Improved Mosquito Housing and Saliva Collection Method Enhances Safety While Facilitating Longitudinal Assessment of Individual Mosquito Vector Competence for Arboviruses.

Background: Assessing the potential for mosquitoes to transmit medically important arboviruses is essential for understanding their threat to human populations. Currently, vector competence studies are typically performed by collecting saliva using a glass capillary tube system which involves sacrificing the mosquito at the time of saliva collection allowing only a single data point. These techniques also require handling infected mosquitoes and glass capillaries, constituting a safety risk. Materials and Methods: To improve the efficiency and safety of assessing vector competence, a novel containment and saliva collection approach for individually housed mosquitoes was developed. The improved housing, allowing longitudinal tracking of individual mosquitoes, consists of a 12-well Corning polystyrene plate sealed with a three-dimensional printed lid that holds organdy netting firmly against the rims of the wells. Results: This method provides excellent mosquito survival for five species of mosquitoes, with at least 79% of each species tested surviving for more than 2 weeks, comparable to the carton survival rates of ≥76%. When the plate housing system was used to assess vector infection, replication of West Nile virus (WNV) in mosquito tissues was similar to traditional containment mosquito housing. Mosquito saliva was collected using either blotting paper pads or traditional glass capillaries to assay viral transmission. The blotting paper collection showed similar or better sensitivity than the capillary method; in addition, longitudinal saliva samples could be collected from individual mosquitoes housed in the 12-well plates. Conclusions: The improved housing and saliva collection technique described herein provides a safer and more informative method for determining vector competence in mosquitoes.

Identification of mosquito proteins that differentially interact with alphavirus nonstructural protein 3, a determinant of vector specificity.

Chikungunya virus (CHIKV) and the closely related onyong-nyong virus (ONNV) are arthritogenic arboviruses that have caused significant, often debilitating, disease in millions of people. However, despite their kinship, they are vectored by different mosquito subfamilies that diverged 180 million years ago (anopheline versus culicine subfamilies). Previous work indicated that the nonstructural protein 3 (nsP3) of these alphaviruses was partially responsible for this vector specificity. To better understand the cellular components controlling alphavirus vector specificity, a cell culture model system of the anopheline restriction of CHIKV was developed along with a protein expression strategy. Mosquito proteins that differentially interacted with CHIKV nsP3 or ONNV nsP3 were identified. Six proteins were identified that specifically bound ONNV nsP3, ten that bound CHIKV nsP3 and eight that interacted with both. In addition to identifying novel factors that may play a role in virus/vector processing, these lists included host proteins that have been previously implicated as contributing to alphavirus replication.

Metabolomic Insights into Human Arboviral Infections: Dengue, Chikungunya, and Zika Viruses.

The global burden of arboviral diseases and the limited success in controlling them calls for innovative methods to understand arbovirus infections. Metabolomics has been applied to detect alterations in host physiology during infection. This approach relies on mass spectrometry or nuclear magnetic resonance spectroscopy to evaluate how perturbations in biological systems alter metabolic pathways, allowing for differentiation of closely related conditions. Because viruses heavily depend on host resources and pathways, they present unique challenges for characterizing metabolic changes. Here, we review the literature on metabolomics of arboviruses and focus on the interpretation of identified molecular features. Metabolomics has revealed biomarkers that differentiate disease states and outcomes, and has shown similarities in metabolic alterations caused by different viruses (e.g., lipid metabolism). Researchers investigating such metabolomic alterations aim to better understand host⁻virus dynamics, identify diagnostically useful molecular features, discern perturbed pathways for therapeutics, and guide further biochemical research. This review focuses on lessons derived from metabolomics studies on samples from arbovirus-infected humans.

Baculovirus Inhibitor-of-Apoptosis Op-IAP3 Blocks Apoptosis by Interaction with and Stabilization of a Host Insect Cellular IAP.

UNLABELLED: Baculovirus-encoded inhibitor of apoptosis (IAP) proteins likely evolved from their host cell IAP homologs, which function as critical regulators of cell death. Despite their striking relatedness to cellular IAPs, including the conservation of two baculovirus IAP repeat (BIR) domains and a C-terminal RING, viral IAPs use an unresolved mechanism to suppress apoptosis in insects. To define this mechanism, we investigated Op-IAP3, the prototypical IAP from baculovirus OpMNPV. We found that Op-IAP3 forms a stable complex with SfIAP, the native, short-lived IAP of host insect Spodoptera frugiperda. Long-lived Op-IAP3 prevented virus-induced SfIAP degradation, which normally causes caspase activation and apoptosis. In uninfected cells, Op-IAP3 also increased SfIAP steady-state levels and extended SfIAP's half-life. Conversely, SfIAP stabilization was lost or reversed in the presence of mutated Op-IAP3 that was engineered for reduced stability. Thus, Op-IAP3 stabilizes SfIAP and preserves its antiapoptotic function. In contrast to SfIAP, Op-IAP3 failed to bind or inhibit native Spodoptera caspases. Furthermore, BIR mutations that abrogate binding of well-conserved IAP antagonists did not affect Op-IAP3's capacity to prevent virus-induced apoptosis. Remarkably, Op-IAP3 also failed to prevent apoptosis when endogenous SfIAP was ablated by RNA silencing. Thus, Op-IAP3 requires SfIAP as a cofactor. Our findings suggest a new model wherein Op-IAP3 interacts directly with SfIAP to maintain its intracellular level, thereby suppressing virus-induced apoptosis indirectly. Consistent with this model, Op-IAP3 has evolved an intrinsic stability that may serve to repress signal-induced turnover and autoubiquitination when bound to its targeted cellular IAP. IMPORTANCE: The IAPs were first discovered in baculoviruses because of their potency for preventing apoptosis. However, the antiapoptotic mechanism of viral IAPs in host insects has been elusive. We show here that the prototypical viral IAP, Op-IAP3, blocks apoptosis indirectly by associating with unstable, autoubiquitinating host IAP in such a way that cellular IAP levels and antiapoptotic activities are maintained. This mechanism explains Op-IAP3's requirement for native cellular IAP as a cofactor and the dispensability of caspase inhibition. Viral IAP-mediated preservation of the host IAP homolog capitalizes on normal IAP-IAP interactions and is likely the result of viral IAP evolution in which degron-mediated destabilization and ubiquitination potential have been reduced. This mechanism illustrates another novel means by which DNA viruses incorporate host death regulators that are modified for resistance to host regulatory controls for the purpose of suppressing host cell apoptosis and acquiring replication advantages.

Insect inhibitor-of-apoptosis (IAP) proteins are negatively regulated by signal-induced N-terminal degrons absent within viral IAP proteins.

UNLABELLED: Inhibitor-of-apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. We report here that the N-terminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the life span of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independent of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, our findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs that are not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signal-responsive degrons that control IAP turnover. Rapid response pathways that trigger IAP degradation and initiate apoptosis independent of canonical prodeath gene (Reaper-Grim-Hid) expression may provide important innate immune advantages. Furthermore, the elimination of these response motifs within viral IAPs, including those of baculoviruses, explains their unusual stability and their potent antiapoptotic activity. IMPORTANCE: Apoptosis is an effective means by which a host controls virus infection. In insects, inhibitor-of-apoptosis (IAP) proteins act as regulatory sentinels by responding to cellular signals that determine the fate of infected cells. We discovered that lepidopteran (moth and butterfly) IAPs, which are degraded upon baculovirus infection, are controlled by a conserved phosphorylation-sensitive degron within the IAP N-terminal leader. The degron likely responds to virus-induced kinase-specific signals for degradation through SKP1/Cullin/F-box complex-mediated ubiquitination. Such signal-induced destruction of cellular IAPs is distinct from degradation caused by well-known IAP antagonists, which act to expel IAP-bound caspases. The major implication of this study is that insects have multiple signal-responsive mechanisms by which the sentinel IAPs are actively degraded to initiate host apoptosis. Such diversity of pathways likely provides insects with rapid and efficient strategies for pathogen control. Furthermore, the absence of analogous degrons in virus-encoded IAPs explains their relative stability and antiapoptotic potency.

Baculovirus F-box protein LEF-7 modifies the host DNA damage response to enhance virus multiplication.

The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein.