymphocyte phenotypes in the CNS revealed elevation of CD19⁺B cells for 4 wpi, CD138 plasma cells at 12 wpi, and CD4⁺and CD8⁺T cells for at least 12 wpi. T cells recruited to the brain were activated, and regulatory T cells (Tregs) were present for at least 12 wpi. WNV-specific antibody secreting cells were detected in the brain from 2 to 16 wpi, and virus-specific CD8⁺T cells directed against an immunodominant WNV epitope were detected in the brain from 1 to 16 wpi. Furthermore, these WNV-specific immune responses occurred in mice with and without acute clinical disease.</p> <p>Conclusions</p> <p>Virus-specific immune cells persist in the CNS of mice after WNV infection for up to 16 wpi.</p

West Nile Virus Methyltransferase Domain Interacts with Protein Kinase

Background The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5. Methods We used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering. Results We identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication. Conclusions PKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication

Induction of sterilizing immunity against West Nile Virus (WNV), by immunization with WNV-like particles produced in insect cells

No specific vaccine for West Nile virus (WNV) is currently available for human use. In the present study, we describe the generation of WNV-like particles (WNV-LPs) in insect cells by use of recombinant baculoviruses expressing the WNV structural proteins prME or CprME. BALB/c mice immunized with purified WNV-LPs developed WNV-specific antibodies that had potent neutralizing activities. Mice immunized with prME-like particles (prME-LPs) showed no morbidity or mortality after challenge with WNV. Immunization with prMELPs can induce sterilizing immunity without producing any evidence of viremia or viral RNA in the spleen or brain. These results suggest that WNV-LPs hold promise as a vaccine candidate for WNV infection

Mosquitoes Inoculate High Doses of West Nile Virus as They Probe and Feed on Live Hosts

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (101.2–104.3 PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 104.3 PFU and 105.0 PFU for Culex tarsalis, 105.9 PFU and 106.1 PFU for Cx. pipiens, 104.7 PFU and 104.7 PFU for Aedes japonicus, and 103.6 PFU and 103.4 PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus (∼102 PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic

Long-term pattern and magnitude of soil carbon feedback to the climate system in a warming world

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here for personal use, not for redistribution. The definitive version was published in Science 358 (2017): 101-105, doi:10.1126/science.aan2874.In a 26-year soil warming experiment in a mid-latitude hardwood forest, we documented changes in soil carbon cycling to investigate the potential consequences for the climate system. We found that soil warming results in a four-phase pattern of soil organic matter decay and carbon dioxide fluxes to the atmosphere, with phases of substantial soil carbon loss alternating with phases of no detectable loss. Several factors combine to affect the timing, magnitude, and thermal acclimation of soil carbon loss. These include depletion of microbially accessible carbon pools, reductions in microbial biomass, a shift in microbial carbon use efficiency, and changes in microbial community composition. Our results support projections of a long-term, self-reinforcing carbon feedback from mid-latitude forests to the climate system as the world warms.This research has been supported by grants from the Department of Energy - DE-SC0010740; DOE DE-SC0016590: and the National Science Foundation - DEB 1237491 (LTER) ; DEB 1456528 (LTREB)

The ISLANDS project I: Andromeda XVI, An Extremely Low Mass Galaxy not Quenched by Reionization

Based on data aquired in 13 orbits of HST time, we present a detailed evolutionary history of the M31 dSph satellite Andromeda XVI, including its life-time star formation history, the spatial distribution of its stellar populations, and the properties of its variable stars. And XVI is characterized by prolonged star formation activity from the oldest epochs until star formation was quenched ~6 Gyr ago, and, notably, only half of the mass in stars of And XVI was in place 10 Gyr ago. And XVI appears to be a low mass galaxy for which the early quenching by either reionization or starburst feedback seems highly unlikely, and thus, is most likely due to an environmental effect (e.g., an interaction), possibly connected to a late infall in the densest regions of the Local Group. Studying the star formation history as a function of galactocentric radius, we detect a mild gradient in the star formation history: the star formation activity between 6 and 8 Gyr ago is significantly stronger in the central regions than in the external regions, although the quenching age appears to be the same, within 1 Gyr. We also report the discovery of 9 RR Lyrae stars, 8 of which belong to And XVI. The RR Lyrae stars allow a new estimate of the distance, (m-M)0= 23.72+/-0.09 mag, which is marginally larger than previous estimates based on the tip of the red giant branch.Comment: Accepted for publication on Ap